U.S. patent application number 15/214400 was filed with the patent office on 2016-11-03 for stabilized step function opsin proteins and methods of using the same.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Karl Deisseroth, Lief Fenno, Ofer Yizhar.
Application Number | 20160316730 15/214400 |
Document ID | / |
Family ID | 46025140 |
Filed Date | 2016-11-03 |
United States Patent
Application |
20160316730 |
Kind Code |
A1 |
Deisseroth; Karl ; et
al. |
November 3, 2016 |
STABILIZED STEP FUNCTION OPSIN PROTEINS AND METHODS OF USING THE
SAME
Abstract
Provided herein are compositions comprising non-human animals
comprising neurons expressing stabilized step function opsin
proteins on neural plasma membranes and methods of using the same
to selectively depolarize neurons residing in microcircuits of the
pre-frontal cortex to affect one or more social behaviors,
communications, and/or conditioned behaviors in the non-human
animal.
Inventors: |
Deisseroth; Karl; (Stanford,
CA) ; Yizhar; Ofer; (Palo Alto, CA) ; Fenno;
Lief; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
University |
Stanford |
CA |
US |
|
|
Family ID: |
46025140 |
Appl. No.: |
15/214400 |
Filed: |
July 19, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13882666 |
Sep 17, 2013 |
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PCT/US11/59390 |
Nov 4, 2011 |
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15214400 |
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61511905 |
Jul 26, 2011 |
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61410704 |
Nov 5, 2010 |
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61410711 |
Nov 5, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01K 2267/03 20130101;
A01K 67/0275 20130101; A01K 2217/206 20130101; C12N 2740/16043
20130101; A01K 2227/105 20130101; C07K 14/405 20130101; C12N 5/0619
20130101; G01N 33/5088 20130101; C12N 2750/14143 20130101; A01K
2267/0393 20130101; G01N 33/5091 20130101 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C12N 5/0793 20060101 C12N005/0793; G01N 33/50 20060101
G01N033/50; C07K 14/405 20060101 C07K014/405 |
Claims
1. A non-human animal comprising a first light-activated cation
channel protein expressed in neurons of the pre-frontal cortex of
the animal, wherein the protein is capable of inducing depolarizing
current in the neurons by light and exhibits rapid step-like
activation in response to a single pulse of light having a first
wavelength and deactivation in response to a pulse of light having
a second wavelength, wherein the depolarizing current in the
neurons is maintained for at least about ten minutes; and wherein
the activation of the protein in the pre-frontal cortex neurons
induces changes in social behaviors, communications, and/or
conditioned behaviors in the animal.
2. The animal of claim 1, wherein the protein comprises the amino
acid sequence of ChR2, ChR1, VChR1, or VChR2 with an amino acid
substitution at a residue corresponding to C128 or D156 of the
amino acid sequence of ChR2.
3. The animal of claim 1, wherein the protein comprises the amino
acid sequence of ChR2, ChM, VChR1, or VChR2 with amino acid
substitutions at a residues corresponding to C128 and D156 of the
amino acid sequence of ChR2.
4. The animal of claim 2 or 3, wherein the residue corresponding to
C128 of the amino acid sequence of ChR2 is substituted to
serine.
5. The animal of claim 2 or 3, wherein the residue corresponding to
D156 of the amino acid sequence of ChR2 is substituted to a
non-acidic amino acid.
6. The animal of claim 2 or 3, wherein the residue corresponding to
D156 of the amino acid sequence of ChR2 is substituted to
alanine.
7. The animal of claim 1, wherein the protein comprises an amino
acid sequence at least 95% identical to the sequence shown in SEQ
ID NO:1, and wherein C128 is substituted with serine and D156 is
substituted with alanine.
8. The animal of any one of claims 1-7, wherein the protein is
expressed by delivering the polynucleotide sequence encoding the
protein using a viral vector.
9. The animal of claim 8, wherein the viral vector is an
adeno-associated viral vector, a retroviral vector, a lentiviral,
an adenoviral vector, or a HSV vector.
10. The animal of any one of claims 1-9, wherein said social
behaviors are selected from the group consisting of allogrooming,
resident-intruder aggression, isolation-induced fighting, sexual
behavior, parental behavior, social recognition, and auditory
communication.
11. The animal of claim 11, wherein the neurons are in the
infralimbic or prelimbic subregions of the medial prefrontal
cortex.
12. The animal of any one of claims 1-11, wherein the protein is
expressed in either inhibitory neurons or excitatory neurons of the
pre-frontal cortex.
13. The animal of claim 12, wherein the excitatory neurons are
pyramidal neurons.
14. The animal of claim 12, wherein the inhibitory neurons are
GABAergic parvalbumin neurons.
15. The animal of claim 14, wherein the animal further comprises a
second light-activated protein expressed in said inhibitory neurons
or said excitatory neurons of the pre-frontal cortex and wherein
the second light activated protein is not expressed in the same
neurons as the first light-activated protein.
16. The animal of claim 15, wherein the second light activated
protein is C1V1.
17. A brain slice comprising neurons of the pre-frontal cortex,
wherein a light-activated protein is expressed in the neurons of
the pre-frontal cortex, wherein the protein is capable of inducing
depolarizing current in the neurons by light and exhibits rapid
step-like activation in response to a single pulse of light having
a first wavelength and deactivation in response to a pulse of light
having a second wavelength; wherein the depolarizing current in the
neurons is maintained for at least about ten minutes.
18. The brain slice of claim 17, wherein the protein is selected
from the group consisting of ChR2, ChR1, VChR1, or VChR2.
19. The brain slice claim 18, wherein the opsin comprises at least
one amino acid substitution at amino acid residues corresponding to
C128 and D156 of the amino acid sequence of ChR2.
20. The brain slice of claim 19, wherein the substitution at the
amino acid residue corresponding to C128 of the amino acid sequence
of ChR2 is a substitution to serine.
21. The brain slice of claim 19, wherein the substitution at the
amino acid residue corresponding to D156 of the amino acid sequence
of ChR2 is a substitution to a non-acidic amino acid.
22. The brain slice of claim 19, wherein the substitution at the
amino acid residue corresponding to D156 of the amino acid sequence
of ChR2 is a substitution to alanine.
23. The brain slice of claim 19, wherein the protein comprises an
amino acid sequence at least 95% identical to the sequence shown in
SEQ ID NO:1, and wherein C128 is substituted with serine and D156
is substituted with alanine.
24. The brain slice of any one of claims 17-23, wherein the protein
is expressed in either inhibitory neurons or excitatory neurons of
the pre-frontal cortex.
25. A method for identifying a chemical compound that inhibits the
depolarization of excitatory or inhibitory neurons in the
prefrontal cortex of a non-human animal, the method comprising: (a)
depolarizing excitatory or inhibitory neurons in the prefrontal
cortex of a non-human animal comprising a first light-activated
protein cation channel protein expressed on the cell membrane of
the neurons of the pre-frontal cortex of the animal, wherein the
protein is capable of mediating a depolarizing current in the
neurons when the neurons are illuminated with light, wherein the
protein exhibits rapid step-like activation in response to a single
pulse of light having a first wavelength and deactivation in
response to a pulse of light having a second wavelength; wherein
the depolarizing current in the neurons is maintained for at least
about ten minutes; wherein the protein comprises the amino acid
sequence of ChR2, ChR1, VChR1, or VChR2 with amino acid
substitutions at amino acid residues corresponding to C128 and D156
of the amino acid sequence of ChR2; wherein the activation of the
protein in the pre-frontal cortex neurons induces changes in social
behaviors, communications, and/or conditioned behaviors in the
animal; (b) measuring an excitatory post synaptic potential (EPSP)
or an inhibitory post synaptic current (IPSC) in response to
selectively depolarizing the excitatory neurons comprising the
light-activated protein; (c) contacting the excitatory or
inhibitory neurons with a chemical compound; and (d) measuring the
excitatory post synaptic potential (EPSP) or the inhibitory post
synaptic current (IPSC) to determine if contacting the excitatory
neurons with the chemical compound inhibits the depolarization of
the neurons.
26. The method of claim 25, wherein the light activated protein
comprises an amino acid sequence at least 95% identical to the
sequence shown in SEQ ID NO:1 and wherein the protein has a D to A
mutation at amino acid 156.
27. The method of claim 25, wherein the single pulse of light
having a first wavelength is for 100 milliseconds or less.
28. The method of claim 25, wherein the first wavelength is blue
light and wherein the second wavelength is green or yellow
light.
29. A method for identifying a chemical compound that restores a
social behavior, communication, and/or conditioned behavior in a
non-human animal, the method comprising: (a) depolarizing
excitatory neurons in the prefrontal cortex of a non-human animal
comprising a light-activated protein cation channel protein
expressed on the cell membrane of the neurons, wherein the protein
is capable of inducing a depolarizing current in the neurons when
the neurons are illuminated with light, wherein the protein
exhibits rapid step-like activation in response to a single pulse
of light having a first wavelength and deactivation in response to
a pulse of light having a second wavelength; wherein the
depolarizing current in the neurons is maintained for at least
about ten minutes; and wherein the protein comprises the amino acid
sequence of ChR2, ChR1, VChR1, or VChR2 with amino acid
substitutions at amino acid residues corresponding to C128 and D156
of the amino acid sequence of ChR2, wherein depolarizing the
excitatory neuron inhibits one or more social behaviors,
communications, and/or conditioned behaviors in the non-human
animal; (b) administering a chemical compound to the non-human
animal; and (c) determining if the administration of the chemical
compound to the non-human animal restores said one or more social
behaviors, communications, and/or conditioned behaviors in the
non-human animal.
30. The method of claim 29, wherein the social behavior is selected
from the group consisting of: allogrooming, resident-intruder
aggression, isolation-induced fighting, sexual behavior, parental
behavior, social recognition, and auditory communication.
31. The method of claim 29, wherein the administration of the
chemical compound decreases or inhibits depolarization of
excitatory neurons.
32. The method of claim 29, wherein the administration of the
chemical compound increases depolarization of inhibitory
neurons.
33. A method of modeling the symptoms of a central nervous system
(CNS) disorder in a patient, the method comprising: (a) identifying
a target neural circuit associated with the CNS disorder; (b)
expressing light-responsive opsins in the target neural circuit;
(c) activating the light-responsive opsins in the target neural
circuit; and (d) monitoring a symptom of the central nervous system
(CNS) disorder.
34. A method of claim 33, wherein activating the light responsive
opsins includes elevating an excitation/inhibition (E/I) balance in
the target neural circuit while preserving the responsiveness of
the targeted neural circuit to intrinsic electrical activity,
wherein symptoms of the CNS disorder are temporally increased.
35. The method of claim 33, wherein the target neural circuit is
located in the prefrontal cortex.
36. The method of claim 33, wherein the step of activating includes
increasing the excitation relative to the inhibition in the target
neural circuit.
37. The method of claim 33, wherein the step of expressing further
includes targeting cell-types of the target neural circuit for
increased expression of exogenous light-responsive ion channels
relative to other cell-types of the target neural circuit.
38. A method of modeling the symptoms of a central nervous system
(CNS) disorder in a patient, the method comprising: modifying an
excitation/inhibition (E/I) balance in a target neural circuit of
the CNS while preserving the responsiveness of the targeted neural
circuit to intrinsic electrical activity, wherein symptoms of the
CNS disorder are temporally increased.
39. The method of claim 38, wherein the step of modifying an E/I
balance includes applying optical stimulus to exogenous
light-responsive ion channels that are expressed in cells of the
targeted neural circuit of the CNS.
40. The method of claim 38, wherein the target neural circuit is
located in the prefrontal cortex.
41. The method of claim 38, wherein the step of modifying includes
increasing the excitation relative to the inhibition in the target
neural circuit.
42. The method of claim 38, further including the step of targeting
cell-types of the target neural circuit for increased expression of
exogenous light-responsive ion channels relative to other
cell-types of the target neural circuit.
43. A method comprising: assessing the effects of a treatment for a
central nervous system (CNS) disorder by expressing
light-responsive opsins in a target neural circuit associated with
the CNS disorder; activating the light-responsive opsins in the
target neural circuit, wherein symptoms of the CNS disorder are
increased; applying the treatment; and monitoring the symptoms of
the CNS disorder.
44. A method for assessing the effects of a treatment for substance
dependence, the method comprising: artificially inducing a central
nervous system (CNS) disorder in an animal by activating the
light-responsive opsins in a target neural circuit associated with
the CNS disorder, wherein symptoms of the CNS disorder are
increased; applying the treatment; and monitoring the symptoms of
the CNS disorder.
45. A system comprising: a set of neurons associated with a CNS
disorder; a drug delivery device for providing drugs to the set of
neurons; and a monitoring device for assessing activity of the set
of neurons in response to the drugs being provided to the set of
neurons.
46. The system of claim 45, wherein the set of neurons include
light-responsive opsins, and wherein the system further includes an
optical delivery system for exciting the neurons by activating the
light-responsive opsins.
47. The system of claim 45, wherein the monitoring device is
further configured to assess the activity of the set of neurons by
monitoring electrical activation of the set of neurons.
48. The system of claim 45, wherein the monitoring device is
further configured to assess the activity of the set of neurons by
monitoring electrical activation of the set of neurons.
49. A method of claim 33, wherein activating the light responsive
opsins includes lowering an excitation/inhibition (E/I) balance in
the target neural circuit while preserving the responsiveness of
the targeted neural circuit to intrinsic electrical activity.
50. A method comprising: elevating an excitation/inhibition (EA)
balance in a targeted neural circuit in a prefrontal cortex of a
subject/patient while preserving the responsiveness of the targeted
neural circuit to intrinsic electrical activity, wherein symptoms
of a disorder are temporally increased; introducing a stimulus to
the subject; and monitoring the symptoms of the disorder.
51. The method of claim 50, further including assessing the effect
of the stimulus on the symptoms.
52. The method of claim 50, wherein the symptoms of the disorder
include symptoms of schizophrenia.
53. The method of claim 50, wherein the symptoms of the disorder
include symptoms of autism.
54. The method of claim 50, wherein the stimulus is a drug-based
treatment.
55. The method of claim 50, wherein the stimulus is a
psychological-based treatment.
56. The method of claim 54, wherein pyramidal cells and parvalbumin
cells in the prefrontal cortex are monitored for an effect of the
drug-based treatment.
57. The method of claim 50, further including determining the
relative effect of excitatory pyramidal neurons and
parvalbumin-expressing inhibitory intemeurons in the prefrontal
cortex on the E/I balance in the targeted neural circuit.
58. The method of claim 57, further including assessing the
relative effect of a drug on the pyramidal neurons and the
parvalbumin intemeurons.
59. The method of claim 50, wherein the stimulus inhibits pyramidal
neurons in the prefrontal cortex.
60. The method of claim 50, wherein the stimulus excites pyramidal
neurons in the prefrontal cortex.
61. The method of claim 50, wherein the stimulus inhibits
inhibitory or parvalbumin-expressing interneurons in the prefrontal
cortex.
62. The method of claim 50, wherein the stimulus excites inhibitory
or parvalbumin-expressing interneurons in the prefrontal
cortex.
63. The method of claim 50, wherein the stimulus excites one of a
set of pyramidal neurons and parvalbumin-expressing interneurons in
the prefrontal cortex and inhibiting the other one of the set of
pyramidal neurons and parvalbumin-expressing interneurons in the
prefrontal cortex.
64. A method comprising: providing a stimulus to a subject, the
subject exhibiting symptoms of a neurological disease; and
assessing the effect of the stimulus on the symptoms.
65. The method of claim 64, wherein the stimulus is a potential
treatment and assessing the effect of the stimulus includes
determining the efficacy of the potential treatment.
66. The method of claim 16, further including modifying the
potential treatment based on the determined efficacy of the
potential treatment, delivering the modified potential treatment to
the subject, and accessing the efficacy of the modified potential
treatment.
67. Devices, reagents, tools, technologies, methods and approaches
for using models of disorders to study the disorders, to identify
phenotypes/endophenotypes, and/or to identify treatments.
68. A method comprising: lowering an excitation/inhibition (E/I)
balance in a targeted neural circuit in a prefrontal cortex of a
subject while preserving the responsiveness of the targeted neural
circuit to intrinsic electrical activity; introducing a stimulus to
the subject; and monitoring the symptoms of the disorder.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 61/410,704 filed on Nov. 5, 2010, U.S. Provisional
Patent Application No. 61/410,711 filed on Nov. 5, 2010, and U.S.
Provisional Patent Application No. 61/511,905 filed on Jul. 26,
2011, the disclosures of each of which are incorporated by
reference herein in their entireties.
TECHNICAL FIELD
[0002] This application pertains to compositions comprising
non-human animal cells expressing stabilized step function opsin
(SSFO) proteins on their plasma membranes and methods of using the
same to selectively depolarize neurons residing in microcircuits of
the pre-frontal cortex to affect one or more social behaviors,
communications, and/or conditioned behaviors in the non-human
animal.
BACKGROUND
[0003] Optogenetics is the combination of genetic and optical
methods used to control specific events in targeted cells of living
tissue, even within freely moving mammals and other animals, with
the temporal precision (millisecond-timescale) needed to keep pace
with functioning intact biological systems. The hallmark of
optogenetics is the introduction of fast light-activated channel
proteins to the plasma membranes of target neuronal cells that
allow temporally precise manipulation of neuronal membrane
potential while maintaining cell-type resolution through the use of
specific targeting mechanisms. Among the microbial opsins which can
be used to investigate the function of neural systems are the
channelrhodopsins (ChR2, ChR1, VChR1, and SFOs) used to promote
depolarization in response to light. In just a few short years, the
field of optogenetics has furthered the fundamental scientific
understanding of how specific cell types contribute to the function
of biological tissues such as neural circuits in vivo. Moreover, on
the clinical side, optogenetics-driven research has led to insights
into Parkinson's disease and other neurological and psychiatric
disorders.
[0004] However, in spite of these advances, the neurophysiological
substrates of most psychiatric disorders remain poorly understood,
despite rapidly emerging information on genetic factors that are
associated with complex behavioral phenotypes such as those
observed in autism and schizophrenia (Cichon et al., The American
Journal of Psychiatry 166(5):540 (2009); O'Donovan et al., Human
Genetics 126(1): 3 (2009)). One remarkable emerging principle is
that a very broad range of seemingly unrelated genetic
abnormalities can give rise to the same class of psychiatric
phenotype (such as social behavior dysfunction; Folstein &
Rosen-Sheidley, Nature Reviews 2(12):943 (2001)). This surprising
pattern has pointed to the need to identify simplifying
circuit-level insights that could unify diverse genetic factors
under a common pathophysiological principle.
[0005] One such circuit-level hypothesis is that elevation in the
ratio of cortical cellular excitation and inhibition (cellular E/I
balance) could give rise to the social and cognitive deficits of
autism (Rubenstein, Current Opinion in Neurology 23(2):118;
Rubenstein & Merzenich, Genes, Brain, and Behavior 2(5):255
(2003)). This hypothesis could potentially unify diverse streams of
pathophysiological evidence, including the observation that many
autism-related genes are linked to gain-of-function phenotypes in
ion channels and synaptic proteins (Bourgeron, Current Opinion in
Neurobiology 19 (2), 231 (2009)) and that .about.30% of autistic
patients also show clinically apparent seizures (Gillberg &
Billstedt, Acta Psychiatrica Scandinavica, 102(5):321 (2000)).
However, it has not been clear if such an imbalance (to be relevant
to disease symptoms) would be operative on the chronic (e.g. during
development) or the acute timescale. Furthermore, this hypothesis
is by no means universally accepted, in part because it has not yet
been susceptible to direct testing. Pharmacological and electrical
interventions lack the necessary specificity to selectively favor
activity (in a manner fundamentally distinct from receptor
modulation) of neocortical excitatory cells over inhibitory cells,
whether in the clinical setting or in freely behaving experimental
mammals during social and cognitive tasks. It is perhaps related to
challenges such as this that the social and cognitive deficits of
autism and schizophrenia have proven largely unresponsive to
conventional psychopharmacology treatments in the clinic.
[0006] Existing optogenetic methods are also inadequate for this
purpose; driving coordinated spikes selectively in excitatory or
inhibitory cells with a channelrhodopsin is feasible, but not well
suited to the sparse coding and asynchronous firing patterns of
neocortical pyramidal cells. Moreover, the continuous presence of
an optical fiber and other hardware poses challenges for prolonged
behavioral tests with fast and spatially complex movements typical
of social behavior and cognitive measures (for example in
contextual conditioning). Instead, selectively favoring excitation
of one population over another with a bistable step-function opsin
(SFO) gene product could partially address these challenges, since
the targeted population would not be driven with coordinated
spikes, but merely sensitized to native inputs that can be sparse
and asynchronous. Use of SFOs also has the potential to address the
hardware challenge, since the orders-of-magnitude greater light
sensitivity characteristic of SFOs could in theory allow non-brain
penetrating light delivery, and the persistent action of the
bistable SFOs after light-off could allow hardware-free behavioral
testing. However, the known SFOs (C128A,S,T and D156A) are not
stable enough to produce constant photocurrent after a single light
flash over the many minutes required for complex behavioral
testing.
[0007] What is needed, therefore, is an optogenetic tool which
would permit direct testing of the E/I balance hypothesis in the
prefrontal cortex both in vitro and in vivo in freely-moving mice.
Such a light-activated protein could permit investigation of the
effect of bi-directional modulation of prefrontal cellular E/I
balance on both conditioned and innate behaviors relevant for
cognitive and social dysfunction, as well as probe the resulting
effects on circuit physiology and quantitative transmission of
information.
BRIEF SUMMARY OF THE INVENTION
[0008] Provided herein are animal cells, non-human animals, brain
slices comprising cells expressing stabilized step function opsin
proteins on their plasma membranes and methods of using the same to
selectively depolarize neurons residing in microcircuits of the
pre-frontal cortex.
[0009] Accordingly, provided herein are non-human animals
comprising a first light-activated cation channel protein expressed
in neurons of the pre-frontal cortex of the animal, wherein the
protein is capable of inducing depolarizing current in the neurons
by light and exhibits rapid step-like activation in response to a
single pulse of light having a first wavelength and deactivation in
response to a pulse of light having a second wavelength, wherein
the depolarizing current in the neurons is maintained for at least
about ten minutes; and wherein the activation of the protein in the
pre-frontal cortex neurons induces changes in social behaviors,
communications, and/or conditioned behaviors in the animal.
[0010] In some aspects, there is provided a brain slice comprising
neurons of the pre-frontal cortex, wherein a light-activated
protein is expressed in the neurons of the pre-frontal cortex,
wherein the protein is capable of inducing depolarizing current in
the neurons by light and exhibits rapid step-like activation in
response to a single pulse of light having a first wavelength and
deactivation in response to a pulse of light having a second
wavelength; wherein the depolarizing current in the neurons is
maintained for at least about ten minutes.
[0011] In another aspect, there is provided a method for
identifying a chemical compound that inhibits the depolarization of
excitatory or inhibitory neurons in the prefrontal cortex of a
non-human animal, the method comprising: (a) depolarizing
excitatory or inhibitory neurons in the prefrontal cortex of a
non-human animal comprising a first light-activated protein cation
channel protein expressed on the cell membrane of the neurons of
the pre-frontal cortex of the animal, wherein the protein is
capable of mediating a depolarizing current in the neurons when the
neurons are illuminated with light, wherein the protein exhibits
rapid step-like activation in response to a single pulse of light
having a first wavelength and deactivation in response to a pulse
of light having a second wavelength; wherein the depolarizing
current in the neurons is maintained for at least about ten
minutes; wherein the protein comprises the amino acid sequence of
ChR2, ChR1, VChR1, or VChR2 with amino acid substitutions at amino
acid residues corresponding to C128 and D156 of the amino acid
sequence of ChR2; wherein the activation of the protein in the
pre-frontal cortex neurons induces changes in social behaviors,
communications, and/or conditioned behaviors in the animal; (b)
measuring an excitatory post synaptic potential (EPSP) or an
inhibitory post synaptic current (IPSC) in response to selectively
depolarizing the excitatory neurons comprising the light-activated
protein; (c) contacting the excitatory or inhibitory neurons with a
chemical compound; and (d) measuring the excitatory post synaptic
potential (EPSP) or the inhibitory post synaptic current (IPSC) to
determine if contacting the excitatory neurons with the chemical
compound inhibits the depolarization of the neurons.
[0012] In another aspect, there is provided a method for
identifying a chemical compound that restores a social behavior,
communication, and/or conditioned behavior in a non-human animal,
the method comprising: (a) depolarizing excitatory neurons in the
prefrontal cortex of a non-human animal comprising a
light-activated protein cation channel protein expressed on the
cell membrane of the neurons, wherein the protein is capable of
inducing a depolarizing current in the neurons when the neurons are
illuminated with light, wherein the protein exhibits rapid
step-like activation in response to a single pulse of light having
a first wavelength and deactivation in response to a pulse of light
having a second wavelength; wherein the depolarizing current in the
neurons is maintained for at least about ten minutes; and wherein
the protein comprises the amino acid sequence of ChR2, ChR1, VChR1,
or VChR2 with amino acid substitutions at amino acid residues
corresponding to C128 and D156 of the amino acid sequence of ChR2,
wherein depolarizing the excitatory neuron inhibits one or more
social behaviors, communications, and/or conditioned behaviors in
the non-human animal; (c) administering a chemical compound to the
non-human animal; and (d) determining if the administration of the
chemical compound to the non-human animal restores said one or more
social behaviors, communications, and/or conditioned behaviors in
the non-human animal.
[0013] The present disclosure relates to optical control over
nervous system disorders (such as disorders associated with social
dysfunction), as described herein. While the present disclosure is
not necessarily limited in these contexts, various aspects of the
disclosure may be appreciated through a discussion of examples
using these and other contexts.
[0014] Various embodiments of the present disclosure relate to an
optogenetic system or method that correlates temporal, spatio
and/or cell-type control over a neural circuit with measurable
metrics. For instance, various metrics or symptoms might be
associated with a neurological disorder (such as a neurological
disorder exhibiting various symptoms of social dysfunction). The
optogenetic system targets a neural circuit within a
subject/patient for selective control thereof. The optogenetic
system involves monitoring the subject/patient for the metrics or
symptoms associated with the neurological disorder. In this manner,
the optogenetic system can provide detailed information about the
neural circuit, its function and/or the neurological disorder.
[0015] Consistent with the embodiments discussed herein, particular
embodiments relate to studying and probing disorders. Other
embodiments relate to the identification and/or study of phenotypes
and endophenotypes. Still other embodiments relate to the
identification of treatment targets.
[0016] Aspects of the present disclosure are directed toward the
artificial inducement of disorder/disease states on a fast-temporal
time scale. These aspects allow for study of disease states in
otherwise healthy animals. This can be particularly useful for
diseases that are poorly understood and otherwise difficult to
accurately model in live animals. For instance, it can be difficult
to test and/or study disease states due to the lack of available
animals exhibiting the disease state. Moreover, certain embodiments
allow for reversible disease states, which can be particularly
useful for establishing baseline/control points for testing and/or
for testing the effects of a treatment on the same animal when
exhibiting the disease state and when not exhibiting the disease
state. Various other possibilities exist, some of which are
discussed in more detail herein.
[0017] Aspects of the present disclosure are directed to using an
artificially induced disorder/disease state for the study of
disease states in otherwise healthy animals. This can be
particularly useful for diseases that are poorly understood and
otherwise difficult to accurately model in living animals. For
instance, it can be difficult to test and/or study disease states
due to the lack of available animals exhibiting the disease state.
Moreover, certain embodiments allow for reversible disease states,
which can be particularly useful in establishing baseline/control
points for testing and/or for testing the effects of a treatment on
the same animal when exhibiting the disease state and when not
exhibiting the disease state.
[0018] Certain aspects of the present disclosure are directed to a
method that includes modifying (e.g., elevating or lowering) an
excitation/inhibition (E/I) balance in a targeted neural circuit in
a prefrontal cortex of a subject/patient. For instance, the E/I
balance is changed to a level that preserves the responsiveness of
the targeted neural circuit to intrinsic electrical activity while
symptoms of a disorder are temporally increased. While the E/I
balance is changed, a stimulus is introduce to the subject/patient
and the symptoms of the disorder are monitored. The subject can be
a test animal that is healthy, or an animal model of a disorder.
The result of the manipulation is either a transient recapitulation
of disease symptoms (in an otherwise healthy animal) or alleviation
of symptoms (in an animal model of a neurological disorder). In
certain more specific embodiments, the monitoring of the symptoms
also includes assessing the efficacy of the stimulus in mitigating
the symptoms of the disorder. Various other possibilities exist,
some of which are discussed in more detail herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Various example embodiments may be more completely
understood in consideration of the following description and the
accompanying drawing, in which:
[0020] FIG. 1: Kinetic and absorbance properties of a fully
stabilized SFO. (a) Normalized absorbance spectra of dark-adapted
wild type ChR2, ChR2-C128S, ChR2-D156A and ChR2-C128S/D156A (SSFO).
(b-d) Absorption spectra recorded after illumination with 450 nm
light for 30 seconds. Absorption difference spectra taken from the
corresponding absorption spectra are shown in the insets. Spectra
were collected at the indicated times after the end of
illumination; note prominent recovery after 3 min in the single
mutants, in contrast to the double mutant. (e) Simplified
photocycle scheme; in C128/D156 mutants the transition P520 to P480
is likely slowed down or blocked, avoiding the desensitized state
Des470 which cannot be reactivated with 470 nm light. (f-h)
Monochromatic absorption changes recorded at the indicated
wavelength before, during and after illumination with 450 nm light
for all three variants, highlighting the distinct stability of the
double mutant.
[0021] FIG. 2 depicts Stable step-modulation of neural activity in
multiple cell types in vitro and in vivo. (a) Activation (top left)
and deactivation (bottom left) spectra recorded from cultured
neurons expressing ChR2 (C128S/D156A). Gray horizontal bars
indicate light pulses and trace colors indicate wavelength of light
used in each light pulse; summary spectra (right) for measurements
of activation and deactivation of ChR2(C128S/D156A) are shown. (b)
Monoexponential fits of photocurrent decay in cells expressing
ChR2(C128S/D 156A) (black; -t=29.3 min) or ChR2(D156A) (gray;
-t=6.9 min) (c) Representative whole-cell patch clamp recording of
photocurrent in cultured hippocampal neuron expressing
ChR2(C128S/D156A; "SSFO"). Bars indicate activation and
deactivation light pulses; recording carried out in the
naturalistic setting of incoming synaptic excitatory postsynaptic
currents (epscs). (d) Whole-cell photocurrent responses of a
cultured neuron expressing SSFO to 470 nm light pulses of indicated
power (left). Pulse lengths were 2 s (gray horizontal bar traces)
or 5s (black horizontal bar traces). Dashed lines mark light pulse
termination. Time constants for activation (T) are displayed on a
log-log plot versus light power (n=27 recordings from 5 cells;
middle). Regardless of light power, the calculated number of
incident photons arriving at each cell for photocurrents to reach
the exponential curve constant (63% of Imax) for that cell was
constant (right). Each point represents a photon number from a
single recording at a given light power (Methods). (e) Optrode
recording configuration. 470 nm and 561 nm lasers were coupled to
an optical fiber through a fiber coupler. A tungsten electrode was
attached to the optical fiber with a 400 .mu.m 1 lm projection past
the fiber tip and advanced into the brain. (f) Activation of
excitatory neurons using CaMKIIa-SSFO in anaesthetized animals
stably elevates neuronal activity within the injected loci. Starred
example trace is plotted below the instantaneous spike-rate heat
maps calculated with 2s moving average. Each heat-map line
represents one sweep at indicated depth (3 sweeps at each site);
470 nm activation pulse and 561 nm deactivation pulses are
indicated by blue and green bars, respectively. (g) Activation of
PV-positive interneurons with PV::Cre/DIO-SSFO inhibits local
network activity within the injected loci. Starred example trace is
plotted below the instantaneous spike rate heat maps. (h) Average
spike rates of traces showing significant differences in activity
pre- and post-stimulation before activation, after activation, and
after deactivation in CK-SSFO (squares) and PV::Cre/DIO-SSFO
(circles) animals (n=2 mice, >5 recording sites per animal). (i)
Representative 10-min long recording demonstrating sustained
activity of SSFO. Instantaneous spike-rate heat maps are shown for
activity of isolated single units indicated as Neuron 1 and Neuron
2; waveforms of indicated units are plotted next to corresponding
traces.
[0022] FIG. 3 depicts elevated, but not reduced, prefrontal E/I
balance leads to behavioral impairment. (a) Wild-type or PV::Cre
transgenic mice injected with control CaMKII.alpha.-eYFP,
CaMKII.alpha.-SSFO, or DIO-SSFO virus in mPFC and chronically
implanted with fiber optic connector were subjected to fear
conditioning and social exploration tests. (b) Confocal image from
a mouse injected with CaMKII.alpha.-SSFO-eYFP virus shows
expression in prelimbic (PL) and infralimbic (IL) cortex. (c)
Representative images of prefrontal slices from PV::Cre/DIO-SSFO
and CaMKII.alpha.-SSFO mice stained for c-fos 90 min following a 2s
470 nm light pulse; Bar=25 um. Graph shows average c-fos positive
cell counts in mPFC of CaMKII.alpha.-SSFO, and PV::Cre/DIO-SSFO
animals. (d) Summary data for social exploration in control,
CaMKII.alpha.-SSFO, and PV::Cre/DIO-SSFO mice of a juvenile
intruder in the home cage. CaMKII.alpha.-SSFO mice showed a
significant reduction in social exploration. (e) Mice administered
one 2s 470 nm pulse of light prior to fear conditioning were tested
the next day for freezing in response to the conditioned context or
to a conditioned auditory cue; CaMKII.alpha.-SSFO mice were
significantly impaired in freezing response to both conditioned
stimuli. On the following day, mice were reconditioned without
optical stimulation and freezing was evaluated 24 h later. All mice
showed similar freezing behavior in the absence of light. (f)
Open-field exploration is indistinguishable in CaMKII.alpha.-SSFO
(blue) and CaMKII.alpha.-EYFP (gray) control mice, before (Test 1)
and after (Test light activation. Example track from animal
expressing CaMKII.alpha.-SSFO for Test 1 (top) and Test two
(bottom) are shown. (g) Exploration of a novel object over a
10-minute period is similar in mice expressing CaMKII.alpha.-SSFO
(black) and CaMKII.alpha.-EYFP (gray). (h) Fluorescence images of
coronal sections from wild-type mice injected with
CaMKII.alpha.-SSFO in PFC (top) or V1 (bottom). (i) Social behavior
in the 3-chamber test is impaired following a 2 s 470 nm light
pulse in mice expressing CaMKII.alpha.-SSFO in PFC (n=6), but not
in control mice (n=8) or mice expressing CaMKII.alpha.-SSFO in V1
(n=8). All bar graphs depict mean.+-.s.e.m. (* p<0.05, **
p<0.005, *** p<0.0005). (j) High magnification confocal
images of a 40 .mu.m coronal brain slice from a PV::Cre mouse
bilaterally injected with Cre-dependent AAV5-EF1 a-DIO-SSFO-EYFP
virus and stained with anti-parvalbumin antibody. Arrows indicate
double-labeled PV neurons identified by membrane-bound EYFP
labeling; arrowhead shows PV-positive neuron that did not express
detectable levels of SSFO-EYFP. (k) Low-power confocal image of the
same slice shown in (j), demonstrating spatially restricted
expression of the DIO-SSFO virus in mPFC. (1) Percent
double-labeled cells out of the entire PV+ cell population, and out
of the entire YFP+ cell population as counted from
high-magnification confocal z-stacks (n=7 slices from 4 mice; total
of 617 PV+ cells counted, 191 YFP+ cells, 169 double-labeled
cells). This number is consistent with .about.40% PV neurons
expressing Cre recombinase in this line and approximately 50%
transduction efficiency of the virus. Since expression of PV is not
uniform across cells, some PV+ neurons might express undetectable
levels of PV but still contain sufficient levels of Cre for
activating DIO-SSFO expression. (m) Quantification of c-fos
immunofluorescence in cortical and subcortical regions from animals
injected unilaterally with CaMKII.alpha.::SSFO-EYFP virus (gray;
n=2 mice) and controls injected unilaterally with
CaMKIl.alpha.::EYFP virus (light gray; n=2 mice). Shown are data
from the ipsilateral (injected) and contralateral (uninjected)
hemispheres. Error bars indicate mean s.e.m p=0.044). (n) Two
representative traces showing open-field exploration in a control
mouse expressing CaMKIla::EYFP in mPFC, pre-activation and
post-activation with a 2 s 473 nm light pulse. Neither locomotion
velocity nor time spent exploring the center of the open field was
altered in CaMKIl.alpha.::SSFO and CaMKII.alpha.:EYFP animals after
a 2 s 473 nm light pulse (bottom; p>0.1, for both compared to
pre-activation; paired t-test), indicating that SSFO activation is
not anxiogenic.
[0023] FIG. 4 depicts SSFO activation in pyramidal cells increases
network activity and impairs information transmission through
principal neurons. (a) Whole cell recording from a layer 2/3
pyramidal neuron expressing SSFO in a prefrontal cortical slice
from a mouse injected with AAV5-CaMKIl.alpha.-SSFO-EYFP. Activation
with 470 nm light triggered depolarization of the recorded cell.
Inset compares expanded 2s periods pre-activation (1),
post-activation (2) and post-deactivation (3). (b) Whole cell
recording in a non-expressing pyramidal neuron from a slice
expressing CaMKII.alpha.::SSFO-EYFP shows increased synaptic
activity (top) following a 1 s 470 nm light pulse, which is
eliminated by excitatory synaptic blockers CNQX (10 .mu.M) and APV
(25 .mu.M; bottom). Inset compares activity pre-activation (1),
post-activation (2), and post-deactivation (3). (c) Sample trace
showing response of a representative pyramidal neuron in a PV::SSFO
slice (expressing DIO-SSFO-EYFP) at baseline and during 5510
activation in PV cells in the slice (between blue and yellow light
pulses). Inset compares three 5s periods before activation (1),
after activation (2), and after deactivation (3). (d) Activity of
PV cells after activation with SSFO.
[0024] FIG. 5 depicts impaired cellular information processing in
elevated but not reduced cellular E/I balance. (a) Representative
traces showing response of a representative
CaMKII.alpha.::SSFO-eYFP expressing cell to injection of an
identical defined pattern of sEPSCs before (top) and after (bottom)
blue light activation. Resting membrane potential for each trace is
indicated. (b) Input-output curve for a pyramidal neuron expressing
SSFO, showing reduced response to higher sEPSC rates after SSFO
activation (pre-stimulation: black; post-stimulation: gray; error
bars show s.e.m). (c) Cell-by-cell reduction in transmitted mutual
(EPSC-spike) information in 6 individual pyramidal cells expressing
SSFO following the is 470 nm pulse. Average MI is shown in black
(mean.+-.s.e.m; p=0.0063, Student's t-test; reduction in mutation
information between spike rate and injected sEPSC rate obtained
within 125 ms windows). (d) Representative traces showing responses
of a pyramidal neuron from a slice expressing DIO-SSFO-eYFP to an
identical injection of sEPSCs as in a before (top) and after
(bottom) blue light activation. Resting membrane potential for each
trace is indicated. (e) Input-output curve for a pyramidal neuron
in a PV::SSFO slice, showing linear reduction in gain after SSFO
activation in PV neurons (pre-stimulation: black; post-stimulation:
blue; error bars show s.e.m). (f) Cell-by-cell summary data showing
no significant reduction in pyramidal cell transmitted information,
despite spike suppression, after a 1 s 470 nm pulse that triggered
activation of DIO-SSFO in PV neurons. Mean MI is shown in black.
(g) Mean mutual information across cells in baseline vs.
SSFO-activated conditions across a range of time bin widths used
for calculating mutual information. For these comparisons, the bin
width of input sEPSC rate was kept constant at 50 Hz. Asterisks
indicate the significance of the change in mutual information in
SSFO-activated conditions (h) Comparison of mean change in mutual
information (SSFO-activation minus baseline) in cells recorded from
slices expressing CaMKII.alpha.::SSFO or PV::SSFO. Asterisks
indicate the significance of the difference in magnitude of the
change in mutual information for CaMKII.alpha.::SSFO vs. PV::SSFO.
(i) Same as in (g), but with varying input sEPSC rate bins. Here
the time bin width was kept constant at 125 ms. (j) Same as in (h),
but with varying input sEPSC rate bins. All bar graphs depict
mean.+-.s.e.m. (* p<0.05; ** p<0.01).
[0025] FIG. 6 depicts elevated cellular E/I balance in mPFC drives
baseline gamma rhythmically in freely-moving, socially impaired
mice. (a) Wild-type mice injected with CaMKII.alpha.::SSFO or
CaMKII.alpha.::EYFP were implanted with a non-brain-penetrating
fiberoptic connector via a small craniotomy at the time of virus
injection. (b) Representative image of viral expression of
SSFO-eYFP in PL cortex in a mouse implanted with
non-brain-penetrating fiberoptic connector. (c) c-fos positive cell
counts in PFC of control (CaMKII.alpha.::EYFP) mice or
CaMKII.alpha.::SSFO mice, 90 min after activation with a 2 s 470 nm
light pulse. (d) Freezing behavior assessed in
non-brain-penetrating implanted mice that received a 2s 470 nm
light pulse immediately prior to the conditioning session. Freezing
was measured immediately following the conditioning session
(Immediate), 24 h later (Test 1), and then 24 h following a second
fear conditioning session in which no light was delivered (Test 2).
(e) Social exploration was measured either with no light activation
(Test 1) or following a 2s 470 nm light pulse (Test 2). (f)
Implantable chronic multisite optrode (CMO) for awake, behaving
recordings in mouse M2 and PFC. Arrowheads indicate wire
termination sites; arrow shows cleaved end of fiberoptic connector.
(g) Electrolytic lesions mark the sites from which recordings were
taken in a mouse expressing CaMKII.alpha.::SSFO. (h) Social
exploration (left) and novel object exploration (right) before
(gray left vertical bar) and after (blue right vertical bar)
activation with 470 nm light in the three mice in which CMO
recordings were conducted (n=3 mice). (i) Multiunit activity from
two channels simultaneously recorded during an
activation/deactivation protocol. Blue light and yellow light were
delivered as indicated. Channels with significant multiunit
modulation (bottom) were selected for spectral analysis. (j)
Average increase in MUA rate on channels within the expressing
region (blue right vertical bar; n=4 channels in 3 mice), compared
with channels that were outside the expressing region (gray left
vertical bar; n=4 channels in 3 mice). (k) LFP wavelet spectrogram
from an un-modulated channel Example traces are shown for the
baseline, activation and deactivation periods. Average wavelet
spectra for the three indicated periods (n=5 trials in 1 mouse) and
population data of power change in 3 frequency ranges (inset; n=3
mice) are shown. (1) LFP wavelet spectrogram from a modulated
channel. Example traces are shown for the baseline, activation and
deactivation periods. Average wavelet spectra for the three
indicated periods (n=5 trials in 1 mouse) and population data
(inset; n=3 mice) are shown, demonstrating a specific increase in
gamma rhythmicity after SSFO activation in PFC pyramidal neurons.
All bar graphs depict mean.+-.s.e.m. Power spectra in (k), (1) are
averaged from 5 trials, shaded areas indicate standard deviation
across recordings. (* p<0.05; ** p<0.005).
[0026] FIG. 7 depicts locomoter behavior in a novel open field
behavioral test. (a) Open-field behavior of mice expressing
CaMKII.alpha.::SSFO in mPFC pre-activation (dark gray bars; 2.5
min) and post-activation (light gray bars; 2.5 min) with 1 s 473 nm
light. Track length, % time in center, and % time in the periphery
are shown (n=3 mice). A yellow light pulse was applied after the
second 2.5 min period to deactivate SSFO. (b) Average power
spectra, measured pre-activation (black), post-activation (dark
gray) and post-deactivation (light gray) from channels determined
to arise from electrodes placed in the virus-expressing mPFC region
(n=3 mice, shaded areas indicate s.e.m across mice). (c) Average
power spectra measured from channels in i during the social
behavior test in trials without light activation of SSFO (gray) and
with activation (light gray). (d) As in (b), for novel-object
exploration experiments (n=3 mice, shaded areas indicate s.e.m
across mice). Note that unmodulated channels did not show
significant changes in power spectrum following light
activation.
[0027] FIG. 8 depicts increase in power at gamma frequency under
high light density. (a) voltage clamp experiment with corresponding
spectra for IPSCs recorded at 0 mV and for EPSCs at -60 mV (b)
Change in power of synaptic activity within the indicated frequency
bins recorded in mPFC pyramidal neurons during a 20s pulse of 560
nm light at the indicated light power densities. Power differences
are shown between baseline (pre-light) period to light-on period
when voltage clamping the cells to -60 mV or 0 mV, or in current
clamp (CC) mode. Strongest gamma-modulation is evident at the
highest light power density, and is strongest in 0 mV and CC
recordings. (c) Relative gamma power for the three light powers in
the three recording configurations from (b).
[0028] FIG. 9 depicts inhibition of PFC excitatory or inhibitory
cells. (a) Wild-type mice bilaterally injected with
CaMKIl.alpha.::eNpHR3.0, PV::Cre mice bilaterally injected with
EF1.alpha.::D10-eNpHR3.0, and control mice bilaterally injected
with CaMKIl.alpha.-EYFP were tested in social exploration in the
home cage (a; n=6 for all conditions) and the three-chamber social
test (b; n=3, 5, and 6, respectively). Social behavior in the home
cage was not affected under these conditions (a; p>0.5 for both
NpHR3.0 groups compared with controls, unpaired t-test) and all
three groups showed similar social preference in the three chamber
social test (b; p>0.5 for both NpHR3.0 groups compared with
controls, unpaired t-test) and significantly preferred the social
chamber (b; p<0.05, paired t-test). Due to expression
penetrance, the inhibition of PV cells in these experiments is
expected to leave activity in the vast majority of inhibitory
neurons (and even PV neurons) unchanged.
[0029] FIG. 10 depicts combinatorial optogenetics in behaving
mammals: rescue of elevated E/I-balance social behavior. (a) Action
spectra of SSFO and C1V1-E122T/E162T (C1V1). Vertical lines
indicate stimulation wavelengths used in the experiments. (b)
Experiment design and pulse patterns; no-light control was used for
baseline behavior; 2 s 470 nm light was used to activate SSFO,
transiently activating C1V1 only during the light pulse; 10 Hz 470
nm was used to co-activate SSFO and C1V1; 10 Hz 590 nm activated
only C1V1. (c) Mice expressing CaMKII.alpha.::SSFO showed
significant social preference at baseline, but exhibited social
dysfunction after either 2 s 470 nm activation or during 10 Hz 470
nm activation. (d) Mice expressing both CaMKII.alpha.::SSFO and
(DIO) PV::C1V1 showed impaired social behavior after a 2 s 470 nm
pulse, but displayed restored social behavior during the 10 Hz 470
nm light stimulation. Activation of C1V1 alone with 10 Hz 590 nm
pulses did not impair social behavior.
[0030] FIG. 11 depicts combinatorial optical control of mPFC
cellular E/I balance: control experiments. Diagrams illustrate the
light-stimulation protocols used in 4 different experiments using
CaMKIl.alpha.::YFP mice. In all four experiments, light stimulation
had no effect on the significant preference of these control mice
to spend time in the chamber in which the novel conspecific mouse
was located (n=8 mice).
[0031] FIG. 12 depicts a flow diagram for testing of a disease
model, consistent with various 10 embodiments of the present
disclosure.
[0032] FIG. 13 depicts a model for assessing treatments of various
nervous system disorders, consistent with an embodiment of the
present disclosure.
[0033] While the present disclosure is amenable to various
modifications and alternative forms, specifics thereof have been
shown by way of example in the drawing and will be described in
detail. It should be understood, however, that the intention is not
to limit the present disclosure to the particular embodiments
described. On the contrary, the intention is to cover all
modifications, equivalents, and alternative falling within the
scope of the present disclosure including aspects defined in the
claims.
DETAILED DESCRIPTION
[0034] This invention provides, inter alia, animal cells, non-human
animals, and brain slices comprising cells expressing stabilized
step function opsin proteins on their plasma membranes, and methods
of using the stabilized step function opsin proteins to selectively
depolarize excitatory or inhibitory neurons residing in the same
microcircuit in the pre-frontal cortex. The step function opsins,
or SFOs, are ChR2 light-activated cation channel proteins that can
induce prolonged stable excitable states in neurons upon exposure
to blue light and then be reversed upon exposure to green or yellow
light. The SFOs were developed to implement bistable changes in
excitability of targeted populations operating on timescales up to
4 orders of magnitude longer than that of wild type (wt) ChR2 for
more stable state modulation (SFOs: up to 10-100 seconds). While
these opsin genes delivered a new kind of optogenetic control
complementary to that of conventional channelrhodopsins designed to
control individual action potentials, the timescale was still not
suitable for evaluating prolonged and complex mammalian behaviors
over many minutes.
[0035] Subsequent work by the inventors has further developed the
initial SFO concept, with mutation of the C128 proton networking
partner D156 for additional extension of the photocycle and
lifetime of the open state. This "stabilized step function opsin"
(SSFO) protein possesses unique physiochemical properties which
permit experimental manipulation of cortical E/I elevations and the
ability to monitor gamma oscillations in cortical slices. This new
tool, known as a stabilized step function opsin (SSFO), enables
stable circuit modulation for time periods that are sufficient for
temporally precise and complex behavioral experiments over many
minutes in the absence of ongoing light activation, external fiber
optic attachments, and even without any optical hardware brain
penetration at all. Additionally, due to the phenomena of photon
integration--a property that renders neurons expressing the SSFO
extremely light sensitive--cells expressing these proteins on their
plasma membranes are able to be activated with light pulses that
can have a light power density in the low .mu.W/mm.sup.2 range and
at least 3 mm deep into brain tissue from the light source. These
unique light-sensitive step function opsin proteins can be
expressed in either excitatory or inhibitory neural circuits, such
as in the prefrontal cortex of nonhuman animals, which can then be
depolarized in response to light having particular wavelengths,
thus permitting experimental manipulation of cortical E/I balances.
Furthermore, brain slices from non-human animals containing
cortical excitatory or inhibitory neurons expressing the stabilized
step function opsin proteins disclosed herein can be used to search
for chemical compounds which can selectively inhibit the
depolarization of either excitatory or inhibitory neurons residing
within a neural circuit. These cortical neurons may be responsible
for or involved with the social and cognitive behavioral defects
associated with neurological disorders such as schizophrenia and/or
autism spectrum disorder.
[0036] General Techniques
[0037] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, cell biology, biochemistry, nucleic acid chemistry,
and immunology, which are well known to those skilled in the art.
Such techniques are explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, second edition (Sambrook et
al., 1989) and Molecular Cloning: A Laboratory Manual, third
edition (Sambrook and Russel, 2001), (jointly referred to herein as
"Sambrook"); Current Protocols in Molecular Biology (F. M. Ausubel
et al., eds., 1987, including supplements through 2001); PCR: The
Polymerase Chain Reaction, (Mullis et al., eds., 1994); Harlow and
Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor
Publications, New York; Harlow and Lane (1999) Using Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (jointly referred to herein as "Harlow and Lane"),
Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry,
John Wiley & Sons, Inc., New York, 2000), Handbook of
Experimental Immunology, 4.sup.th edition (D. M. Weir & C. C.
Blackwell, eds., Blackwell Science Inc., 1987); and Gene Transfer
Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds.,
1987).
DEFINITIONS
[0038] As used herein, the singular form "a", "an", and "the"
includes plural references unless indicated otherwise.
[0039] An "animal" can be a vertebrate, such as any common
laboratory model organism, or a mammal. Mammals include, but are
not limited to, humans and non-human primates, farm animals, sport
animals, pets, mice, rats, and other rodents.
[0040] An "amino acid substitution" or "mutation" as used herein
means that at least one amino acid component of a defined amino
acid sequence is altered or substituted with another amino acid
leading to the protein encoded by that amino acid sequence having
altered activity or expression levels within a cell.
[0041] It is intended that every maximum numerical limitation given
throughout this specification includes every lower numerical
limitation, as if such lower numerical limitations were expressly
written herein. Every minimum numerical limitation given throughout
this specification will include every higher numerical limitation,
as if such higher numerical limitations were expressly written
herein. Every numerical range given throughout this specification
will include every narrower numerical range that falls within such
broader numerical range, as if such narrower numerical ranges were
all expressly written herein.
[0042] SSFO Proteins and Cells Expressing the Same
[0043] Previously described SFOs capitalize on slower channel
deactivation kinetics, as introduced by mutation of ChR2-C128,
which was chosen based on the homology between channelrhodopsin-2
(ChR2) and bacteriorhodopsin (BR), in which similar mutations led
to moderate slowing of the photocycle. T90, the BR homolog of
ChR2-C128, is hydrogen-bonded to D115 of BR; these two amino acids
are thought to work in concert to stabilize the all-trans
conformation of the retinal chromophore, and ChR2-D156 is the
homolog of BR D115. If C128 and D156 modulate ChR2 closure solely
via their presumptive shared hydrogen bond, then a combination
mutation of these two residues would not be expected to generate
significantly greater effects on channel kinetics than either
mutation alone. However, contrary to expectations, neurons
expressing the ChR2-C128S/D156A double mutant gave rise to
sustained photocurrents that were far more stable than those from
cells expressing either single mutant alone.
[0044] In some aspects, the invention includes proteins comprising
substituted or mutated amino acid sequences, wherein the mutant
protein retains the characteristic light-activatable nature of the
precursor SFO protein but may also possess altered properties in
some specific aspects. For example, the mutant light-activated SFO
proteins described herein may exhibit an increased level of
expression both within an animal cell or on the animal cell plasma
membrane; an increased level of sustained photocurrents in response
to a first wavelength of light; a faster but less complete
deactivation when exposed to a second wavelength of light; and/or a
combination of traits whereby the SFO protein possess the
properties of low desensitization, fast deactivation, and/or strong
expression in animal cells.
[0045] Light-activated SFO proteins comprising amino acid
substitutions or mutations include those in which one or more amino
acid residues have undergone an amino acid substitution while
retaining the ability to respond to light and the ability to
control the polarization state of a plasma membrane. For example,
light-activated proteins comprising amino acid substitutions or
mutations can be made by substituting one or more amino acids into
the amino acid sequence corresponding to SEQ ID NO:1, SEQ ID NO:2,
SEQ ID NO:3, or SEQ ID NO:4. In some embodiments, the invention
includes proteins comprising altered amino acid sequences in
comparison with the amino acid sequence in SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, or SEQ ID NO:4, wherein the altered
light-activated stabilized step function opsin protein retains the
characteristic light-activated nature and/or the ability to
regulate ion flow across plasma membranes of the protein with the
amino acid sequence represented in SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3, or SEQ ID NO:4 but may have altered properties in some
specific aspects.
[0046] Amino acid substitutions in a native protein sequence may be
conservative or non-conservative and such substituted amino acid
residues may or may not be one encoded by the genetic code. The
standard twenty amino acid "alphabet" is divided into chemical
families based on chemical properties of their side chains. These
families include amino acids with basic side chains (e.g., lysine,
arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), beta-branched side
chains (e.g., threonine, valine, isoleucine) and side chains having
aromatic groups (e.g., tyrosine, phenylalanine, tryptophan,
histidine). A "conservative amino acid substitution" is one in
which the amino acid residue is replaced with an amino acid residue
having a chemically similar side chain (i.e., replacing an amino
acid possessing a basic side chain with another amino acid with a
basic side chain). A "non-conservative amino acid substitution" is
one in which the amino acid residue is replaced with an amino acid
residue having a chemically different side chain (i.e., replacing
an amino acid having a basic side chain with an amino acid having
an aromatic side chain). The amino acid substitutions may be
conservative or non-conservative. Additionally, the amino acid
substitutions may be located in the SFO retinal binding pocket, in
one or more of the SFO intracellular loop domains, and/or in both
the retinal binding pocket or the intracellular loop domains.
[0047] Provided herein, therefore, are light-activated stabilized
step function opsin proteins that may have specific amino acid
substitutions at key positions throughout the retinal binding
pocket of the protein. For information regarding the retinal
binding pocket of light sensitive polypeptides, see Greenhalgh et
al., J. Biol. Chem., 268, 20305-20311 (1993), the disclosure of
which is hereby incorporated herein in its entirety. In some
embodiments, the SFO protein can have a mutation at amino acid
residue C128 of SEQ ID NO:1. In some embodiments, the SFO protein
can have a mutation at amino acid residue D156 of SEQ ID NO:1. In
other embodiments, the SFO protein can have a mutation at both
amino acid residues C128 and D156 of SEQ ID NO:1 (SSFO). In some
embodiments, each of the disclosed mutant stabilized step function
opsin proteins can have specific properties and characteristics for
use in depolarizing the membrane of an animal cell in response to
light.
[0048] Accordingly, in one aspect there is provided a
light-activated SSFO protein expressed on a cell plasma membrane
capable of mediating a depolarizing current in the cell when the
cell is illuminated with light, wherein the protein exhibits rapid
step-like activation in response to a single pulse of light having
a first wavelength and deactivation in response to a pulse of light
having a second wavelength; wherein the depolarizing current in the
cell is maintained for up to about five, about ten, about fifteen,
or about twenty minutes. In some embodiments, the protein comprises
the amino acid sequence of ChR2, ChR1, VChR1, or VChR2 with amino
acid substitutions at amino acid residues corresponding to C128 and
D156 of the amino acid sequence of ChR2 (See, e.g., FIG. 1B of
International Patent Application Publication No. WO 2009/131837,
which is incorporated by reference herein, illustrating
conservation of amino acid residues corresponding to C128 and D156
of the amino acid sequence of ChR2 between several species of
channelrhopsin cation channels; see also Kianianmomeni et al.,
Plant Physiol., 2009, 151:347-356, which is incorporated by
reference herein in its entirety). In other embodiments, the
light-activated SSFO protein can comprise an amino acid sequence at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identical to the sequence shown in SEQ ID NO:1 without the signal
peptide sequence. In other embodiments, the light-activated SSFO
protein can comprise an amino acid sequence at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
sequence shown in SEQ ID NO:1. In other embodiments, the
light-activated SSFO protein can comprise an amino acid sequence at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identical to the sequence shown in SEQ ID NO:2. In other
embodiments, the light-activated SSFO protein can comprise an amino
acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100% identical to the sequence shown in SEQ ID NO:3. In
another embodiment, the light-activated SSFO protein can comprise
an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID
NO:4. In some embodiments, the signal peptide sequence in the SSFO
proteins is deleted or substituted with a signal peptide sequence
from a different protein. In some embodiments, the substitution at
amino acid residues corresponding to C128 and D156 of the amino
acid sequence of ChR2 are conservative amino acid substitutions. In
other embodiments, the substitution at amino acid residues
corresponding to C128 and D156 of the amino acid sequence of ChR2
are non-conservative amino acid substitutions. In some embodiments,
the substitution at the amino acid residue corresponding to C128 of
the amino acid sequence of ChR2 is a substitution to serine. In
other embodiments, the substitution at the amino acid residue
corresponding to D156 of the amino acid sequence of ChR2 is a
substitution to a non-acidic amino acid. In another embodiment, the
substitution at the amino acid residue corresponding to D156 of the
amino acid sequence of ChR2 is a substitution to alanine. In some
embodiments, the protein can further comprise a C-terminal
fluorescent protein. In some specific embodiments, the C-terminal
fluorescent protein can be enhanced yellow fluorescent protein
(EYFP), green fluorescent protein (GFP), cyan fluorescent protein
(CFP), or red fluorescent protein (RFP). In some embodiments, the
second light-activated protein can be capable of mediating a
hyperpolarizing current in the cell when the cell is illuminated
with light. In some embodiments the second light-activated protein
can be NpHR, eNpHR2.0, eNpHR3.0, eNpHR3.1, GtR3, or a C1V1 chimeric
protein as described in International Patent Application No:
PCT/US2011/028893 and U.S. Provisional Patent Application Nos.
61/410,736 and 61/410,744, the disclosure of each of which is
incorporated by reference herein in their entirety.
[0049] In some embodiments, the C1V1 chimeric protein comprises a
light-activated protein expressed on the cell membrane, wherein the
protein is a chimeric protein derived from VChR1 from Volvox
carteri and ChR1 from Chlamydomonas reinhardti, wherein the protein
comprises the amino acid sequence of VChR1 having at least the
first and second transmembrane helices replaced by the first and
second transmembrane helices of ChR1; is responsive to light; and
is capable of mediating a depolarizing current in the cell when the
cell is illuminated with light. In some embodiments wherein the
protein further comprises a replacement within the intracellular
loop domain located between the second and third transmembrane
helices of the chimeric light responsive protein, wherein at least
a portion of the intracellular loop domain is replaced by the
corresponding portion from the ChR1. In another embodiment, the
portion of the intracellular loop domain of the C1V1 chimeric
protein is replaced with the corresponding portion from the ChR1
extending to amino acid residue A145 of the ChR1. In other
embodiments, the C1V1 chimeric protein further comprises a
replacement within the third transmembrane helix of the chimeric
light responsive protein, wherein at least a portion of the third
transmembrane helix is replaced by the corresponding sequence of
ChR1. In another embodiment, the portion of the intracellular loop
domain of the C1V1 chimeric protein is replaced with the
corresponding portion from the ChR1 extending to amino acid residue
W163 of the ChR1.
[0050] In some embodiments of the stabilized step function opsin
proteins provided herein, the light having a first wavelength can
be blue light. In other embodiments, said light having a first
wavelength can be about 445 nm. In another embodiment, said light
having a second wavelength can be green light or yellow light. In
other embodiments, said light having a second wavelength can be
about 590 nm. In other embodiments, said light having a second
wavelength can be between about 390-400 nm, inclusive, as well as
every number within this range. In some embodiments, the
light-activated stabilized step function opsin proteins described
herein can be activated by light pulses that can have a duration
for any of about 1 millisecond (ms), about 2 ms, about 3, ms, about
4, ms, about 5 ms, about 6 ms, about 7 ms, about 8 ms, about 9 ms,
about 10 ms, about 15 ms, about 20 ms, about 25 ms, about 30 ms,
about 35 ms, about 40 ms, about 45 ms, about 50 ms, about 60 ms,
about 70 ms, about 80 ms, about 90 ms, about 100 ms, about 200 ms,
about 300 ms, about 400 ms, about 500 ms, about 600 ms, about 700
ms, about 800 ms, about 900 ms, about 1 sec, about 1.25 sec, about
1.5 sec, or about 2 sec, inclusive, including any times in between
these numbers. In some embodiments, the light-activated stabilized
step function opsin proteins described herein can be activated by
light pulses that can have a light power density of any of about 1
.mu.W mm.sup.-2, about 2 .mu.W mm.sup.-2, about 3 .mu.W mm.sup.-2,
about 4 .mu.W mm.sup.-2, about 5 .mu.W mm.sup.-2, about 6 .mu.W
mm.sup.-2, about 7 .mu.W mm.sup.-2, about 8 .mu.W mm.sup.-2, about
9 .mu.W mm.sup.-2, about 10 .mu.W mm.sup.-2, about 11 .mu.W
mm.sup.-2, about 12 .mu.W mm.sup.-2, about 13 .mu.W mm.sup.-2,
about 14 .mu.W mm.sup.-2, about 15 .mu.W mm.sup.-2, about 16 .mu.W
mm.sup.-2, about 17 .mu.W mm.sup.-2, about 18 .mu.W mm.sup.-2,
about 19 .mu.W mm.sup.-2, or about 20 .mu.W mm.sup.-2, inclusive,
including any values between these numbers. In other embodiments,
the light-activated proteins can be activated by light pulses that
can have a light power density of any of about 1 mW mm.sup.-2,
about 2 mW mm.sup.-2, about 3 mW mm.sup.-2, about 4 mW mm.sup.-2,
about 5 mW mm.sup.-2, about 6 mW mm.sup.-2, about 7 mW mm.sup.-2,
about 8 mW mm.sup.-2, about 9 mW mm.sup.-2, about 10 mW mm.sup.-2,
about 11 mW mm.sup.-2, about 12 mW mm.sup.-2, about 13 mW
mm.sup.-2, about 14 mW mm.sup.-2, about 15 mW mm.sup.-2, about 16
mW mm.sup.-2, about 17 mW mm.sup.-2, about 18 mW mm.sup.-2, about
19 mW mm.sup.-2, about 20 mW mm.sup.-2, about 21 mW mm.sup.-2,
about 22 mW mm.sup.-2, about 23 mW mm.sup.-2, about 24 mW
mm.sup.-2, or about 25 mW mm.sup.-2, inclusive, including any
values between these numbers.
[0051] In some embodiments, the light-activated stabilized step
function opsin proteins described herein can maintain a sustained
photocurrent for about 20 minutes. In other embodiments, the
light-activated stabilized step function opsin proteins described
herein can maintain a sustained photocurrent for any of about 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27,
28, 29, or 30 minutes, inclusive, including for any times in
between these numbers. In other embodiments, the photocycle
progression of any of the light-activated stabilized step function
opsin proteins described herein is completely blocked after the
protein is illuminated with said single pulse of light having a
first wavelength.
[0052] In some aspects of the light-activated stabilized step
function opsin proteins described herein, the cell can be an animal
cell. In some embodiments, the animal cell can be a neuronal cell,
a cardiac cell, or a stem cell. In some embodiments, the animal
cell can be a neuronal cell. In other embodiments, the animal cells
comprise neurons that effect social behavior when depolarized. In
some embodiments, the neuronal cell is a neuron that changes innate
social behavior and/or conditioned behavior when depolarized. In
other embodiments, the animal cells comprise neurons that give rise
to the social and cognitive defects in autism and/or schizophrenia
when depolarized. In other embodiments, the neuronal cell can be an
excitatory neuron located in the pre-frontal cortex of a non-human
animal. In other embodiments, the excitatory neuron can be a
pyramidal neuron. In some embodiments the neuronal cell can be an
inhibitory neuron located in the pre-frontal cortex of a non-human
animal. In still other embodiments, the inhibitory neuron can be a
parvalbumin neuron. In some embodiments, the inhibitory and
excitatory neurons can be in a living non-human animal.
[0053] In other aspects of the light-activated stabilized step
function opsin proteins, the cells can be neurons in a living brain
slice from a non-human animal. In some embodiments, the brain
slices are coronal brain slices. In some embodiments, the brain
slices are from the pre-frontal cortex of a non-human animal. In
other embodiments, the brain slices comprise neurons that effect
social behavior when depolarized. In some embodiments, the brain
slices comprise neurons that change innate social behavior and/or
conditioned behavior when depolarized. In other embodiments, the
brain slices comprise neurons that give rise to the social and
cognitive defects in autism and/or schizophrenia when
depolarized.
[0054] In some aspects, the stabilized step function opsin proteins
described herein may be modified by the addition of one or more
amino acid sequence motifs which enhance transport to the plasma
membranes of mammalian cells. Light-activated opsin proteins are
derived from evolutionarily simpler organisms and therefore may not
be expressed or tolerated by mammalian cells or may exhibit
impaired subcellular localization when expressed at high levels in
mammalian cells. Consequently, in some embodiments, the stabilized
step function opsin proteins described herein may be fused to one
or more amino acid sequence motifs selected from the group
consisting of a signal peptide, an endoplasmic reticulum (ER)
export signal, a membrane trafficking signal, and an N-terminal
golgi export signal. The one or more amino acid sequence motifs
which enhance the light-activated stabilized step function opsin
proteins transport to the plasma membranes of mammalian cells can
be fused to the N-terminus, the C-terminus, or to both the N- and
C-terminal ends of the light-activated protein. Optionally, the
light-activated protein and the one or more amino acid sequence
motifs may be separated by a linker. In some embodiments, the
stabilized step function opsin protein is modified by the addition
of a trafficking signal (ts) which enhances transport of the
protein to the cell plasma membrane. In some embodiments, the
trafficking signal is derived from the amino acid sequence of the
human inward rectifier potassium channel K.sub.ir2.1. In some
embodiments, the trafficking signal comprises the amino acid
sequence KSRITSEGEYIPLDQIDINV. In other embodiments, the
light-activated stabilized step function opsin protein is modified
by the addition of a signal peptide (e.g., which enhances transport
to the plasma membrane). The signal peptide may be fused to the
C-terminus of the core amino acid sequence or may be fused to the
N-terminus of the core amino acid sequence. In some embodiments,
the signal peptide is linked to the core amino acid sequence by a
linker. The linker can comprise any of 5, 10, 20, 30, 40, 50, 75,
100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino
acids in length. In some embodiments, the signal peptide comprises
the amino acid sequence
MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNG. In other
embodiments, the light-activated stabilized step function opsin
protein is modified by the addition of an endoplasmic reticulum
(ER) export signal. The ER export signal may be fused to the
C-terminus of the core amino acid sequence or may be fused to the
N-terminus of the core amino acid sequence. In some embodiments,
the ER export signal is linked to the core amino acid sequence by a
linker. The linker can comprise any of 5, 10, 20, 30, 40, 50, 75,
100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino
acids in length. In some embodiments, the ER export signal
comprises the amino acid sequence FXYENE, where X can be any amino
acid. In some embodiments, the ER export signal comprises the amino
acid sequence VXXSL, where X can be any amino acid. In some
embodiments, the ER export signal comprises the amino acid sequence
FCYENEV.
[0055] Animal Cells, Non-Human Animals, and Brain Slices
[0056] Provided herein are cells comprising the light activated
chimeric proteins disclosed herein. In some embodiments, the cells
are animal cells. In some embodiments, the animal cells comprise
the protein corresponding to SEQ ID NO:1. In other embodiments, the
animal cells comprise the stabilized step function opsin proteins
disclosed herein. In one embodiment, the animal cell can be a
neuronal cell. In some embodiments, the animal cells are from the
pre-frontal cortex of a non-human animal. In other embodiments, the
animal cells comprise neurons that effect social behavior when
depolarized. In some embodiments, the neuronal cell is a neuron
that changes innate social behavior and/or conditioned behavior
when depolarized. In other embodiments, the animal cells comprise
neurons that give rise to the social and cognitive defects in
autism and/or schizophrenia when depolarized. In some embodiments
the neuronal cell can be an excitatory neuron located in the
pre-frontal cortex of a non-human animal. In other embodiments, the
excitatory neuron can be a pyramidal neuron. In some embodiments
the neuronal cell can be an inhibitory neuron located in the
pre-frontal cortex of a non-human animal. In still other
embodiments, the inhibitory neuron can be a parvalbumin neuron.
[0057] Also provided herein are non-human animals comprising the
proteins disclosed herein. In some embodiments, the non-human
animals comprise the protein corresponding to SEQ ID NO:1. In some
embodiments, the animals comprise the stabilized step function
opsin proteins disclosed herein. In some embodiments, the animals
comprising the stabilized step function opsin proteins disclosed
herein are transgenically expressing said stabilized step function
opsin proteins. In other embodiments, the animals comprising the
stabilized step function opsin proteins described herein have been
virally transfected with a vector carrying the stabilized step
function opsin proteins such as, but not limited to, an adenoviral
vector. In some embodiments, the animals comprising the stabilized
step function opsin proteins disclosed herein exhibit changes in
behavior when said stabilized step function opsin proteins are
depolarized by activation with light. In other embodiments, the
animals comprising the stabilized step function opsin proteins
disclosed herein exhibit changes in innate and learned social
behaviors when said stabilized step function opsin proteins are
depolarized by activation with light. In other embodiments, the
animals comprising the stabilized step function opsin proteins
disclosed herein exhibit changes in conditioned behaviors when said
stabilized step function opsin proteins are depolarized by
activation with light.
[0058] Provided herein are living brain slices from a non-human
animal comprising the stabilized step function opsin proteins
described herein. In some embodiments, the brain slices are from
non-human animals transgenically expressing the stabilized step
function opsin proteins described herein. In other embodiments, the
brain slices are from non-human animals that have been virally
transfected with a vector carrying said stabilized step function
opsin proteins such as, but not limited to, an adenoviral vector.
In some embodiments, the brain slices are coronal brain slices. In
some embodiments, the brain slices are from the pre-frontal cortex
of a non-human animal. In other embodiments, the brain slices
comprise neurons that effect social behavior when depolarized. In
some embodiments, the brain slices comprise neurons that change
innate social behavior and/or conditioned behavior when
depolarized. In other embodiments, the brain slices comprise
neurons that give rise to the social and cognitive defects in
autism and/or schizophrenia when depolarized. In some embodiments,
the brain slices are any of about 100 .mu.m, about 150 .mu.m, about
200 about 250 .mu.m, about 300 .mu.m, about 350 .mu.m, about 400
.mu.m, about 450 .mu.m, or about 500 .mu.m thick, inclusive,
including any thicknesses in between these numbers.
[0059] Polynucleotides, Promoters, and Vectors
[0060] Provided herein are isolated polynucleotides that encode
stabilized step function opsin proteins that have at least one
activity of a step function opsin protein. The disclosure provides
isolated, synthetic, or recombinant polynucleotides comprising a
nucleic acid sequence having at least about 70%, e.g., at least
about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%; 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99%, or complete (100%) sequence identity to the
nucleic acid of SEQ ID NO:2 over a region of at least about 10,
e.g., at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150,
200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,
850, 900, 950, or 1000 nucleotides.
[0061] The disclosure specifically provides a polynucleotide
comprising a nucleic acid sequence encoding a stabilized step
function opsin protein and/or a mutant variant thereof. For
example, the disclosure provides an isolated polynucleotide
molecule, wherein the polynucleotide molecule encodes a protein
comprising an amino acid sequence with at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino
acid sequence of SEQ ID NO:1. The disclosure also provides an
isolated polynucleotide molecule, wherein the polynucleotide
molecule encodes a protein comprising an amino acid sequence with
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to the amino acid sequence of SEQ ID NO:2. The
disclosure moreover provides an isolated polynucleotide molecule,
wherein the polynucleotide molecule encodes a protein comprising an
amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% sequence identity to the amino acid
sequence of SEQ ID NO:3. The disclosure additionally provides an
isolated polynucleotide molecule, wherein the polynucleotide
molecule encodes a protein comprising an amino acid sequence with
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to the amino acid sequence of SEQ ID NO:4.
[0062] The disclosure also provides expression cassettes and/or
vectors comprising the above-described nucleic acids. Suitably, the
nucleic acid encoding a stabilized step function opsin protein of
the disclosure is operably linked to a promoter. Promoters are well
known in the art. Any promoter that functions in the host cell can
be used for expression of SSFO and/or any variant thereof of the
present disclosure. Initiation control regions or promoters, which
are useful to drive expression of a SSFO protein or variant thereof
in a specific animal cell are numerous and familiar to those
skilled in the art. Virtually any promoter capable of driving these
nucleic acids can be used.
[0063] Specifically, where recombinant expression of SSFO proteins,
such as the proteins described herein, in an excitatory neural cell
is desired, a human calmodulin-dependent protein kinase II alpha
(CaMKII.alpha.) promoter may be used. In other embodiments, an
elongation factor 1a (EF-1a) promoter in conjunction with a
Cre-inducible recombinant AAV vector can be used with
parvalbumin-Cre transgenic mice to target expression SSFO proteins
to inhibitory neurons.
[0064] Also provided herein are vectors comprising the
polynucleotides disclosed herein encoding a stabilized step
function opsin proteins or any variant thereof. The vectors that
can be administered according to the present invention also include
vectors comprising a polynucleotide which encodes an RNA (e.g., an
mRNA) that when transcribed from the polynucleotides of the vector
will result in the accumulation of light-activated stabilized step
function opsin proteins on the plasma membranes of target animal
cells. Vectors which may be used, include, without limitation,
lentiviral, HSV, adenoviral, and andeno-associated viral (AAV)
vectors. Lentiviruses include, but are not limited to HIV-1, HIV-2,
SIV, FIV and EIAV. Lentiviruses may be pseudotyped with the
envelope proteins of other viruses, including, but not limited to
VSV, rabies, Mo-MLV, baculovirus and Ebola. Such vectors may be
prepared using standard methods in the art.
[0065] In some embodiments, the vector is a recombinant AAV vector.
AAV vectors are DNA viruses of relatively small size that can
integrate, in a stable and sitespecific manner, into the genome of
the cells that they infect. They are able to infect a wide spectrum
of cells without inducing any effects on cellular growth,
morphology or differentiation, and they do not appear to be
involved in human pathologies. The AAV genome has been cloned,
sequenced and characterized. It encompasses approximately 4700
bases and contains an inverted terminal repeat (ITR) region of
approximately 145 bases at each end, which serves as an origin of
replication for the virus. The remainder of the genome is divided
into two essential regions that carry the encapsidation functions:
the left-hand part of the genome, that contains the rep gene
involved in viral replication and expression of the viral genes;
and the right-hand part of the genome, that contains the cap gene
encoding the capsid proteins of the virus.
[0066] AAV vectors may be prepared using standard methods in the
art. Adeno-associated viruses of any serotype are suitable (see,
e.g., Blacklow, pp. 165-174 of "Parvoviruses and Human Disease" J.
R. Pattison, ed. (1988); Rose, Comprehensive Virology 3:1, 1974; P.
Tattersall "The Evolution of Parvovirus Taxonomy" in Parvoviruses
(J R Kerr, S F Cotmore. M E Bloom, R M Linden, C R Parrish, Eds.) p
5-14, Hudder Arnold, London, U K (2006); and D E Bowles, J E
Rabinowitz, R J Samulski "The Genus Dependovirus" (J R Kerr, S F
Cotmore. M E Bloom, R M Linden, C R Parrish, Eds.) p 15-23, Hudder
Arnold, London, UK (2006), the disclosures of which are hereby
incorporated by reference herein in their entireties). Methods for
purifying for vectors may be found in, for example, U.S. Pat. Nos.
6,566,118, 6,989,264, and 6,995,006 and International Patent
Application Publication No.: WO/1999/011764 titled "Methods for
Generating High Titer Helper-free Preparation of Recombinant AAV
Vectors", the disclosures of which are herein incorporated by
reference in their entirety. Preparation of hybrid vectors is
described in, for example, PCT Application No. PCT/US2005/027091,
the disclosure of which is herein incorporated by reference in its
entirety. The use of vectors derived from the AAVs for transferring
genes in vitro and in vivo has been described (See e.g.,
International Patent Application Publication Nos: WO 91/18088 and
WO 93/09239; U.S. Pat. Nos. 4,797,368, 6,596,535, and 5,139,941;
and European Patent No: 0488528, all of which are herein
incorporated by reference in their entirety). These publications
describe various AAV-derived constructs in which the rep and/or cap
genes are deleted and replaced by a gene of interest, and the use
of these constructs for transferring the gene of interest in vitro
(into cultured cells) or in vivo (directly into an organism). The
replication defective recombinant AAVs according to the invention
can be prepared by co-transfecting a plasmid containing the nucleic
acid sequence of interest flanked by two AAV inverted terminal
repeat (ITR) regions, and a plasmid carrying the AAV encapsidation
genes (rep and cap genes), into a cell line that is infected with a
human helper virus (for example an adenovirus). The AAV
recombinants that are produced are then purified by standard
techniques.
[0067] In some embodiments, the vector(s) for use in the methods of
the invention are encapsidated into a virus particle (e.g. AAV
virus particle including, but not limited to, AAV1, AAV2, AAV3,
AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13,
AAV14, AAV15, and AAV16). Accordingly, the invention includes a
recombinant virus particle (recombinant because it contains a
recombinant polynucleotide) comprising any of the vectors described
herein. Methods of producing such particles are known in the art
and are described in U.S. Pat. No. 6,596,535.
[0068] For the animal cells described herein, it is understood that
one or more vectors may be administered to neural cells, heart
cells, or stem cells. If more than one vector is used, it is
understood that they may be administered at the same or at
different times to the animal cells.
[0069] Methods of the Invention
[0070] Provided herein are methods for depolarizing excitatory or
inhibitory neurons residing in a microcircuit by expressing in
those neurons the light-activated stabilized step function opsin
proteins described herein. In some aspects, there is a provided a
method for using the stabilized step function opsin proteins
described herein by activating proteins with light. The stabilized
step function opsin proteins disclosed herein can be expressed in
an excitatory neuron or in an inhibitory neuron. In other
embodiments, method for using the stabilized step function opsin
proteins disclosed herein can be in a living non-human animal or in
a living brain slice from a non-human animal. In other aspects,
there is provided a method for identifying a chemical compound that
inhibits the depolarization of excitatory neurons in the prefrontal
cortex of a non-human animal. In other aspects, there is provided a
method for identifying a chemical compound that restores an innate
social behavior and/or communication in a non-human animal.
[0071] Methods for Using SSFO Proteins
[0072] Provided herein are methods for using the stabilized step
function opsin proteins disclosed herein comprising activating the
proteins with light having a first wavelength. In some embodiments,
the proteins can be activated with light having a first wavelength
that can be blue light. In other embodiments, said light having a
first wavelength can be about 445 nm.
[0073] In another aspect of the methods for using the compositions
disclosed herein, the stabilized step function opsin proteins
disclosed herein can be deactivated with light having a second
wavelength. In some embodiments, said light having a second
wavelength can be green light or yellow light. In other
embodiments, said light having a second wavelength can be about 590
nm. In other embodiments, said light having a second wavelength can
be between about 390-400 nm, inclusive, as well as every number
within this range.
[0074] In some aspects of the methods provided herein, the
stabilized step function opsin proteins can be activated by light
pulses that can have a duration for any of about 1 millisecond
(ms), about 2 ms, about 3, ms, about 4, ms, about 5 ms, about 6 ms,
about 7 ms, about 8 ms, about 9 ms, about 10 ms, about 15 ms, about
20 ms, about 25 ms, about 30 ms, about 35 ms, about 40 ms, about 45
ms, about 50 ms, about 60 ms, about 70 ms, about 80 ms, about 90
ms, about 100 ms, about 200 ms, about 300 ms, about 400 ms, about
500 ms, about 600 ms, about 700 ms, about 800 ms, about 900 ms,
about 1 sec, about 1.25 sec, about 1.5 sec, or about 2 sec,
inclusive, including any times in between these numbers. In some
embodiments of the methods provided herein, the stabilized step
function opsin proteins can be activated by light pulses that can
have a light power density of any of about 1 .mu.W mm.sup.-2, about
2 .mu.W mm.sup.-2, about 3 .mu.W mm.sup.-2, about 4 .mu.W
mm.sup.-2, about 5 .mu.W mm.sup.-2, about 6 .mu.W mm.sup.-2, about
7 .mu.W mm.sup.-2, about 8 .mu.W mm.sup.-2, about 9 .mu.W
mm.sup.-2, about 10 .mu.W mm.sup.-2, about 11 .mu.W mm.sup.-2,
about 12 .mu.W mm.sup.-2, about 13 .mu.W mm.sup.-2, about 14 .mu.W
mm.sup.-2, about 15 .mu.W mm.sup.-2, about 16 .mu.W mm.sup.-2,
about 17 .mu.W mm.sup.-2, about 18 .mu.W mm.sup.-2, about 19 .mu.W
mm.sup.-2, or about 20 .mu.W mm.sup.-2, inclusive, including any
values between these numbers. In other embodiments, the
light-activated stabilized step function opsin proteins can be
activated by light pulses that can have a light power density of
any of about 1 mW mm.sup.-2, about 2 mW mm.sup.-2, about 3 mW
mm.sup.-2, about 4 mW mm.sup.-2, about 5 mW mm.sup.-2, about 6 mW
mm.sup.-2, about 7 mW mm.sup.-2, about 8 mW mm.sup.-2, about 9 mW
mm.sup.-2, about 10 mW mm.sup.-2, about 11 mW mm.sup.-2, about 12
mW mm.sup.-2, about 13 mW mm.sup.-2, about 14 mW mm.sup.-2, about
15 mW mm.sup.-2, about 16 mW mm.sup.-2, about 17 mW mm.sup.-2,
about 18 mW mm.sup.-2, about 19 mW mm.sup.-2, about 20 mW
mm.sup.-2, about 21 mW mm.sup.-2, about 22 mW mm.sup.-2, about 23
mW mm.sup.-2, about 24 mW mm.sup.-2, or about 25 mW mm.sup.-2,
inclusive, including any values between these numbers.
[0075] In some embodiments, the light-activated stabilized step
function opsin proteins of the methods described herein can
maintain a sustained photocurrent for about 10 minutes or longer.
In other embodiments, the light-activated stabilized step function
opsin proteins described herein can maintain a sustained
photocurrent for any of about 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, or 30 minutes,
inclusive, including for any times in between these numbers. In
other embodiments, the methods provided herein comprise completely
blocking the photocycle progression of any of the light-activated
stabilized step function opsin proteins described herein after the
protein is illuminated with a single pulse of light having a first
wavelength.
[0076] In some aspects of the methods described herein, the animal
cell can be a neuronal cell, a cardiac cell, or a stem cell. In
some embodiments, the animal cell can be a neuronal cell. In other
embodiments, the neuronal cell can be an excitatory neuron located
in the pre-frontal cortex of a non-human animal. In other
embodiments, the excitatory neuron can be a pyramidal neuron. In
other embodiments, the animal cells comprise neurons that effect
social behavior when depolarized. In some embodiments, the neuronal
cell is a neuron that changes innate social behavior and/or
conditioned behavior when depolarized. In other embodiments, the
animal cells comprise neurons that give rise to the social and
cognitive defects in autism and/or schizophrenia when depolarized.
In some embodiments the neuronal cell can be an inhibitory neuron
located in the pre-frontal cortex of a non-human animal. In still
other embodiments, the inhibitory neuron can be a parvalbumin
neuron. In some embodiments, the inhibitory and excitatory neurons
can be in a living non-human animal. In other embodiments, the
inhibitory and excitatory neurons can be in a brain slice from a
non-human animal.
[0077] Methods or Identifying a Chemical Compound that Inhibits the
Depolarization of Excitatory or Inhibitory Neurons in the
Prefrontal Cortex
[0078] Provided herein is a method for identifying a chemical
compound that inhibits the depolarization of excitatory or
inhibitory neurons in the prefrontal cortex of a non-human animal,
the method comprising: (a) depolarizing an excitatory or inhibitory
neuron in the prefrontal cortex of a non-human animal or a living
tissue slice from a non-human animal comprising a light-activated
protein cation channel expressed on the cell membrane capable of
mediating a depolarizing current in the cell when the cell is
illuminated with light, wherein the protein exhibits rapid
step-like activation in response to a single pulse of light having
a first wavelength and deactivation in response to a pulse of light
having a second wavelength; wherein the depolarizing current in the
cell is maintained for up to about twenty minutes; and wherein the
protein comprises the amino acid sequence of ChR2, ChR1, VChR1, or
VChR2 with amino acid substitutions at amino acid residues
corresponding to C128 and D156 of the amino acid sequence of ChR2;
(b) measuring an excitatory post synaptic potential (EPSP) or an
inhibitory post synaptic current (IPSC) in response to selectively
depolarizing the excitatory or inhibitory neuron comprising the
light-activated protein; (c) contacting the excitatory neuron with
a chemical compound; and (d) measuring the excitatory post synaptic
potential (EPSP) or an inhibitory post synaptic current (IPSC) to
determine if contacting the excitatory neuron with the chemical
compound inhibits the depolarization of the neuron. In some
embodiments, the proteins can be activated with light having a
first wavelength that can be blue light. In other embodiments, said
light having a first wavelength can be about 445 nm. In other
embodiments, said light having a second wavelength can be green
light or yellow light. In other embodiments, said light having a
second wavelength can be about 590 nm. In still other embodiments,
said light having a second wavelength can be between about 390-400
nm, inclusive, as well as every number within this range. In some
embodiments, the chemical compound can be a member of a
combinatorial chemical library.
[0079] In some aspects of the methods provided herein, the
light-activated stabilized step function opsin proteins can be
activated by light pulses that can have a duration for any of about
1 millisecond (ms), about 2 ms, about 3, ms, about 4, ms, about 5
ms, about 6 ms, about 7 ms, about 8 ms, about 9 ms, about 10 ms,
about 15 ms, about 20 ms, about 25 ms, about 30 ms, about 35 ms,
about 40 ms, about 45 ms, about 50 ms, about 60 ms, about 70 ms,
about 80 ms, about 90 ms, about 100 ms, about 200 ms, about 300 ms,
about 400 ms, about 500 ms, about 600 ms, about 700 ms, about 800
ms, about 900 ms, about 1 sec, about 1.25 sec, about 1.5 sec, or
about 2 sec, inclusive, including any times in between these
numbers. In some embodiments of the methods provided herein, the
light-activated stabilized step function opsin proteins can be
activated by light pulses that can have a light power density of
any of about 1 .mu.W mm.sup.-2, about 2 .mu.W mm.sup.-2, about 3
.mu.W mm.sup.-2, about 4 .mu.W mm.sup.-2, about 5 .mu.W mm.sup.-2,
about 6 .mu.W mm.sup.-2, about 7 .mu.W mm.sup.-2, about 8 .mu.W
mm.sup.-2, about 9 .mu.W mm.sup.-2, about 10 .mu.W mm.sup.-2, about
11 .mu.W mm.sup.-2, about 12 .mu.W mm.sup.-2, about 13 .mu.W
mm.sup.-2, about 14 .mu.W mm.sup.-2, about 15 .mu.W mm.sup.-2,
about 16 .mu.W mm.sup.-2, about 17 .mu.W mm.sup.-2, about 18 .mu.W
mm.sup.-2, about 19 .mu.W mm.sup.-2, or about 20 .mu.W mm.sup.-2,
inclusive, including any values between these numbers. In other
embodiments, the light-activated stabilized step function opsin
proteins can be activated by light pulses that can have a light
power density of any of about 1 mW mm.sup.-2, about 2 mW mm.sup.-2,
about 3 mW mm.sup.-2, about 4 mW mm.sup.-2, about 5 mW mm.sup.-2,
about 6 mW mm.sup.-2, about 7 mW mm.sup.-2, about 8 mW mm.sup.-2,
about 9 mW mm.sup.-2, about 10 mW mm.sup.-2, about 11 mW mm.sup.-2,
about 12 mW mm.sup.-2, about 13 mW mm.sup.-2, about 14 mW
mm.sup.-2, about 15 mW mm.sup.-2, about 16 mW mm.sup.-2, about 17
mW mm.sup.-2, about 18 mW mm.sup.-2, about 19 mW mm.sup.-2, about
20 mW mm.sup.-2, about 21 mW mm.sup.-2, about 22 mW mm.sup.-2,
about 23 mW mm.sup.-2, about mW mm.sup.-2, or about 25 mW
mm.sup.-2, inclusive, including any values between these
numbers.
[0080] In some aspects of the methods described herein, the animal
cell can be a neuronal cell, a cardiac cell, or a stem cell. In
some embodiments, the animal cell can be a neuronal cell. In other
embodiments, the neuronal cell can be an excitatory neuron located
in the pre-frontal cortex of a non-human animal. In other
embodiments, the excitatory neuron can be a pyramidal neuron. In
some embodiments the neuronal cell can be an inhibitory neuron
located in the pre-frontal cortex of a non-human animal. In still
other embodiments, the inhibitory neuron can be a parvalbumin
neuron. In some embodiments, the inhibitory and excitatory neurons
can be in a living non-human animal. In other embodiments, the
inhibitory and excitatory neurons can be in a brain slice from a
non-human animal. In other embodiments, the brain slices comprise
neurons that effect social behavior when depolarized. In some
embodiments, the neuronal cell is a neuron that changes innate
social behavior and/or conditioned behavior when depolarized.
[0081] In other embodiments, the brain slices comprise neurons that
give rise to the social and cognitive defects in autism and/or
schizophrenia when depolarized.
[0082] Methods for Identifying a Chemical Compound that Restores an
Innate Social Behavior and/or Communication in a Non-Human
Animal
[0083] Provided herein are method for identifying a chemical
compound that restores one or more social behaviors,
communications, and/or conditioned behaviors in the non-human
animal, the method comprising: (a) depolarizing an excitatory
neuron in the prefrontal cortex of a non-human animal comprising a
light-activated protein cation channel expressed on the cell
membrane capable of mediating a depolarizing current in the cell
when the cell is illuminated with light, wherein the protein
exhibits rapid step-like activation in response to a single pulse
of light having a first wavelength and deactivation in response to
a pulse of light having a second wavelength; wherein the
depolarizing current in the cell is maintained for up to about
twenty minutes; and wherein the protein comprises the amino acid
sequence of ChR2, ChR1, VChR1, or VChR2 with amino acid
substitutions at amino acid residues corresponding to C128 and D156
of the amino acid sequence of ChR2, wherein depolarizing the
excitatory neuron inhibits one or more one or more social
behaviors, communications, and/or conditioned behaviors in the
non-human animal; (b) administering a chemical compound to the
non-human animal; and (c) determining if the administration of the
chemical compound to the non-human animal restores said one or more
social behaviors, communications, and/or conditioned behaviors in
the non-human animal. In some aspects, the social behavior is an
innate social behavior and is selected from the group consisting
of: allogrooming, resident-intruder aggression, isolation-induced
fighting, sexual behavior, parental behavior, social recognition,
and auditory communication. Information pertaining to innate social
behavioral tests for mice and other lab models can be found in
Crawley, Social Behavior Tests for Mice, Laboratory of Behavioral
Neuroscience, National Institute of Mental Health, (Bethesda, Md.;
2007), the disclosure of which is hereby incorporated herein by
reference in its entirety. In other embodiments, the behavior is a
conditioned behavior, such as, but not limited to, a conditioned
fear response. In some embodiments, the non-human animal is not
constrained by any hardware during steps (b) through (c). In some
embodiments, the hardware is a light source attached to a fiber
optic cable. In other embodiments, the non-human animal is
separated from hardware immediately after the stabilized step
function opsin protein is activated in response to said single
pulse of light having a first wavelength. In some embodiments, the
animal cell is located on the surface of a biological tissue. In
some embodiments, the tissue is neural tissue or brain tissue. In
some embodiments, the chemical compound can be a member of a
combinatorial chemical library.
[0084] In some embodiments, the non-human animals of the methods
provided herein comprise the protein corresponding to SEQ ID NO:1.
In other embodiments, the animals comprise the stabilized step
function opsin proteins disclosed herein. In some embodiments, the
animals comprising the stabilized step function opsin proteins
disclosed herein are transgenically expressing said stabilized step
function opsin proteins. In other embodiments, the animals
comprising the stabilized step function opsin proteins described
herein have been virally transfected with a vector carrying the
stabilized step function opsin proteins such as, but not limited
to, an adenoviral vector or an andeno-associated viral vector. In
some embodiments, the animals comprising the stabilized step
function opsin proteins disclosed herein exhibit changes in
behavior when said stabilized step function opsin proteins are
depolarized by activation with light. In other embodiments, the
animals comprising the stabilized step function opsin proteins
disclosed herein exhibit changes in innate and learned social
behaviors when said stabilized step function opsin proteins are
depolarized by activation with light. In other embodiments, the
animals comprising the stabilized step function opsin proteins
disclosed herein exhibit changes in conditioned behaviors when said
stabilized step function opsin proteins are depolarized by
activation with light.
EXEMPLARY EMBODIMENTS
[0085] The present disclosure is believed to be useful for optical
control over nervous system disorders. Specific applications of the
present invention relate to optogenetic systems or methods that
correlate temporal, spatio, and/or cell-type control over a neural
circuit with measurable metrics. As many aspects of the example
embodiments disclosed herein relate to and significantly build on
previous developments in this field, the following discussion
summarizes such previous developments to provide a solid
understanding of the foundation and underlying teachings from which
implementation details and modifications might be drawn including
those found in Yizhar et al., Nature, 2011, 477(7363):171-8, the
disclosure of which in incorporated by reference herein in its
entirety. It is in this context that the following discussion is
provided and with the teachings in the references incorporated
herein by reference. While the present invention is not necessarily
limited to such applications, various aspects of the invention may
be appreciated through a discussion of various examples using this
context.
[0086] Various embodiments of the present disclosure relate to an
optogenetic system or method that correlates temporal control over
a neural circuit with measurable metrics. For instance, various
metrics or symptoms might be associated with a neurological
disorder exhibiting various symptoms of social dysfunction. The
optogenetic system targets a neural circuit within a
subject/patient for selective control thereof. The optogenetic
system involves monitoring the subject/patient for the metrics or
symptoms associated with the neurological disorder. In this manner,
the optogenetic system can provide detailed information about the
neural circuit, its function and/or the neurological disorder.
[0087] FIG. 12 depicts a flow diagram for testing of a disease
model, consistent with various embodiments of the present
disclosure. At 102, one or more disease models are identified or
selected. The disease models can be for one or more central nervous
system (CNS) disorders. The models can include various disorders,
diseases or even general characteristics of patients (e.g., mood,
memory, locomotion or social behavior). At 104, one or more CNS
targets are identified. As used herein, the CNS targets include the
properties of the stimulus to be provided as part of assessing,
testing or otherwise related to the disease model. Non-limiting
examples of targets can be spatial targets, cell type targets,
temporal targets and combinations thereof.
[0088] The properties of the targets 106-118 can then be used to
select a particular opsin from the optogenetic toolkit 120. The
optogenetic toolkit 120 includes a variety of different opsins,
which can be aligned with one or more of the properties 106-118.
Various non-limiting examples of the opsins are discussed herein.
The selected opsin(s) 122 can be those opsins that most closely
match the CNS target(s) and/or stimulus properties. For example, a
desired target may be the modification of excitation/inhibition
(E/I) balance within a portion of the brain over an extended period
of time. As discussed herein, the opsin C1V1 (discussed in more
detail herein) and its variants could be selected. Thereafter, the
selected opsin(s) are expressed in a target CNS location/cell-type
124. The disease module is then tested 126, e.g., through optical
stimulus of the expressed opsin(s).
[0089] Embodiments of the present disclosure are directed toward
control over the cellular excitation/inhibition (E/I) balance
within neocortical microcircuitry. Such E/I balance control can be
particularly useful for modeling and/or treatment of social and
cognitive deficits (e.g., autism and schizophrenia) that are linked
to elevations in excitation.
[0090] Embodiments of the present disclosure are directed toward
the use of opsins for providing a mechanism for inducing an
elevated cellular E/I balance with specific spatial and temporal
control. This can include expression of light-sensitive opsins in
excitatory neurons linked with one or more severe neuropsychiatric
diseases.
[0091] Various embodiments relate to tools and methods for
controlling the E/I balance in freely moving mammals, which can be
particularly useful for exploring underlying circuit physiology
mechanisms. Particular aspects of the present disclosure relate to
increasing the excitability of excitatory neurons, relative to the
excitability of inhibitory neurons with selective spatial control.
This can be particularly useful for increasing the susceptibility
of the excitatory neurons to intrinsic stimulus and thereby
preserving natural firing patterns. In some implementations, this
excitation is reversible.
[0092] Certain embodiments are directed toward the use of ion
channels that are optically controllable. When expressed in a
neuron, the ion channels are designed to increase the
susceptibility of the neurons to intrinsic stimulus to maintain the
increased susceptibility for extended periods of time. Embodiments
of the present disclosure relate to SSFOs (stabilized step-function
opsins) that are stable enough to produce constant photocurrent
after a single light flash over many minutes, and the use thereof
for complex behavioral testing. In particular implementations, the
increased susceptibility can be maintained from many minutes after
optical stimulus is applied.
[0093] Various embodiments are directed toward treatments, modeling
and other aspects that relate to the discovery that impairments in
specific social interaction and cognition behaviors in freely
moving mice can be induced from targeted elevation in the E/I
balance.
[0094] Other embodiments are directed towards treatments, modeling
and other aspects that relate to the discovery that no such
behavioral effects are seen when selectively providing the same
excitability advantage to inhibitory neurons, irrespective of
profound effects on local circuit activity.
[0095] Still other embodiments of the present disclosure are
directed toward treatments, modeling and other aspects that relate
to the discovery that the dominant circuit-level effect of the
behaviorally significant E/I balance intervention is a specific
elevation in baseline gamma-band (around 40-60 Hz) recurrent
synaptic excitation, analogous to the elevated gamma rhythms seen
at baseline in autism and schizophrenia, with concomitant
quantitative impairment in microcircuit information
transmission.
[0096] Embodiments of the present disclosure relate to the use of
opsins to drive E/I elevations and monitor gamma oscillations in
cortical slices. Particular embodiments are directed toward the use
of C1V1 (discussed in more detail herein) and its variants, which
can be particularly useful for driving E/I elevations and
monitoring gamma oscillations in cortical slices, with 1) high
potency to enable dose-response tests; 2) low desensitization to
allow for step-like changes in E/I balance; and 3) red-shifted
excitation to allow separable driving of different populations
within the same preparation.
[0097] Embodiments of the present disclosure relate to control over
elevated (or lowered) cellular E/I balance. This can be
particularly useful for studying, testing and treatment relating to
medication-unresponsive social and cognitive impairment in
neurological disorders, such as autism and schizophrenia.
Particular aspects relate to studying and distinguishing the long
term effects on the development and maturation of the circuit
relative to the immediate effects of E/I abnormalities with regard
to the function of the neural circuits involved. Other aspects are
directed toward the confirmation of elevated cellular E/I balance
as a core component of cognitive defects observed in the various
disease models and patients (human or otherwise). Particular
embodiments provide timing and specificity sufficient for testing
the elevated cellular E/I balance hypothesis in the mammalian brain
(e.g., the prefrontal cortex), and identified circuit-physiology
manifestations.
[0098] A particular aspect relates to the use of the double-mutant
SSFO (discussed in more detail herein), which can be particularly
useful for providing stable circuit modulation for time periods
that are sufficient for temporally precise and complex behavioral
experiments. For instance, the modulation and behavioral
experiments circuit modulation can span several minutes in the
absence of ongoing light activation, external fiber optic
attachments and/or optical-hardware brain penetration (e.g., using
a light delivery device entirely external to the brain). Particular
implementations use a property of photon integration, which can
facilitate activation of cells with low light intensity (e.g., in
the low-gm/mm.sup.2). This activation can occur with relatively
deep penetration of light into brain tissue (e.g., 3 mm or more
relative to the light source). SSFO activation in excitatory (but
not inhibitory) neurons can be used to produce profound and
reversible impairments in social and cognitive function. In certain
implementations, the impairments can be produced with little, if
any, motor abnormalities or altered fear/anxiety behaviors.
[0099] Embodiments of the present disclosure also relate to the use
of SSFO for in vitro probing of changes in circuit properties. For
instance, SSFO's can be used to elevate cellular E/I balance and to
measure the transfer functions of pyramidal neurons. Experimental
results suggest that such elevation saturates the transfer
functions of pyramidal neurons at low excitatory post-synaptic
current (EPSC) rates, impairing information transmission within
cortical circuitry, in contrast to consequences of reduction in E/I
balance.
[0100] These and other aspects can be particularly useful for
addressing the symptomatic and treatment challenges in
medication-unresponsive disorders like autism, e.g., relative
to
elevations in Ed balance and situations in which the brain appears
hyper-excitable and impaired in its ability to process
information.
[0101] Consistent with an experimental embodiment, a comparison was
performed between light-evoked activity in C1V1-E162T-expressing
(discussed in more detail herein) and nonexpressing pyramidal cells
(PYR cells). PYR cells expressing C1V1-E162T spiked in response to
2 ms 561 nm light pulses, while the same stimulation paradigm
reliably evoked excitatory postsynaptic potentials (EPSPs) in
non-expressing cells within the same slices.
[0102] Particular embodiments of the present disclosure are
directed toward the use of SSFO gene product to selectively favor
excitation of one neural population over another. The selective
favoring of the targeted population can be configured to prevent
the SSFOs from overriding intrinsic excitation inputs to the
targeted population. In this manner, the targeted population would
not be driven with coordinated spikes directly caused by the
opsins. Rather, the targeted population would exhibit an increased
sensitivity to native inputs, which can be sparse and
asynchronous.
[0103] Embodiments of the present disclosure are directed toward
the use of SFOs to address various the hardware challenges. For
instance, the significant increase in light sensitivity (e.g.,
orders-of-magnitude greater) can facilitate the use alternative
light delivery mechanisms, and hardware-free behavioral
testing.
[0104] Aspects of certain embodiments of the present disclosure are
directed toward identification and modification of specific
portions of light-gated channels. These modifications involve
identifying key portions of the channels. The channels can be
identified using high resolution imaging of the tertiary structure
of the channel. Alternatively, knowledge of the structure of
similar channels can be used. The following description provides
details of a specific experimental implementation and methodology.
The present disclosure is not limited to any one implementation and
can be implemented for a number of different molecular
modifications at various locations consistent with the teachings
herein.
[0105] Specific aspects of the present disclosure relate to
microbial opsin genes adapted for neuroscience, allowing
transduction of light pulse trains into millisecond-timescale
membrane potential changes in specific cell types within the intact
mammalian brain (e.g., channelrhodopsin (ChR2), Volvox
channelrhodopsin (VChR1), and halorhodopsin (NpHR)). ChR2 is a
rhodopsin derived from the unicellular green algae Chlamydomonas
reinhardtii. The term "rhodopsin" as used herein is a protein that
comprises at least two building blocks, an opsin protein, and a
covalently bound cofactor, usually retinal (retinaldehyde). The
rhodopsin ChR2 is derived from the opsin Channelopsin-2 (Chop2),
originally named Chlamyopsin-4 (Cop4) in the Chlamydomonas genome.
The temporal properties of one depolarizing channelrhodopsin, ChR2,
include fast kinetics of activation and deactivation, affording
generation of precisely timed action potential trains. For
applications seeking long timescale activation, it has been
discovered that the normally fast off-kinetics of the
channelrhodopsins can be slowed. For example, certain
implementations of channelrhodopsins apply 1 mW/mm.sup.-2 light for
virtually the entire time in which depolarization is desired, which
can be less than desirable.
[0106] Much of the discussion herein is directed to ChR2. Unless
otherwise stated, the disclosure includes a number of similar
variants. Examples include, but are not limited to, Chop2,
ChR2-310, Chop2-310, and Volvox channelrhodopsin (VChR1). For
further details on VChR1, reference can be made to "Red-shifted
optogenetic excitation: a tool for fast neural control derived from
Volvox carteri," Nat Neurosci., June 2008, 11(6):631-3. Epub 2008
Apr. 23, which is fully incorporated herein by reference. In other
implementations similar modifications can be made to other opsin
molecules. For instance, modifications/mutations can be made to
ChR2 or VChR1 variants. Moreover the modified variants can be used
in combination with light-activated ion pumps.
[0107] Embodiments of the present disclosure include relatively
minor amino acid variants of the naturally occurring sequences. In
one instance, the variants are greater than about 75% homologous to
the protein sequence of the naturally occurring sequences. In other
variants, the homology is greater than about 80%. Yet other
variants have homology greater than about 85%, greater than 90%, or
even as high as about 93% to about 95% or about 98%. Homology in
this context means sequence similarity or identity, with identity
being preferred. This homology can be determined using standard
techniques known in the sequence analysis. The compositions of
embodiments of the present disclosure include the protein and
nucleic acid sequences provided herein, including variants which
are more than about 50% homologous to the provided sequence, more
than about 55% homologous to the provided sequence, more than about
60% homologous to the provided sequence, more than about 65%
homologous to the provided sequence, more than about 70% homologous
to the provided sequence, more than about 75% homologous to the
provided sequence, more than about 80% homologous to the provided
sequence, more than about 85% homologous to the provided sequence,
more than about 90% homologous to the provided sequence, or more
than about 95% homologous to the provided sequence.
[0108] As used herein, stimulation of a target cell is generally
used to describe modification of properties of the cell. For
instance, the stimulus of a target cell may result in a change in
the properties of the cell membrane that can lead to the
depolarization or polarization of the target cell. In a particular
instance, the target cell is a neuron and the stimulus affects the
transmission of impulses by facilitating or inhibiting the
generation of impulses (action potentials) by the neuron.
[0109] For further details on light-responsive opsins, reference
can be made to PCT publication No. WO 2010/056970, entitled
"Optically-Based Stimulation of Target Cells and Modifications
Thereto," to Deisseroth et al., which is fully incorporated herein
by reference.
[0110] Embodiments of the present disclosure are directed towards
implementation of bistable changes in excitability of targeted
populations. This includes, but is not necessarily limited to, the
double-mutant ChR2-C128S/D156A. This double-mutant ChR2-C128S/D156A
has been found to be well-tolerated in cultured hippocampal neurons
and preserved the essential SFO properties of rapid step-like
activation with single brief pulses of blue light, and deactivation
with green or yellow light. In particular, the activation spectrum
of ChR2-C128S/D156A peaks at 445 nm. A second deactivation peak was
found at 390-400 nm, with faster but less complete deactivation by
comparison with the 590 nm deactivation peak. Peak photocurrents in
cells expressing ChR2-C128S/D156A were found to be robust, and
comparable to those of ChR2-D156A (231.08.+-.31.19 s.e.m; n=9 cells
and 320.96.+-.78.26 s.e.m; n=7 cells, respectively). Other
embodiments are directed toward a similar mutation in VChR1. For
instance, the mutation in VChR1 could be provided at C123S/D151A,
to provide a red-shifted photocurrent with slow kinetics comparable
to ChR2.
[0111] Individual transfected and patch-clamped neurons were next
activated with 100 ms pulses of 470 nm light, and to ensure over
very long recordings that current decay would not be attributable
to cell rundown, each cell was deactivated with prolonged 590 nm
light pulses at distinct intervals to determine the magnitude of
remaining SFO current at each time point. Surprisingly, neurons
expressing ChR2-C128S/D156A gave rise to sustained photocurrents
that were more stable than those from cells expressing either
single mutant alone. Fitting a mono-exponential decay curve to the
ratio of Ideactivation/Iactivation over time revealed a spontaneous
decay time constant of 29.3 min for ChR2-C128S/D156A, indicating
that the C128 and D156 mutations act synergistically to delay the
decay of the open state of ChR2.
[0112] Consistent with the required improvement for the anticipated
application to complex mammalian behaviors, significant portions of
the double-mutant SFO current were still present up to 20 minutes
after the single photoactivation pulse. Based on these surprisingly
slow decay kinetics, the double-mutant gene is referred to as SSFO
(for stabilized step-function opsin) gene. SSFO is also used as
shorthand for the active protein. Both residues likely are involved
in ChR2 channel closure (gating), and both mutations likely
stabilize the open state configuration of the channel.
[0113] Without being limited by theory, aspects of the present
disclosure relate to the discovery that SSFO may be completely
blocked in photocycle progression, and may therefore represent the
maximal stability possible with photocycle engineering. For
instance, in contrast to ChR2-C128X and ChR2-D156A, the SSFO
photocycle does not appear to access additional inactive
deprotonated side products which likely split off the photocycle at
later photocycle stages not reached in this mutant, in turn making
the SSFO even more reliable for repeated use in vivo than the
parental single mutations.
[0114] Embodiments of the present disclosure are directed toward
the sensitivity of the SSFO to light. For instance,
channelrhodopsins with slow decay constants effectively act as
photon integrators. This can be particularly useful for
more-sensitive, less-invasive approaches to optogenetic circuit
modulation, still with readily titratable action on the target
neuronal population via modulation of light pulse length. It has
been discovered that, even at extraordinarily low light intensities
(as low as 8 .mu.W/mm.sup.2), hundreds of picoamps of whole-cell
photocurrents could be obtained from neurons expressing SSFO, which
increased with monoexponential kinetics in response to 470 nm light
during the entire time of illumination. Other aspects relate to the
use of activation time constants that are linearly correlated with
the activation light power on a log-log scale, which is indicative
of a power-law relationship and suggesting that the SSFO is a pure
integrator, with total photon exposure over time as the only
determinant of photocurrent. For instance, it is believed that the
number of photons per membrane area required for photocurrents to
reach a given sub-maximal activation (time to T) is constant
regardless of activation light power.
[0115] Example embodiments of the present disclosure relate to the
use of a hybrid ChR1/VChR1 chimera that contains no ChR2 sequence
at all, is derived from two opsins genes that do not express well
individually, and is herein referred to as C1V1. Embodiments of the
present disclosure also relate to improvements of the membrane
targeting of VChR1 through the addition of a membrane trafficking
signal derived from the Ki.sub.r2.1 channel. Confocal images from
cultured neurons expressing VChR1-EYFP revealed a large proportion
of intracellular protein compared with ChR2; therefore, membrane
trafficking signal derived from the Ki.sub.r2.1 channel was used to
improve the membrane targeting of VChR1. Membrane targeting of this
VChR1-is-EYFP was slightly enhanced compared with VChR1-EYFP;
however, mean photocurrents recorded from cultured hippocampal
neurons expressing VChR1ts-EYFP were only slightly larger than
those of VChR1-EYFP.
[0116] Accordingly, embodiments of the present disclosure relate
VChR1 modified by exchanging helices with corresponding helices
from other ChRs. For example, robust improvement has been
discovered in two chimeras where helices 1 and 2 were replaced with
the homologous segments from ChM. It was discovered that whether
splice sites were in the intracellular loop between helices 2 and 3
(at ChR1 residue A1a145) or within helix 3 (at ChR1 residue
Trp163), the resulting chimeras were both robustly expressed and
showed similarly enhanced photocurrent and spectral properties.
This result was unexpected as ChR1 is only weakly expressed and
poorly integrated into membranes of most mammalian host cells. The
resulting hybrid ChR1IVChR1 chimera is herein referred to as
C1V1.
[0117] Aspects of the present disclosure relate to the expression
of C1V1 in cultured hippocampal neurons. Experimental tests have
shown a number of surprising and useful results, which are
discussed in more detail hereafter. C1V1-EYFP exhibits surprisingly
improved average fluorescence compared with VChR1-EYFP. Whole cell
photocurrents in neurons expressing C1V1 were much larger than
those of VChR1-EYFP and VChR1-ts-EYFP, and ionic selectivity was
similar to that of ChR2 and VChR1. The addition of the Kir2.1
trafficking signal between C1V1 and YFP further enhanced
photocurrents by an additional 41% (C1V1-ts-EYFP mean photocurrents
were extremely large, nearly tenfold greater than wild type (WT)
VChR1). Mean fluorescence levels closely matched the measured
photocurrents (mean fluorescence 9.3.+-.1, 19.6.+-.3.4, 19.8.+-.2.8
and 36.3.+-.3.8 for VChR1-EYFP, VChR1-ts-EYFP, C1V1-EYFP and
C1V1-ts-EYFP, respectively), suggesting that the increase in
photocurrent sizes resulted mainly from the improved expression of
these channels in mammalian neurons. Total somatic fluorescence
(measured as integrated pixel density) was linearly correlated with
photocurrent size in individual recorded/imaged cells across the
different constructs (VChR1, VChR1-ts-EYFP, C1V1, C1V1-ts-EYFP).
This suggests (without being limited by theory) that the increased
photocurrent of C1V1 results from functional expression changes in
neurons.
[0118] Various embodiments of the present disclosure relate to
opsins with fast decay constants. This property can be particularly
useful for providing precise control over spiking, e.g., in order
to interfere minimally with intrinsic conductance, trigger single
spikes per light pulse and/or minimize plateau potentials during
light pulse trains Experimental results suggest that the
light-evoked photocurrents recorded in C1V1-ts-EYFP decayed with a
time constant similar to that of VChR1. Aspects of the present
disclosure are therefore directed toward modifications in the
chromophore region to improve photocycle kinetics, reduced
inactivation and/or possible further red-shifted absorption.
[0119] One embodiment is directed toward a corresponding ChETA
mutation E162T, which experiments suggest provides an accelerated
photocycle (e.g., almost 3-fold); reference can be made to
Gunaydin, et al., Ultrafast optogenetic control, Nat Neurosci,
2010, and which is fully incorporated herein by reference.
Surprisingly, this mutation was shown to shift the action spectrum
hypsochromic to 530 nm, whereas analogous mutations in ChR2 or
other microbial rhodopsins have caused a red-shift. Another
embodiment is directed toward a mutation of glutamate-122 to
threonine (C1V1-E122T). Experimental tests showed that C1V1-E122T
is inactivated only by 26% compared to 46% inactivation of ChR2; in
addition, the spectrum was further red-shifted to 546 nm.
[0120] Another embodiment of the present disclosure is directed
toward a double mutant of C1V1 including both E122T and E162T
mutations. Experimental tests have shown that the inactivation of
the current was even lower than in the E122T mutant and the
photocycle was faster compared to E162T. This suggests that
multiple useful properties of the individual mutations were
conserved together in the double mutant.
[0121] Embodiments of the present disclosure include the expression
of various light-responsive opsins in neurons. Experimental tests
of C1V1 opsin genes in neurons were carried out by generating
lentiviral vectors encoding C1V1-ts-EYFP and various point mutation
combinations discussed herein. The opsins were then expressed in
cultured hippocampal neurons and recorded whole-cell photocurrents
under identical stimulation conditions (2 ms pulses, 542 nm light,
5.5 mW/mm.sup.-2). Photocurrents in cells expressing C1V1,
C1V1-E162T and C1V1-E122T/E162T were all robust and trended larger
than photocurrents of ChR2-H134R. The experiments also included a
comparison of integrated somatic YFP fluorescence and photocurrents
from cells expressing C1V1-E122T/E162T and from cells expressing
ChR2-H134R. Surprisingly, C1V1-E122T/E162T cells showed stronger
photocurrents than ChR2-H134R cells at equivalent fluorescence
levels. This suggests that C1V1 could possess a higher unitary
conductance compared with ChR2-H134R. The test results suggest that
the kinetics of C1V1-E122T were slower than those of
C1V1-E122T/E162T and that cells expressing C1V1-E122T responded
more strongly to red light (630 nm) than cells expressing the
double mutant. This can be particularly useful for generating
optogenetic spiking in response to red-light.
[0122] Consistent with various embodiments of the present
disclosure, inhibitory and/or excitatory neurons residing within
the same microcircuit are be targeted with the introduction of
various opsins. Experimental tests were performed by separately
expressed C1V1-E122T/E162T and ChR2-H134R under the
CaMKII.alpha.promoter in cultured hippocampal neurons. Cells
expressing C1V1-E122T/E162T spiked in response to 2 ms green light
pulses (560 nm) but not violet light pulses (405 nm). In contrast,
cells expressing ChR2-H134R spiked in response to 2 ms 405 nm light
pulses, but not in response to 2 ms 561 nm light pulses.
[0123] Various embodiments of the present disclosure relate to
independent activation of two neuronal populations within living
brain slices. Experimental tests were performed by
CaMKIIa-C1V1-E122T/E162Tts-eYFP and EF1a-DIO-ChR2-H134R-EYFP in
mPFC of PV::Cre mice. In non-expressing PYR cells, 405 nm light
pulses triggered robust and fast inhibitory postsynaptic currents
due to direct activation of PV cells, while 561 nm light pulses
triggered only the expected long-latency polysynaptic IPSCs arising
from C1V1-expressing pyramidal cell drive of local inhibitory
neurons.
[0124] Consistent with other embodiments of the present disclosure,
excitation of independent cellular elements can be performed in
vivo. Experimental tests were performed using optrode recordings.
To examine the inhibitory effect of PV cell activity on pyramidal
neuron spiking, an experimental protocol was used in which 5 Hz
violet light pulses (to activate ChR2 in PV cells) preceded 5 Hz
green light pulses (to activate C1V1 in excitatory pyramidal
neurons) with varying inter-pulse intervals. The test results
suggest that when violet and green light pulses were separated by
100 ms, responses to green light pulses were not affected by the
violet pulses. However, as delays between violet and green pulses
were reduced, green light-induced events became more readily
inhibited until being effectively/completely abolished when light
pulses were presented simultaneously.
[0125] As discussed herein, various embodiments of the present
disclosure relate to an optogenetic system or method that
correlates temporal, spatio and/or cell-type control over a neural
circuit with measurable metrics. Consistent with the other
embodiments discussed herein, particular embodiments relate to
studying and probing disorders. A non-exhaustive list of example
embodiments and experimental results consistent with such
embodiments is provided in Yizhar et al., Nature, 2011,
477(7363):171-8, the disclosure of which in incorporated by
reference herein in its entirety. The references listed therein may
assist in providing general information regarding a variety of
fields that may relate to one or more embodiments of the present
disclosure, and further may provide specific information regarding
the application of one or more such embodiments, to which one or
more references as follows may be applicable. Accordingly, each of
these references is fully incorporated herein by reference.
[0126] Various embodiments described above and shown in the figures
may be implemented together and/or in other manners. One or more of
the items depicted in the drawings/figures can also be implemented
in a more separated or integrated manner, or removed and/or
rendered as inoperable in certain cases, as is useful in accordance
with particular applications. In view of the description herein,
those skilled in the art will recognize that many changes may be
made thereto without departing from the spirit and scope of the
present disclosure.
[0127] The present disclosure is believed to be useful as it
relates to control over nervous system disorders, such as disorders
associated with social dysfunction, as described herein. Specific
applications of the present invention relate to optogenetic systems
or methods that correlate temporal, spatio and/or
cell-type-specific control over a neural circuit with measurable
metrics. As many aspects of the example embodiments disclosed
herein relate to and significantly build on previous developments
in this field, the following discussion summarizes such previous
developments to provide a solid understanding of the foundation and
underlying teachings from which implementation details and
modifications might be drawn, including those found in the attached
Appendix. It is in this context that the following discussion is
provided and with the teachings in the references incorporated
herein by reference. While the present invention is not necessarily
limited to such applications, various aspects of the invention may
be appreciated through a discussion of various examples using this
context.
[0128] FIG. 13 depicts a model for assessing stimuli and/or
potential treatments for various nervous system disorders. Baseline
observations 220 are taken 202 of behavior and/or cellular response
for a subject/patient. A target cell population is chosen and
modified to express a light-responsive molecule. In particular
implementations, the target cell population is selected to provide
control over the E/I balance in the prefrontal cortex of a
subject's brain, as discussed in more detail herein. The
excitation/inhibition (E/I) balance within the target cell
population can then modified 204 (e.g., elevated or lowered) by
exposing the modified target cell population to light. The light
can be provided within a predetermined range based on absorption
characteristics of the light-responsive molecule. Observations 220
of behavior and/or cellular response of the subject are again
taken. These observations provide a reference point for how the
subject acts under no stimuli or treatment.
[0129] To assess potential treatments, a stimulus and/or potential
treatment is chosen 206 for the subject. Non-limiting examples of
stimuli and treatments include pharmacological/drugs 208,
behavioral 210 and/or electrical stimulus 212. The
stimuli/treatments can then be assessed 214 by observing the
subject's behavior in response to the treatment and/or the target
cell population's behavior in response to the treatment. Based on
the observations, a determination can be made regarding the need
for additional stimulus or treatment 216, or the desire to test
additional and/or different treatments. After the observations 220
have been collected, the observations 220 from various treatments
can be compared 218 to each other as well as the baseline
observation and the observations of behavior after E/I elevation.
The comparison of the observations 220 can be used to assess the
efficacy of various potential treatments.
[0130] In certain more specific embodiments, the elevation of the
E/I balance results in social and cognitive deficits as compared to
the behaviors during baseline observations. The purposeful and
controlled elevation of the E/I balance allows for the testing of
potential treatments in mammalian test subjects such as mice that
do not otherwise exhibit symptoms of the disease being modeled.
[0131] Aspects of the present disclosure relate to assessing the
effect of various stimuli on symptoms of neurological diseases. As
discussed throughout this disclosure, modification of the E/I
balance in the prefrontal cortex of a subject's brain results in
the symptoms similar to those of various neurological disorders,
such as autism and schizophrenia. In certain aspects of the present
disclosure, the neural circuit identified as effecting E/I balance
is manipulated using one or more techniques including
pharmacological, electrical, magnetic, surgical and optogenetic
methods. The effect of the manipulation of the symptoms displayed
is monitored.
[0132] In certain more specific aspects, the manipulation of
pyramidal neurons and parvalbumin-expressing inhibitory
interneurons is used to model disease states, and to identify new
treatments for known diseases. For example, the E/I balance in the
prefrontal cortex is elevated (or lowered) and then a potential
treatment is administered to the subject. The effect of the
treatment on either the observed symptoms or on the neural circuit
(or both) can be monitored. The information obtained from
monitoring the symptoms and/or the neural circuit can be used to
provide a better understanding of the neural pathways causing the
observed symptoms. The information may also be used to determine
the efficacy of the potential treatment. Based on the efficacy, or
lack thereof, of the potential treatment, modifications can be made
resulting in a new potential treatment to be tested.
[0133] In certain embodiments of the present disclosure, a stimulus
is provided to a subject that exhibits symptoms of a neural disease
such as schizophrenia or autism, for example. The stimulus can be
pharmacological, electrical, magnetic, surgical, optogenetic or
behavioral, for example.
[0134] Consistent with various embodiments of the present
disclosure, control over the neural circuit can include inhibition
or excitation, which can each include coordinated firing, and/or
modified susceptibility to external circuit inputs. For instance,
inhibition can be accomplished using a light-responsive opsin, such
as an ion pump (e.g., NpHR and NpHR variants). Such ion pumps move
the membrane potential of the neuron away from its threshold
voltage to dissuade or inhibit action potentials. In another
instance, excitation can be accomplished using a light-responsive
opsin, such as an ion channel (e.g., ChR2 and ChR2 variants). Such
ion channels can cause the membrane potential to move toward and/or
past the threshold voltage, thereby exciting or encouraging action
potentials. Consistent with various embodiments, a light-responsive
opsin can be used to (temporarily) shift the resting potential of a
neuron to increase or decrease its susceptibility to external
circuit inputs. These various options can also be used in
combination.
EXAMPLES
Example 1
Creation and Characterization of the Stabilized Step Function
Opsin
[0135] Long-timescale (indeed bistable) optogenetic tools were
initially developed that operate on timescales up to 4 orders of
magnitude longer than that of wild type (wt) ChR2 (SFO or step
function opsin gene products; -r-off=2.5-102 seconds); these
mutations at the C128 position of ChR2 led to increased light
sensitivity that scaled with the deactivation time constant.
Subsequent work further developed the initial SFO concept, with
mutation of the C128 proton networking partner D156 (FIG. 1A) for
extension of the photocycle and lifetime of the open state.
However, neither class of mutation gives rise to full stability on
the mammalian-behavioral timescale-both showing substantial decay
during the first 5-10 min--and extended illumination of
SFO-expressing neurons in some cases can lead to channelrhodopsin
inactivation caused by deprotonation of the chromophore and
accumulation of a photocycle side product in a side reaction from a
late photocycle intermediate. Therefore, the generation of a
blue-light activated SFO suitably stable for combinatorial
optogenetics in mammalian systems by mutating both C128 and D156
was attempted, hypothesizing that the combined mutant could
potentially exhibit sufficient stabilization of the open state.
Since the SFOs are activated with blue light but in fact can be
deactivated with yellow light, if this additional property were
maintained such a stable SFO would also deliver lateral-inhibition
in the spectral domain that could further enhance combinatorial
control.
[0136] Materials and Methods
[0137] ChR2(D156A) and SSFO were generated by introducing point
mutations into the pLentiCaMKII.alpha.-ChR2-EYFP-WPRE vector using
site-directed mutagenesis (Quikchange II XL; Stratagene). The
membrane trafficking signal was derived from the Kir2.1 channel.
Mutations were confirmed by sequencing the coding sequence and
splice sites. For AAV-mediated gene delivery, opsin-EYFP fusions
along with the CaMKII.alpha. promoter were subcloned into a
modified version of the pAAV2-MCS vector. Cre dependent opsin
expression was achieved by cloning the opsin-EYFP cassette in the
reverse orientation between pairs of incompatible lox sites (loxP
and 1ox2722) to generate a doublefloxed inverted open reading frame
(D10) under the control of the elongation factor 1a (EF-1.alpha.)
promoter. All constructs are available from the Deisseroth Lab
(www.optogenetics.org).
[0138] For heterologous expression of ChRs in Pichia pastoris cells
(strain 1168H, purchased from Invitrogen), human codon-optimized
synthetic ChR-fragment encoding amino acids 1-315 (see accession
no. AF461397) was cloned in the pPICZ vector (Invitrogen) via its
EcoRI and NotI restriction sites. The C-terminal polyhistidine tag
encoded on the vector was modified to a 12His sequence. Mutants of
ChR were generated by site-directed mutagenesis (QuickChange kit,
Stratagene). Transformation, cell culture and protein purification
were performed. After induction of protein expression for 24 h,
cells were harvested and gently lysed using a high pressure
homogenizer (Avastin). The membrane fraction was collected,
homogenized and solubilized in 1% (w/v) dodecylmaltoside. After
binding of ChR protein to a Ni-NTA resin (Qiagen) and washing of
the column with 200 mM imidazole, ChR was eluted with 500 mM
imidazole. Fractions that contained the protein were pooled,
desalted (Float-a-lyzer, Roth) and concentrated (Amicon Ultra,
Millipore) to an optical density of 1 at 480 nm. Spectra were
recorded in a Cary 50 Bio spectrophotometer (Varian Inc.).
[0139] Results
[0140] The ChR2 mutants C128S, D156A, and the double mutant
128S/156A were generated and purified from Pichia pastoris to first
measure intrinsic open-state stability in the absence of
potentially confounding cellular properties. Absorption spectra
showed expected rapid changes in response to brief light delivery
that largely recovered within 3 minutes for the single mutants
C128S (FIG. 1B, F) and D156A (FIG. 1C, G). However, in contrast to
both single mutants, the double mutant C128S/D156A showed
remarkably complete stability of the activated state, with
essentially no detectable return to the dark state even after 30
minutes (FIG. 1D, H). The characteristic two peaks of these
absorption spectra can be ascribed to formation of the conducting
state and a deprotonated species (P390; FIG. 1B, C) with some
interesting differences among the variants. First, a reduced red
shift of the conducting state relative to the dark state was noted
for the double mutant compared with C128S (FIG. 1A, D), raising the
concerning question of how effective the important property of
inactivation with redshifted light would be for the double mutant.
On the potentially beneficial side, it was also noted that a
reduced contribution from the nonconducting (P390) state relative
to the conducting state existed in the double mutant compared with
C128S (FIG. 1B, D), a useful property that may predict reduced
accumulation of nonconducting channels and that suggests a late
step of the photocycle that could deplete the conducting state
(e.g. P520-*P480 desensitized state (Des480); FIG. 1E) may be
almost completely blocked (FIG. 1E). The unique stability of the
double mutant C1 28S/D 156A is further illustrated by continuous
monochromatic absorbance measurements of all three mutants over 35
minutes of recording (FIG. 1H).
Example 2
Validation of Activation in Neurons and In Vivo
[0141] The double mutant therefore appeared to have markedly
distinct and near-optimal stability on the mammalian behavioral
timescale, but with potentially reduced crucial capability for
redshifted light deactivation; all of these issues required
validation in neurons and in vivo.
[0142] Materials and Methods.
[0143] Whole Cell Patch-Clamp Electrophysiology Hippocampal and
Cortical Neurons
[0144] Primary hippocampal cultures were isolated from PO
Sprague-Dawley rats, plated on Matrigel (Invitrogen)-coated glass
coverslips and treated with FUDR to inhibit glia overgrowth.
Endotoxin-free plasmid DNA was transfected in cultured neurons
using a HEPES buffered Saline/CaPO.sub.4 mix. Electrophysiological
recordings from individual neurons identified by fluorescent
protein expression were obtained in Tyrode media ([mM] 150 NaCl, 4
KCl, 2 MgCl.sub.2, 2 MgCl.sub.2, 10 D-glucose, 10 HEPES, pH 7.35
with NaOH) using a standard internal solution ([mM] 130 KGluconate,
10 KCl, 10 HEPES, 10 EGTA, 2 MgCl.sub.2, pH 7.3 with KOH) in 3-5 MO
glass pipettes. For cortical slice physiology, acute 300 .mu.m
coronal slices from 8-9 week old wild-type C57BL/6J or PV::Cre mice
previously injected with virus were obtained in ice-cold sucrose
cutting solution ([mM] 11 D-glucose, 234 sucrose, 2.5 KCl, 1.25
NaH.sub.2PO.sub.4, 10 MgSO.sub.4, 0.5 CaCl.sub.2, 26 NaHCO3) using
a Vibratome (Leica). Slices were recovered in oxygenated Artificial
Cerebrospinal Fluid (ACSF; [mM] 124 NaCl, 3 KCl, 1.3 MgCl.sub.2,
2.4 CaCl.sub.2, 1.25 NaH.sub.2PO.sub.4, 26 NaHCO.sub.3, 10
D-glucose) at 32.degree. C. for one hour. Individual neuron patches
were obtained after identifying fluorescent protein expression from
indicated prefrontal cortical layer under constant ACSF perfusion.
Filtered light from a broad-wavelength xenon lamp source (Sutter
Instruments DG-4) was coupled to the fluorescence port of the
microscope (Leica DM-LFSA). Band pass filters (Semrock) had 20 nm
bandwidth, and were adjusted with additional neutral density
filters (ThorLabs) to equalize light power output across the
spectrum. While handling cells or tissues expressing SSFO, care was
taken to minimize light exposure to prevent activation by ambient
light. Before each experiment, a 20s pulse of 590 nm light was
applied to convert all of the SSFO channels to the dark state and
prevent run-down of photocurrents. For acquisition of SSFO
activation and deactivation spectra, cultured neurons in voltage
clamp mode were recorded. For recording activation spectra, a 1 s
pulse of varying wavelength was applied, followed by a 10 s 590 nm
pulse. Deactivation spectra were acquired by first applying a 1 s
470 nm pulse to activate SSFO, followed by a 10 s pulse of varying
wavelength. Net activation or deactivation was calculated by
dividing the photocurrent change after the first or second pulse,
respectively, by the maximum photocurrent change induced by the
peak wavelength for that cell. Negative values in deactivation
spectra resulted from traces in which, for example, a 10 s 470 nm
pulse led to a slight increase in photocurrent rather than
deactivate the channels. This could be the result of the relatively
wide (20 nm) band-pass filter width used for these recordings with
the Sutter DG-4. Intermediate wavelengths (between 470 nm and 520
nm) are expected to have a mixed effect on the channel population
for the same reasons.
[0145] Cultured cell images were acquired on the same microscope
using a Retiga Exi CCD camera (Qimaging, Inc.) at 100 ms exposure
with 30 gain. Illumination power density was 12 mW mm.sup.-2 at 500
nm with a standard EYFP filter set. Quantification of fluorescence
was performed with ImageJ software by marking a region containing
the soma and proximal neurites and calculating for each cell the
total integrated pixel intensity in that region, rather than
average fluorescence, since photocurrents are likely to be related
to the total number of membrane-bound channels rather than average
channel expression per area. Photon flux calculations for SSFO
integration properties were conducted by calculating the photon
flux through the microscope objective at each light power, and then
dividing to reach the photon flux across the cell surface, based on
the diameter of the recorded cells and approximating cell shape as
a spheroid.
[0146] Viral Gene Transfection
[0147] Both Lentiviral- and AAV-mediated gene delivery were used
for heterologous expression of opsins in mice. Indicated opsins
were driven by either Human calmodulin-dependent protein kinase II
alpha (CaMKII.alpha.) promoter to target cortical excitatory
neurons or Elongation Factor 1a (EF-1a) in conjunction with a
Cre-inducible cassette and followed by the Woodchuck hepatitis
virus posttranscriptional regulatory element (WPRE). Cre-inducible
recombinant AAV vector was produced by the University of North
Carolina Vector Core (Chapel Hill, N.C., USA) and used in
conjunction with parvalbumin::Cre transgenic mice to target
parvalbumin positive interneurons. Briefly, SSFO-eYFP was inserted
in the reverse orientation between pairs of incompatible lox sites
(loxP and lox2722). AAV constructs were subcloned into a modified
version of the pAAV2-MCS, serotyped with AAV5 coat proteins and
packaged by the viral vector core at the University of North
Carolina. The final viral concentration of AAV vectors was
1*10.sup.12 genome copies (gc)/mL. Lentiviral constructs were
generated as reported. All constructs are available from the
Deisseroth Lab (www.optogenetics.org). Stereotactic viral
injections were carried out under protocols approved by Stanford
University. Juvenile (4-6 weeks) mice kept under isoflurane
anesthesia were arranged in a stereotactic frame (Kopf Instruments)
and leveled using bregma and lambda skull landmarks. Craniotomies
were performed so as to cause minimal damage to cortical tissue.
Infralimbic prefrontal cortex (IL; from bregma: 1.8 mm anterior,
0.35 mm lateral, -2.85 mm ventral) was targeted using a 10 uL
syringe and 35 g beveled needle (Word Precision Instruments). Virus
was infused at a rate of 0.111 L/min. Subjects injected with virus
for behavioral studies were additionally implanted with a chronic
fiber optic coupling device to facilitate light delivery either
with or without an attached penetrating cerebral fiber for local
delivery to target cortical region as noted (Doric Lenses, Canada).
Penetrating fibers were stereotactically inserted to a depth of
-2.5 mm from the same anterior and lateral coordinates and affixed
using adhesive luting cement (C&B MetaBond) prior to adhesive
closure of the scalp (Vetbond, 3M) Animals were administered
analgesic relief following recovery from surgery.
[0148] Results
[0149] As with wild-type ChR2, C128 mutants, and D156 mutants, it
was found that the double-mutant ChR2-C128S/D156A expressed well in
cultured hippocampal neurons and preserved the essential SFO
properties of rapid step-like activation with single brief pulses
of blue light, and deactivation with green or yellow light. Indeed,
despite the reduced redshift in the double-mutant open-state
absorbance, complete deactivation could be still achieved with
redshifted light (in this case with yellow light, optimally at 590
nm), essential for potential combinatorial control purposes.
Deactivation was also possible with 390 nm light, at a faster rate
than yellow light due to the substantial presence of the P390
species, but was also incomplete due to the residual absorption of
the dark state at this wavelength (FIG. 1A). Moreover, following
deactivation with 390 nm light, reactivation with 470 nm was less
effective than following 590 nm deactivation, pointing to a likely
photochemical inactivation with UV light due to trapping in a
deprotonated/desensitized isoform that is not reached after
redshifted-light deactivation (illustrated in FIG. 1E), and again
supporting the use of yellow light deactivation to potentially
enhance spectral separation.
[0150] Peak photocurrents in cells expressing ChR2-C128S/D156A were
comparable to those of ChR2-D156A (231.08.+-.31.19; n=9 cells and
320.96.+-.78.26; n=7 cells, respectively p=0.26, unpaired t-test).
Consistent with the spectroscopic data, neurons expressing
ChR2-C128S/D156A gave rise to sustained photocurrents that were far
more stable than those from cells expressing either single mutant
alone (FIG. 2B). Fitting a monoexponential decay curve to the ratio
of deactivation/activation as a function of time revealed an
apparent spontaneous decay time constant of 29.3 min for
ChR2-C128S/D156A (r.sup.2=0.9139) that was 4.2-fold longer than for
D156A (6.9 min, r.sup.2=0.8357; FIG. 2B) in side-by-side
comparison. Indeed, given the fact that spectroscopy revealed
essentially no reversion to the dark state on this timescale,
remaining decay might be attributable in part to cell-dictated
properties such as protein turnover. Consistent with the required
improvement for the anticipated application to complex mammalian
behaviors, FIG. 2C shows a typical long whole-cell recording with
both blue light activation and yellow light deactivation in the
setting of incoming asynchronous synaptic activity. Based on these
surprisingly prolonged temporal properties, the double-mutant gene
is referred to as SSFO (for stabilized step-function opsin) gene,
and for simplicity use SSFO as shorthand for the protein as
well.
[0151] Channelrhodopsins with such slow decay constants could
enable the transduced cell to act as a photon integrator, with
effective light sensitivity (i.e. photocurrent amplitude per photon
absorbed by the cell) scaling with T.sub.off. SSFO could therefore
enable more-sensitive, less-invasive approaches to optogenetic
circuit modulation, but still with temporally precise onset and
offset of action and with readily titratable effects on the
targeted neuronal population via modulation of light pulse length.
Indeed, it was found that with extraordinarily low light
intensities (as low as 8 .mu.W mm.sup.-2), hundreds of picoamps of
whole-cell photocurrent could be obtained from neurons expressing
SSFO (FIG. 2D). Photocurrents increased with monoexponential
kinetics in response to 470 nm light during the entire time of
illumination (FIG. 2D, left), and activation time constants were
linearly dependent on activation light power on a log-log scale
until the channel-intrinsic millisecond-scale was approached,
suggesting that the SSFO achieves the status of a pure integrator,
with total photon exposure over time as the only determinant of
cellular photocurrent (FIG. 2D, middle; n=27 recordings from 5
cells). However, this also means that the opsin expressing tissue
must be kept in complete darkness before experiments are initiated
(trivial for mammalian in vivo experiments but requiring more
attention for in vitro work). When data were represented as the
total number of photons (delivered to a single neuronal soma and
integrated over time) required for photocurrents to reach a fixed
fraction of Imax for the recorded cell, this characteristic number
of photons was constant regardless of activation light power FIG.
2D, right; 9.1.times.108.+-.1.6.times.10.sup.8 photons; n=27
recordings from 5 cells), again demonstrating the pure photon
integration property of the SSFO.
[0152] To validate this new optogenetic tool in vivo, the
capability of the SSFO to achieve stable cell-type specific
modulation in vivo in mammals was explored, using the regulation of
cortical excitation and inhibition as an experimental system. As
readout, optrode recordings in anesthetized mice expressing SSFO in
the prelimbic (PL) and infralimbic (IL) subregions of the medial
prefrontal cortex (mPFC; FIG. 2E) were performed. To modulate
excitation, SSFO-eYFP in pyramidal neurons under the control of the
excitatory neuron-specific CaMKIIa promoter was first expressed.
Second, to modulate inhibition, SSFO-eYFP in PV::Cre transgenic
mice was expressed using a double-floxed inverted open reading
frame (DIO) virus; in these mice, SSFO was only expressed in the
GABAergic Cre-positive parvalbumin neurons. To map optical
modulation, recordings were made at progressively more ventral
sites in mice injected with AAV5-CaMKIIa::SSFO-EYFP in medial
prefrontal cortex (mPFC), using an advancing two-laser optrode
(FIG. 2E) and a blue/green activation/deactivation laser protocol
(FIG. 2F-G). Multiunit activity in mPFC of these mice was
significantly and stably increased only in the transduced region,
in response to a 1 s pulse of 473 nm light (95 mW mm.sup.-2,
corresponding to 10 mW mm.sup.-2 at the electrode tip). This
increased activity was effectively terminated with a 2s 561 nm
light pulse (112 mW mm.sup.-2; FIG. 2F). Significant increases in
multiunit spike rate (Hz) were restricted to mPFC (FIG. 2) and no
significant reductions in spike rate were observed in any of the
recording sites following blue light stimulation. In mPFC recording
sites (but not in sites dorsal to mPFC) the average multiunit spike
rates were light-modulated as expected; in traces that showed
significant modulation of activity, before activation, after
activation, and after deactivation spike rates were 2.60.+-.0.39
Hz, 33.82.+-.4.83 Hz and 5.04.+-.1.23 Hz, respectively (FIG. 2H;
n=46 recordings in 2 mice; p=3e-8 after activation and p=0.048
after deactivation, both compared with pre-activation baseline;
Student's paired t-test).
[0153] Conversely, in PV::Cre mice injected with
AAV5-EF1a-DIO-::SSFO-eYFP in mPFC, multiunit activity was decreased
after an identical 1 s pulse of 470 nm light and returned to
baseline levels following the 2s 561 nm pulse (FIG. 2G). In these
mice, decreases in multiunit spike rate were also highly restricted
to mPFC (n=5 out of 54 recording sites along the full dorsoventral
track) and no significant increase in spike rate was observed in
any of the recording sites following blue light stimulation. In
traces that showed significant modulation of activity, the average
multiunit spike rates before activation, after activation, and
after deactivation were 14.82.+-.1.26 Hz, 3.66.+-.0.58 Hz and
9.69.+-.1.77 Hz, respectively (FIG. 2H; p=0.002 after activation
and p=0.088 after deactivation, both compared with pre-activation
baseline; Student's paired t-test). Again befitting the predicted
high stability of the SSFO photocurrent, it was found that
modulation of firing rates in vivo was stably sustained after the
brief pulse for many minutes (FIG. 21).
Example 3
Effects of SSFO on Behavior and Circuit Dynamics in Freely Moving
Mice
[0154] Having established that SSFO can be used to bi-directionally
modulate prefrontal excitability on behaviorally-relevant time
scales SSFO was used to examine the effects of elevated cellular
E/I balance on behavior and circuit dynamics in freely moving mice
(FIG. 3). SSFO was expressed either in prefrontal cortical
excitatory neurons using the excitatory neuron-specific
CaMKII.alpha. promoter, or in inhibitory parvalbumin
(PV)-expressing neurons using a double-floxed, inverted
open-reading-frame (DIO) virus in conjunction with PV::Cre
transgenic mice (FIG. 3J-L). Virus was injected in mPFC as
described above, followed by a chronic fiber-optic implant that
projected past the skull immediately dorsal to mPFC for light
delivery (FIG. 3A, B).
[0155] Materials and Methods
[0156] Mutual Information Calculations
[0157] To study the effects of SSFO on sEPSC-spike rate
information, whole-cell patch recordings were conducted from
visually identified pyramidal cells in layer V of mPFC. Using
current clamp, a single pyramidal cell was stimulated with a train
of simulated EPSC waveforms. Individual sEPSC events had peak
current magnitudes of 200 pA and decayed with a time constant of 2
ms. Each experiment was divided into 10 sweeps, each 10 seconds
long and separated by 5 seconds to minimize rundown. Each sweep was
divided into 500 ms segments. The total number of sEPSCs in each
500 ms segment was randomly chosen from a uniform distribution
between 0 and 250. Then, the times of the sEPSCs within the 500 ms
segment were randomly selected from a uniform distribution
extending across the entire segment, simulating excitatory input
from a population of unsynchronized neurons. Empirically, these
stimulation parameters reliably drove pyramidal neurons at firing
rates from 0-30 Hz. In conditions marked as baseline, a 10 sec
pulse of 590 nm light was delivered to completely inactivate the
opsin before running the sEPSC protocol. In conditions where the
opsin was activated, a 1 sec pulse of 470 nm light preceded the
sEPSC protocol.
[0158] To understand the net effect of altered EA balance on
information processing, the mutual information between each
neuron's input sEPSC rate and output spike rate was computed, which
captures relevant changes in the shape of the JO curve and in the
response variability. First, the joint distribution of sEPSC rate
and spike rate was estimated by binning in time, sEPSC rate, and
spike rate and building a joint histogram. Time bins were 125 ms
wide, and sEPSC rate was divided into 10 equally spaced bins from 0
to 500 Hz, although the mutual information results were consistent
across a wide range of binning parameters. Spike rate was binned
using the smallest meaningful bin width given the time bin width
(e.g. 8 Hz bin width for 125 ms time bins). From this joint
histogram, mutual information was computed equaling the difference
between response entropy and noise entropy. Response entropy
quantifies the total amount of uncertainty in the output spike rate
of the neuron. Noise entropy quantifies the uncertainty that
remains in the output spike rate given the input rate. Note that
the maximum information that neural responses can transmit about
the input stimulus is the entropy of the stimulus set. For 10
equally spaced input sEPSC rate bins and a uniform distribution of
input rate over these bins, the entropy of the input rate is
log.sub.2(10)=3.322 bits. Mutual information calculated from
undersampled probability distributions can be biased upwards.
Consequently, all reported values of mutual information, response
entropy and noise entropy were corrected for bias due to
undersampling. This correction is done by computing values from
smaller fractions (from one-half to one-eighth) of the full data
and extrapolating to the limit of infinite data. Using 125 ms time
windows, the correction factors were always less than 0.07
bits.
[0159] Also estimated was the input-output transfer function for
each neuron by averaging the output spike rate across time bins
with similar input sEPSC rates. The shape of the input-output
function was quantified by computing the dynamic range and
saturation point of each neuron, treating the baseline and
opsin-activated conditions separately. Dynamic range was defined as
the difference between maximal and minimal output spiking rate
across the range of input sEPSC rates. Saturation point was defined
as the lowest input sEPSC rate which drove the neuron at 90% of its
maximal output spike rate within that condition. A reduced
saturation point cannot result from a multiplicative reduction in
gain or dynamic range, but instead indicates that the input-output
function becomes flatter at higher input sEPSC rates.
[0160] Behavioral Testing
[0161] All animals undergoing behavioral experiments were
acclimated to a 12-hour reverse light/dark cycle. Prior to
behavioral testing, animals were allowed to acclimate to the room
in which experiments were to be conducted for at least 1 hour
before the experiments started.
[0162] The fear conditioning apparatus consisted of a square
conditioning cage (18.times.18.times.30 cm) with a grid floor wired
to a shock generator and a scrambler, surrounded by an acoustic
chamber (Coulburn instruments, PA, USA). The apparatus was modified
to enable light delivery during training and/or testing. To induce
fear-conditioning mice were placed in the cage for 120 seconds, and
then a pure tone (2.9 kHz) was played for 20 sec, followed by a 2
sec foot-shock (0.5 mA). This procedure was then repeated, and
immediate freezing behavior was monitored for an additional 30 sec
after the delivery of the second shock before the mice were
returned to their home cage. Fear conditioning was assessed 24
hours later by a continuous measurement of freezing (complete
immobility), the dominant behavioral fear response. To test
contextual fear conditioning mice were placed in the original
conditioning cage and freezing was measured for 5 min. To test
auditory-cued fear conditioning mice were placed in a different
context--a pyramid-shaped cage with a smooth floor. As a control
for the influence of the novel environment, freezing was measured
for 2.5 min in this new cage, and then a 2.9 kHz tone was played
for 2.5 min, during which conditioned freezing was measured. Light
stimulation through the fiberoptic connector was administered by
delivering light through a custom patch-cord connected to a 473 nm
laser. The light pulse was delivered for 2 seconds at a power of 98
mW mm.sup.-2 at the fiber tip. The results of the contextual- and
cued-conditioning tests were analyzed by a Student's t-test.
[0163] Social interaction in the home cage was analyzed. Briefly, a
single mouse in the homecage was allowed to freely roam in the
absence of the cage top for one minute. A novel juvenile (3-4 week
old) male intruder was introduced to the opposite corner as the
resident male subject and allowed to roam freely for two minutes.
Total physical interaction between the two mice was quantified
visually, scoring social interaction as the time during which the
resident mouse actively explored the intruder. Stimulation trials
were conducted with the addition of a two second pulse of 473 nm
light delivered via a fiber optic cable (Doric Lenses) coupled to a
chronically implanted fiber optic cable or chronically implanted
non-invasive skull fiber coupling device as indicated. Fiber was
decoupled prior to experimentation and one-minute acclimation
period.
[0164] The three-chamber social test was conducted. The test mice
were introduced into the center chamber of the three-chambered
apparatus and allowed to acclimate for 10 minutes with the doors to
the two side chambers closed. Light pulses were applied at the
beginning and end of the 10 minute acclimation period. At the end
of the acclimation period a novel conspecific male mouse was
introduced to the "social" chamber, inside a wire mesh cup (Galaxy
Pencil/Utility cup, Spectrum Diversified Designs). In the other
(non-social) chamber, an identical empty cup was placed. The
designations of the social and non-social chambers were randomly
chosen in each test to prevent chamber bias. Between tests, the
chambers were cleaned with 20% ethanol and allowed to dry
completely before initiating the next test. The time spent in the
non-social, center, and social chambers was quantified using
automated tracking software Viewer II (BiObserve, Fort Lee, N.J.).
Mice not exhibiting social exploration preference at baseline were
excluded from analysis.
[0165] The novel object exploration experiment was performed in the
same three-chamber apparatus used for the social behavior tests,
and using the same general method. Mice were placed in the center
chamber with the doors to both side chambers closed. Light pulses
were delivered during the 10 minute acclimation period, after which
the doors were opened and the mice were allowed to explore the
entire apparatus. In place of the wire mesh cups, novel objects
were presented at random in either of the two end-chambers.
Exploration of the novel objects was scored over a period of 10
minutes for each mouse as the time in which the mouse spent
actively exploring the object. Objects used were either plastic
balls, cubes or porcelain figurines, all of approximately similar
size. Objects were thoroughly cleaned between tests to prevent odor
traces.
[0166] The open-field chamber (50.times.50 cm) was divided into a
central field (center, 23.times.23 cm) and an outer field
(periphery). Individual mice were placed in the periphery of the
field and the paths of the animals were recorded by a video camera.
The total distance traveled was analyzed using the Viewer2 software
(BiObserve, Fort Lee, N.J.). The open field test for each mouse
consisted of a 5-min session divided into two 2.5 minute segments,
with a 2 s 473 nm light pulse delivered between the two segments.
Track length, velocity and % time in the center were scored for
each mouse and averaged across mice for each condition
[0167] The elevated plus maze was made of plastic and consisted of
two light gray open arms (30.times.5 cm), two black enclosed arms
(30.times.5.times.30 cm) extending from a central platform
(5.times.5.times.5 cm) 31 at 90 degrees in the form of a plus. The
maze was placed 30 cm above the floor. For each mouse, a 2 s 473 nm
light pulse was delivered when the mouse was in the home cage. 5
minutes later, the fiberoptic connector was detached and the mice
were individually placed in the center of the maze for a test
duration of 15 minutes. Video tracking software (ViewerII,
BiObserve, Fort Lee, N.J.) was used to track mouse location. All
measurements displayed were relative to the entire mouse body.
[0168] Chronic Electrophysiological Recordings in Awake Mice
[0169] To simultaneously record from sites both within the
virally-transduced tissue and outside of the transduced region, a
novel chronic multisite optrode (CMO) was designed for awake animal
recordings in combination with light delivery. Arrays of four 25
.mu.m tungsten wires were used (California Fine Wire Company,
Grover Beach, Calif.), wound together and cut at approximately 500
gm increments, and coupled these 4-wire bundles to an implantable
fiberoptic lightguide (IFL; Doric Lenses, Quebec, Canada) that
consisted of a 2.5 mm diameter metal ferrule from which a 200
.mu.m-core fiberoptic cable extended. The four-wire bundle was
back-fed into a 250 gm-diameter guide tube into which the
fiberoptic cable was inserted. The wires were connected using gold
pins to a Mill-Max connector, to which a stainless steel ground
wire was also connected. The device was implanted stereotactically
following virus injection (see above) such that the fiber tip only
extended past the skull but not into brain tissue. The ground wire
was inserted through a small craniotomy above cerebellum. Mice were
allowed to recover for two weeks before experiments began.
[0170] To record neural activity during behavior, the mice were
first acclimated over several days to the attachment of the
headstage and the fiberoptic cable. The mice were allowed to
explore the home cage with the headstage attached for 1-2 hours
each day. Recordings were carried out 2-4 weeks after surgery.
Signals were multiplexed at the head-stage into a 3-wire cable that
was passed through an electrical commutator (PlasticsOne),
demultiplexed using a demultiplexing board (Triangle BioSystems,
Inc.) and digitized using Neuralynx Digital Cheetah. The fiberoptic
and electrical commutators were suspended from a weighted arm
(Harvard Apparatus) to allow the mouse to freely explore a large
region (such as in the open field test). This configuration also
prevented both the recorded mouse and juvenile intruders (during
the social interaction test) access to any excess wire or optical
fiber and minimized damage to the hardware. Videos were recorded
using Neuralynx Cheetah software and analyzed offline with Viewer
II (BiObserve, Fort Lee, N.J.) to quantify open-field behavior.
Social interactions and novel object exploration were manually
scored, as in other behavioral experiments. LFPs were filtered at 1
to 500 Hz and sampled at a frequency of 6.5 kHz. Multiunit activity
was recorded at 32 kHz and individual events were collected with a
threshold of 40 .mu.V on all channels.
[0171] Wavelet power spectrograms of LFP recordings were analyzed
as described above by sampling the power spectrum every 2 s for the
duration of the recording. Power was calculated between 2 Hz and
120 Hz with a bin width of 2 Hz. In all mice, the effects of SSFO
activation were recorded using a protocol of 2 minutes baseline
recording, followed by a 1 s 473 nm pulse at an irradiance of 56 mW
mm.sup.-2 at the fiber tip. Following the blue pulse, activity was
recorded for 2 minutes, followed by a 30 s deactivating light pulse
at a wavelength of 594 nm light with similar intensity. Activity
was then recorded for 2 additional minutes. For each mouse this
protocol was repeated at least 4 times, and power spectra for each
of the three periods (pre-activation, post-activation and
post-deactivation) were averaged across the 4 repetitions.
[0172] Social behavior experiments with the electrode-implanted
mice were performed using the home-cage paradigm, as described
above. No-light and light trials were separated by at least 24
hours, using novel juvenile mice in each test. The test consisted
of 2 minutes of baseline recording, then 1 minute of recording
after the 1 s activation light pulse, after which the juvenile
intruder was introduced. Social behavior was scored for 2 minutes,
followed by removal of the juvenile and a 30 s 594 nm light pulse
to deactivate SSFO. Recordings were acquired during the entire time
and analyzed in the same way as described for the home-cage
recordings above. Power spectra for the 2 min social interaction
period were averaged across mice for both the no-light and light
trials. The novel object experiment in these mice was conducted in
an identical manner, replacing the novel juvenile mouse with an
inanimate object.
[0173] Data Analysis
[0174] Statistical significance was calculated using paired or
unpaired two-tailed t-tests, as applicable. Data were analyzed
using Matlab Statistics toolbox or Microsoft Excel.
[0175] Immunohistochemistry
[0176] Animals that had undergone behavioral analysis were
anesthetized with ketamine/xylazine and perfused transcardially
with ice-cold PBS followed by 4% paraformaldehyde in PBS (4% PFA).
Isolated brains were post-fixed in 4% PFA overnight at 4 C and
subsequently immersed in a sterile cryoprotectant consisting of 30%
sucrose in PBS until settling (2 to 3 days at 4.degree. C.). 40
.mu.m coronal slices were collected using a freezing microtome
(Leica), washed in PBS, permeabilized in 0.3% Triton X-100 (PBST)
and blocked in 3% normal donkey serum dissolved in PBS for one hour
at room temperature. Nuclear localization of c-fos was determined
using rabbit anti-c-fos (Calbiochem) on animals that had undergone
1 s 473 nm light stimulation 90 minutes prior to perfusion;
parvalbumin targeting was confirmed using colocalization of mouse
anti-parvalbumin (Sigma Aldrich) and fluorescent protein. Stained
slices were visualized on a Leica SP5 confocal microscope. To
calculate average fluorescence in different anatomical sub-regions,
histology images were analyzed using Image. Individual subregion
images were thresholded at a fixed threshold level. Mean
fluorescence above threshold was calculated and averaged per region
between mice. c-fos counts were performed using standardized
landmarks to identify regions and were anonymized prior to
counting. Counting was done on z-stacks of the entire slice volume.
Data were only compared across experimental conditions in
experiments where c-fos induction was performed on the same day and
in the same physical conditions, and where tissue preparation,
staining and imaging were done under standardized conditions.
[0177] Results
[0178] First, to evaluate the effects of SSFO-induced activity in
neuronal populations on a cellular level, the expression of the
immediate-early gene product c-fos 90 minutes after a 2s pulse of
470 nm light stimulation were examined in vivo (FIG. 3C). The
number of c-fos positive neurons in the entire
prelimbic/infralimbic subfield (delimited in FIG. 3B) was
quantified in the virally-transduced and optically-stimulated
hemisphere. In animals injected with the (control)
CaMKII.alpha.-YFP virus, 335.+-.107 mPFC cells expressed detectable
c-fos at baseline. By comparison, mice expressing SSFO in PV
neurons (PV::SSFO mice) displayed significantly fewer c-fos
expressing cells relative to controls in mPFC (81.+-.7 cells, n=5
mice; p<0.005, two-sided t-test). Remarkably, a large fraction
of these cells were in fact YFP-positive (61.+-.8% out of the total
c-fos positive population; FIG. 3C), indicating that even most of
these active cells are in fact PV-positive neurons directly
activated by the virally-delivered SSFO. In contrast, mice
expressing SSFO in excitatory cells (CaMKII.alpha.::SSFO mice)
showed significant increases in c-fos positive nuclei in both the
virally-transduced hemisphere (1455.+-.305 cells; n=3 mice;
p<0.05, two-sided t-test; FIG. 2C), and the contralateral
hemisphere (617.+-.97 cells; n=3 mice; p<0.05), but not beyond
to other areas of the brain (FIG. 3M), indicating that activation
propagated chiefly locally and to the contralateral hemisphere.
These findings validate the expected targeting, efficacy, and
directionality of SSFO in the awake mouse.
[0179] Three groups of animals to behavioral testing FIG. 3D-G)
CaMKII.alpha.::SSFO mice, PV::SSFO mice, and control mice (either
injected with AAV5-CaMKII.alpha.-eYFP virus or not injected with
virus). Two to four weeks after surgery, conditioned learning and
unconditioned social behavior was tested, as well as exploration of
novel objects and locomotor functioning (FIG. 3D-G); all animals
received a single 1 s pulse of 470 nm light through the implanted
fiberoptic connector, followed by removal of the fiberoptic cable 1
minute before introduction into the behavioral chamber,
capitalizing on the stability of the SSFO.
[0180] Striking deficits were observed in both social behavior and
conditioning, selectively in the mice with elevated cellular E/I
balance (FIG. 3D-G). First unconditioned social exploration of
same-sex juvenile mice that had been introduced into the home cage
of the experimental animal was explored.sup.49. Exploration of the
novel mouse was virtually abolished in the elevated E/I
(CaMKII.alpha.::SSFO) group following a 1 s 470 nm light pulse,
compared with controls (n=8.CaMKII.alpha.::SSFO mice and n=6
controls; p<0.0005, unpaired t-test), while PV::SSFO mice showed
no effect in this behavior (FIG. 3D and 2; n=6 PV::SSFO mice;
p>0.1; unpaired t-test). The same mice were next subjected to a
conditioning protocol performed immediately following delivery of a
1 s 470 nm light pulse. Twenty-four hours later, responses to the
conditioned tone and context were assessed in order to evaluate the
extent to which the mice learned to associate the conditioned and
unconditioned stimuli while under the altered E/I states. The
elevated E/I (CaMKII.alpha.::SSFO) animals showed no conditioned
responses (to either context: p<0.0005 or tone: p<0.05,
compared with controls; two-sided t-test). Moreover, the deficit
was fully reversible; the same animals could be reconditioned 24 hr
later in the absence of SSFO activation, showing fear conditioning
that was indistinguishable from that of the control group when
tested the following day (FIG. 3E; p>0.1 cue and context;
unpaired t-test). In contrast, the PV::SSFO group, in which E/I
balance was reduced, showed no significant impairment in freezing
behavior compared with controls in response to both tone and
context (FIG. 3E; p=0.09 and p=0.56, respectively; two-sided
t-test), just as in the social behavior. The behavioral deficits
associated with elevated E/I balance were not attributable to
changes in motor function since in the same mice, open field
behavior was normal (n=8 CaMKII.alpha.::SSFO mice and n=6
CaMKII.alpha.::YFP mice; FIG. 3F and FIG. 3N).
Example 4
Elevation but not Reduction of Cellular E/I Leads to Quantitative
Reduction in Information Processing
[0181] Next, neurophysiological underpinnings of the behavioral
impairments resulting from prefrontal E/I balance alterations was
investigated. In autism, a finding of 30% co-morbidity with
debilitating seizures has led to the suggestion that
hyperexcitation is involved, and altered cortical excitation or
inhibition have been proposed to underlie some of the core
behavioral deficits in both autism and schizophrenia.
[0182] Materials and Methods
[0183] Acute 300 .mu.m coronal slices isolated from 8-9 week old
wild-type C57BL/6J or PV::Cre mice previously injected with virus
were obtained in ice-cold sucrose cutting solution ([mM] 11
D-glucose, 234 sucrose, 2.5 KCl, 1.25 NaH.sub.2PO.sub.4, 10
MgSO.sub.4, 0.5 CaCl.sub.2, 26 NaHCO.sub.3) using a Vibratome
(Leica). Slices were recovered in oxygenated Artificial
Cerebrospinal Fluid (ACSF; [mM] 124 NaCl, 3 KCl, 1.3 MgCl.sub.2,
2.4 CaCl.sub.2, 1.25 NaH.sub.2PO.sub.4, 26 NaHCO.sub.3, 10
D-glucose) at 32.degree. C. for one hour. Individual neuron patches
were obtained after identifying fluorescent protein expression from
indicated prefrontal cortical layer under constant ACSF perfusion.
Filtered light from a broad-wavelength xenon lamp source (Sutter
Instruments DG-4) was coupled to the fluorescence port of the
microscope (Leica DM-LFSA). Before each experiment, a 20s pulse of
590 nm light was applied to convert all of the SSFO channels to the
dark state and prevent run-down of photocurrents. Cultured cell
images were acquired on the same microscope using a Retiga Exi CCD
camera (Qimaging inc.) at 100 ms exposure with the 30 gain.
Illumination power density was 12 mW mm.sup.-2 at 500 nm with a
standard EYFP filter set. Quantification of fluorescence was done
with ImageJ software by marking a region containing the soma and
proximal neuritis and calculating for each cell the total
integrated pixel intensity in that region, rather than average
fluorescence, since photocurrents are likely to be related to the
total number of membrane-bound channels rather than average channel
expression per area. Photon flux calculations for SSFO integration
properties were done by calculating the photon flux through the
microscope objective at each light power, and then dividing to
reach the photon flux across the cell membrane, based on the
capacitance of individual patched cells.
[0184] For live animal studies, simultaneous optical stimulation
and electrical recording in the prefrontal cortex of wildtype adult
C57/BL6 male mice previously transduced with indicated viral
constructs as described above. Briefly, animals were deeply
anesthetized with isoflurane prior to craniotomy. After aligning
mouse stereotactically and surgically removing skull dorsal to
prefrontal cortex (centered at 1.8 mm anterior, 0.35 mm lateral), a
MO 0.005 inch extracellular tungsten electrode (A-M systems) with
its tip coupled approximately 400 .mu.m below the blunt end of a
0.2 N.A. 200 .mu.m core diameter fiber optic cable (ThorLabs;
"optrode") was stereotactically inserted into the
virally-transduced brain region. Recorded signals were bandpass
filtered between 300 Hz and 20 kHz, AC amplified 10000.times. (A-M
Systems 1800), digitized (Molecular Devices Digidata 1322A) and
recorded using Clampex software (Molecular Devices). Clampex
software was used for both recording field signals and controlling
47 3 nm (OEM Laser Systems) and 561 nm (CrystalLaser) -10 mW solid
state laser diode sources coupled to the optrode.
Electrophysiological recordings were initiated at the Cg/PL
boundary (1.8 mm anterior, 0.35 mm lateral, -2.0 mm ventral) after
lowering isoflurane anesthesia to a constant level of 1%. Optrode
was lowered ventrally in -0.1 mm steps. Events were isolated using
a custom algorithm in Matlab (MathWorks) with the threshold set
above baseline noise (25 to 40 .mu.V). Heatmap images were
generated in Matlab from an unweighted moving average of 2s with
200 ms steps. Moving average value was reset at the onset of
external manipulations (beginning of sweep, initiation of light
pulses).
[0185] Results
[0186] To probe circuit physiology manifestations of the E/I
balance alterations within the prefrontal microcircuit that lead to
the behavioral impairments, acute prefrontal cortical slices from
CaMKII.alpha.::SSFO mice were prepared. Whole-cell recordings were
conducted in the presence of ongoing asynchronous synaptic activity
induced by the cholinergic agonist carbachol at 20 .mu.M52-54;
spiking was never observed with SSFO activation alone. Circuit-wide
SSFO activation with a single blue light pulse had the effect of
depolarizing the recorded SSFO-expressing neurons by 9.8.+-.1.4 mV
(n=7 cells; FIG. 4A), in part by triggering an increase in incoming
synaptic activity (FIG. 4A, inset); both effects were terminated
with yellow light. Spectral analysis of responses to SSFO in both
expressing and non-expressing cells revealed that this increased
activity displayed a broad spectral range with a peak above 20 Hz
(FIG. 4A-B). In contrast, pyramidal cells in slices expressing SSFO
in PV cells showed a robust reduction in synaptic activity and a
reduction in power at low frequencies (FIG. 4C), consistent with
the increased activity of PV cells after activation with SSFO (FIG.
4D).
[0187] Together, these data and the c-fos data in FIG. 3 revealed
that interventions to either elevate or reduce cellular E:I balance
in mPFC robustly influenced neocortical neuronal activity, but
since only elevating cellular E:I balance in mPFC induced
behavioral deficits, it was decided to make an attempt to
understand at a deeper level how information processing in mPFC was
altered in either case. To examine the effects of altered E/I
balance on information transmission in the prefrontal microcircuit,
whole-cell recordings in acute slices from CaMKII.alpha.::SSFO mice
were performed in which opsin-expressing pyramidal neurons were
identified by morphology and fluorescence. Neurons in whole-cell
patch clamp were stimulated with trains of simulated EPSCs designed
to span a wide range of sEPSC rates over time (FIG. 5A) cells
expressing SSFO, blue light activation indeed enhanced excitability
at low sEPSC rates but led to a saturation of the input-output (TO)
curve at higher sEPSC rates (FIG. 5B), thereby causing a
significant reduction in mutual information between the rate of
input EPSCs and the rate of resulting spikes (-0.40.+-.0.09 bits;
p=0.011, paired Student's t-test; FIG. 5C), and demonstrating that
increased cellular E/I balance quantitatively impairs information
processing in neocortical principal cells. Next, to examine the
effects of reduced cellular E/I balance on information processing
in neocortical principal cells, acute slices from PV::SSFO mice and
stimulated non-expressing pyramidal cells with sEPSC trains were
recorded as before (FIG. 5D). Activation of SSFO in PV cells caused
a substantial decrease in the JO curve gain in the recorded
pyramidal cells (FIG. 5E) as expected via synaptic inhibition, but
in this case preserved the overall shape of the JO curve without
saturation and strikingly had no significant effect on mutual
information between the rate of input sEPSCs and the resulting
spike rate in pyramidal cells (FIG. 5F).
[0188] The decrease in information throughput for principal mPFC
cells was significantly larger (4.8-fold, p=0.0144, unpaired
t-test) following light activation in CaMKII.alpha.::SSFO mice
versus PV::SSFO mice across a broad range of both time bin width
(FIG. 5G-H) and input rate bin width (FIG. 5 I-J) used to calculate
mutual information, despite the fact that there was (if anything) a
greater impact on spike rate with the PV::SSFO activation (FIG. 5B,
E). Together these behavioral and informational data illustrate
that, despite the natural intuitive supposition that favoring
inhibition would be more disruptive to information processing, it
is in fact elevations in E/I balance that are detrimental for mPFC
circuit and behavioral performance, consistent with the clinical
association of disorders such as autism with increased-excitability
phenotypes. If the cellular E/I balance-induced social dysfunction
demonstrated here were related to the circuit processes and social
dysfunction seen in severe human neuropsychiatric disease states
such as autism and schizophrenia, an important prediction would be
that characteristic electrophysiological markers of these human
disease states would also be seen in this animal model. Since a
common clinical electrophysiological marker of both autism and
schizophrenia is elevated baseline (non-evoked) gamma power (30-80
Hz), this physiological-marker hypothesis was therefore tested by
measuring this consistent clinical marker in awake, freely-moving
mice with specifically elevated cellular E/I balance.
[0189] Testing for this possibility with the requisite sensitivity
required the additional insertion of multi-site recording
electrodes into mPFC. While the additional presence of such a
device in combination with a penetrating fiberoptic for light
delivery might be too acutely disruptive and spatially invasive for
the small mouse mPFC circuitry, a strategy with two important
features to enable this experiment was developed and implemented.
First, the recording device was designed for chronic implantation,
so that recordings could be carried out in animals habituated to
the recording electrodes. Second, the photon integration properties
of SSFO were capitalized upon to enable not only behavioral testing
without optical hardware, but also (even for deep structures like
IL and PL) without any optical hardware penetration of the brain
itself, at any time. To verify that it is indeed possible to
modulate SSFO-expressing cells in deep cortical structures,
CaMKII.alpha.::SSFO or CaMKII.alpha.::EYFP virus were injected and
implanted fiberoptic connectors extending only past the skull (FIG.
6A), without entering the cortical surface (FIG. 6B). The
directionality of Eli balance elevation in this minimally-invasive
configuration was validated by c-fos analysis in these animals (n=3
CaMKII.alpha.::SSFO and n=4 CaMKII.alpha.::EYFP control mice;
p=0.034, two-sided t-test; FIG. 6C). Elevated cellular E/I balance
during conditioning showed no effect on freezing responses to
footshock (indicating intact sensory perception of the aversive
unconditioned stimulus; FIG. 6D), but showed a marked and fully
reversible effect on contextual (p<0.005; unpaired t-test with
unequal variance) and auditory conditioning (p<0.005; unpaired
t-test with unequal variance; FIG. 6D). Crucially, social behavior
was also impaired in mice receiving noninvasive light stimulation
prior to testing (p<0.005; unpaired t-test; FIG. 6E),
demonstrating the opportunity afforded by the extreme light
sensitivity of the SSFO.
[0190] To obtain direct electrophysiological readouts from these
mice, a novel chronic multisite optrode (CMO) was designed in which
the fiberoptic connector is coupled through a guide tube with 4
25-.mu.m tungsten wires, cut at 0.5 mm distance increments from the
tip of the fiber, to simultaneously sample neural activity at
various depths within the illuminated tissue (FIG. 6F). At the end
of the experiments, electrode positions were marked using
electrolytic lesions (FIG. 6G), which allowed us to identify the
anatomical locations from which individual recordings were taken;
no fiberoptic penetration of the tissue was allowed to occur. In
three mice that were injected with CaMKII.alpha.::SSFO virus and
implanted with the depth-sampling optrode, it was first confirmed
that social behavior was normal at baseline, and impaired following
a 1 s 470 nm pulse (FIG. 6H; p=0.044, paired Student's t-test). The
same animals showed no effect of light on exploration of a novel
object, however, consistent with our previous findings (FIG. 6H;
p=0.82, paired Student's t-test). Additionally, locomotor behavior
in the familiar home-cage (not shown) and in a novel open field
were not significantly altered after the 1 s activation pulse (FIG.
7A) although a trend toward reduced anxiety was apparent (increased
% time in center; FIG. 7A). During these experiments to validate
the behavioral phenotypes in the setting of CMO implantation,
activity was recorded simultaneously on all channels and the
changes resulting from SSFO activation was analyzed.
[0191] Recordings in the animals' home cage were first analyzed
using a protocol that consisted of 2 minutes pre-activation
baseline, a 1 s 470 nm light pulse, 2 minutes of continuous
recording and then a 30 s pulse of 590 nm light to fully deactivate
SSFO. This protocol was repeated 4 times in each mouse and unit
activity traces were averaged across trials (FIG. 6I). In multiunit
recordings from channels within the SSFO-expressing regions,
significant increases in spiking in response to the blue light
pulse (FIG. 6 I-J; 77.+-.18% on modulated channels was observed,
compared with -3.4.+-.4.4% on the unmodulated channels; n=4
modulated and 4 unmodulated channels in 3 mice recorded; p=0.02;
two-sided t-test).
[0192] Also observed were pronounced changes in the local field
potential (LFP) recordings from the modulated channels. Wavelet
spectral analysis was used to generate time-resolved spectrograms
(FIG. 6 K-L; left) of the LFP activity on each channel and
quantified the average change between the pre-activation baseline
and the post-activation period. In unmodulated channels there was
no apparent effect of the activation pulse on the LFP (FIG. 6K,
left), with only a small average decrease in power across all
frequencies in the post-activation and post-deactivation periods
compared with the baseline period (FIG. 6K, right). In contrast,
modulated channels located within virally-transduced regions showed
a marked increase in gamma-band activity (FIG. 6L) after activation
with SSFO, which was sharply temporally delimited to the activation
period and was terminated by the 590 nm deactivation pulse (FIG.
6L, right). The increase in gamma-band activity was associated with
a reduction in lower-frequency power within the same channels that
showed increased gamma activity (FIG. 6L, right; inset). A similar
analysis of the recordings performed during the behavioral
experiments done with these animals showed consistently increased
gamma-band activity in the experiments where a 1 s 470 nm light
pulse was delivered during behavioral testing in the open field
experiment (FIG. 7B), the social exploration test (FIG. 7C) and the
novel object exploration test (FIG. 7D). Together these data reveal
that the physiological biomarker (elevated baseline gamma-band
activity) seen in autism and schizophrenia is conserved with
selectively elevated cellular E/I balance in freely-behaving
mammals with social deficits.
[0193] Finally whether the neocortical circuitry that both induced
and expressed the elevated E/I balance-induced gamma in vivo (FIG.
6) could also give rise to this physiological phenomenon in itself,
in the absence of other brain regions was tested. While acute
slices are more refractory to induction of sharp oscillation
patterns than in vivo preparations, even in this reduced
preparation an 20-80 Hz band power elevations in current-clamp
membrane potential was noted under conditions of moderate
CaMKII::SSFO activation (FIG. 4A-B) and 30-80 Hz gamma elevations
in current-clamp membrane potential using the most potent
channelrhodopsin available (CaMKII::C1V1-E162T).
[0194] At high light power density (12 mW mm-2), the largest
increase in power at gamma frequency (30-80 Hz; FIG. 8B) was
observed. At lower light powers (4.3 mW mm.sup.-2 and 0.6 mW mm-2),
monotonically reduced gamma power along with relatively increased
power at lower frequencies was observed (theta, 8-12 Hz and beta,
15-25 Hz; FIG. 8 B-C). Under voltage clamp conditions,
corresponding spectra both for IPSCs recorded at 0 mV and for EPSCs
at -60 mV were resolved (FIG. 8A). Together these results are
consistent with a monotonic relationship between stable E/I balance
elevation and the physiological biomarker of
intrinsically-generated gamma oscillations in prefrontal
cortex.
[0195] The data presented here point to specific impairments in
social behavior as a result of elevated E/I ratio in mPFC. In
principle an elevated E/I ratio could also be achieved by
inhibiting inhibitory cells, although this loss-of-function
approach would be expected to show effects only in the unlikely
event that there were high stable baseline activity patterns of the
inhibitory cells. Indeed, when AAV5-EF1.alpha.-DIO-eNpHR3.0-EYFP
virus was injected into mPFC in both hemispheres of PV::Cre mice
(generating PV::eNpHR3.0 mice) and implanted bilateral fiberoptic
connectors for the home-cage or three-chamber social exploration
paradigm, no behavioral impairment was found associated with
activation of eNpHR3.0 under these conditions (FIG. 9), as may have
been expected. However, a more important question central to the
elevated cellular E/I ratio hypothesis is the prediction that
increased inhibition could act in the direction of rescuing the
behavioral deficits associated with elevated E/I balance caused by
SSFO activation in excitatory cells (FIG. 3).
[0196] C1V1 is a chimeric light-sensitive protein derived from the
VChR1 cation channel from Volvox carteri and the ChR1 cation
channel from Chlamydomonas Reinhardti. C1V1 and its variants,
permits the experimental manipulation of cortical E/I elevations
and the monitoring of gamma oscillations in cortical slices with
high potency (thus allowing enable dose-response tests), low
desensitization (thus permitting inducement of step-like changes in
E/I balance), and red-shifted excitation (to permit separable drive
of different populations within the same neural circuit). For this
example, C1V1 variant with the highest potency to enable the most
reliable dose-response was selected. To test the above prediction,
a combinatorial optogenetic experiment for freely moving mice was
designed, leveraging the unique spectral and temporal properties of
C1V1 and SSFO to drive pyramidal cells with SSFO and co-activate
(or not) PV cells using C1V1-E122T/E162T for maximal spectral
separation. PV::Cre mice were injected with a combination of
AAV5-CaMKII.alpha.-SSFO and AAV5-EF1.alpha.-DIO-C1V1-E122T/E162T
into mPFC to express SSFO in pyramidal neurons and C1V1 in PV cells
(referred to here as SSFO/C1V1 mice; n=7). A second group of mice
was injected with only CaMKII.alpha.-SSFO virus
(CaMKII.alpha.::SSFO, n=9) and control mice were injected with
CaMKII.alpha.-EYFP (n=10). Two to four weeks later, the mice were
tested in the three-chamber social test under 4 different
illumination paradigms, utilizing the spectrotemporal strategy for
separation between C1V1-E122T/E162T (driven with 590 nm light) and
SSFO (driven for potent currents at the 470 nm peak; FIG. 10A).
Initial characterizations were conducted with no light delivered,
to acquire a baseline social preference (FIG. 10B). In this test,
all mice showed significant preference for the social chamber (FIG.
10B, FIG. 11; CaMKII.alpha.-SSFO mice p=0.002; SSFO/C1V1 mice
p=0.0003; CaMKII.alpha.-EYFP mice p=0.032).
[0197] Next mice in the same paradigm were tested with novel
juvenile mice, while delivering pulsed laser light at 590 nm to
activate only C1V1-E122T/E162T in the PV cells in the SSFO/C1V1
mice (FIG. 10B). In this test, again, all mice showed normal
preference for the novel juvenile mouse (FIG. 10C and FIG. 11;
CaMKII.alpha.-SSFO mice p=0.008; SSFO/C1V1 mice p=0.005;
CaMKII.alpha.-EYFP mice p=0.014), consistent with the earlier
PV::SSFO experiments. In a third test, SSFO was activated with a 2s
470 nm light pulse during the pre-test habituation period (FIG.
10B). In this test, both the CaMKII.alpha.::SSFO group and the
SSFO/C1V1 group showed no preference for the social chamber (FIG.
10C-D; p=0.21 and p=0.87, respectively), a profound social behavior
deficit consistent with our previous observations in
CaMKII.alpha.::SSFO mice (FIG. 3I). Note the importance of
spectrotemporal separation here: while the use of 470 nm light for
maximal drive of SSFO will certainly involve drive of
C1V1-E122T/E162T as well, the contrasting transience of
C1V1-E122T/E162T and the stability of SSFO ensures that the
behavioral testing carried out after the 2s 470 nm light pulse is
in the presence only of SSFO activity. Lastly, it was sought to
rescue the behavioral deficit by compensating cellular Ed balance,
adding to the activation of SSFO in excitatory cells an additional
activation of C1V1-E122T/E162T in inhibitory cells by delivering
pulses of 470 nm light at 10 Hz throughout the behavioral testing
period (FIG. 10A-B). Under these illumination conditions,
CaMKII.alpha.::SSFO mice (with no C1V1-E122T/E162T to be activated,
experiencing a pure elevation in cellular E/I balance) showed
severe social behavior impairment with no significant preference to
the social chamber (FIG. 10C; p=0.59) but in contrast, in the
SSFO/C1V1 mice, preference to the social chamber was restored (FIG.
10D; p=0.005) by this compensatory increased activity of inhibitory
neurons. As expected, control CaMKII.alpha.-EYFP mice showed
significant preference to the social chamber under both the 2s 470
nm and the 10 Hz 470 nm stimulation paradigms (FIG. 11).
[0198] Discussion
[0199] Several lines of evidence have suggested the involvement of
elevated cellular excitation-inhibition (E/I) balance in the
etiology of medication-unresponsive social and
information-processing impairments in autism and schizophrenia. But
it has been difficult to formally test this hypothesis without 1)
selective control over individual cell types; and 2) separating
long-term effects of such control on the development and maturation
of the circuit from immediate effects of E/I abnormalities with
regard to the operation of the neural circuits involved. The tight
interplay and pharmacological complexity of excitation and
inhibition within cortical microcircuitry have precluded the
confirmation of elevated cellular E/I balance as a core component
of behavioral defects observed in the various disease models and
human patients. Here, using two novel optogenetic tools, direct
support for the elevated cellular E/I balance hypothesis was
obtained, and circuit-physiology manifestations of the resulting
social dysfunction were identified.
[0200] To more fully understand the elevated E/I state, the
underlying circuit physiology manifestations were probed both in
vitro and in vivo, which will undoubtedly be complex given the
broad range of circuit phenomena that a cellular E/I balance
elevation could initiate. Cellular E/I balance elevation was found
to alter the transfer functions of principal neurons in a way that
quantitatively impaired information transmission within cortical
circuitry. In marked contrast, reduction in E/I balance (which did
not affect social function despite dramatic effects on principal
cell spike rates) did not impair information transmission and
preserved the overall shape of principal neuron transfer functions.
Also identified was correspondence between a clinical marker of
disease states linked to social dysfunction (elevated baseline
gamma power) and electrophysiological findings during free behavior
in the elevated cellular E/I state. Using a novel chronic multisite
optrode (CMO) device for combined recording and optical modulation
in awake, behaving mice, it was found that the elevated E/I state
is associated with robust, stable gamma oscillations that are
generated by and manifested within the regions directly
experiencing elevated cellular E/I balance. In these mice a
specific impairment in social behavior but no gross changes in
locomotor behavior or exploration of inanimate objects under the
elevated E/I-gamma state was observed.
[0201] The effects of elevated E/I balance on social behavior
showed evidence of specificity for PFC, since increasing the E/I
ratio elsewhere, in primary visual cortex, did not impair social
behavior. The PFC network, with its extensive subcortical
connectivity, might therefore be particularly susceptible to
eliciting psychiatric-related symptoms in the setting of subtle
changes in E/I balance, a notion that is supported by observed of
alterations in PFC inhibitory markers associated with psychiatric
disease and the altered PFC rhythmicity observed in autistic
individuals. Behavioral impairment under conditions in which
PV-positive neurons were inhibited were not observed; notably, the
ability to fully inhibit PV-positive neurons is limited by the
penetrance of expression (DIO::SSFO expressed in .about.25% of
PV-positive cells), and the fact that impact will depend on
baseline activity level of the targeted cells.
[0202] Finally, to attempt to restore the impairment resulting from
elevated Ed balance, a family of novel extensively-engineered red
light-activated channelrhodopsins, collectively termed C1V1
variants were utilized, to independently modulate both excitatory
neurons (using SSFO) and inhibitory PV neurons (using a C1V1
variant). Using a novel form of integrated spectrotemporal
separation of the activity of two optogenetic tools, it was found
that increased cellular inhibition ameliorated social behavior
deficits in mice that had been subjected to elevation of cellular
E/I balance.
[0203] The examples, which are intended to be purely exemplary of
the invention and should therefore not be considered to limit the
invention in any way, also describe and detail aspects and
embodiments of the invention discussed above. The foregoing
examples and detailed description are offered by way of
illustration and not by way of limitation. All publications, patent
applications, and patents cited in this specification are herein
incorporated by reference as if each individual publication, patent
application, or patent were specifically and individually indicated
to be incorporated by reference. In particular, all publications
cited herein are expressly incorporated herein by reference for the
purpose of describing and disclosing compositions and methodologies
which might be used in connection with the invention. Although the
foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the
spirit or scope of the appended claims.
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Sequence CWU 1
1
81310PRTChlamydomonas reinhardtii 1Met Asp Tyr Gly Gly Ala Leu Ser
Ala Val Gly Arg Glu Leu Leu Phe1 5 10 15 Val Thr Asn Pro Val Val
Val Asn Gly Ser Val Leu Val Pro Glu Asp 20 25 30 Gln Cys Tyr Cys
Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn Gly Ala 35 40 45 Gln Thr
Ala Ser Asn Val Leu Gln Trp Leu Ala Ala Gly Phe Ser Ile 50 55 60
Leu Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp Lys Ser Thr Cys Gly65
70 75 80 Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu Met Val Lys Val
Ile Leu 85 90 95 Glu Phe Phe Phe Glu Phe Lys Asn Pro Ser Met Leu
Tyr Leu Ala Thr 100 105 110 Gly His Arg Val Gln Trp Leu Arg Tyr Ala
Glu Trp Leu Leu Thr Cys 115 120 125 Pro Val Ile Leu Ile His Leu Ser
Asn Leu Thr Gly Leu Ser Asn Asp 130 135 140 Tyr Ser Arg Arg Thr Met
Gly Leu Leu Val Ser Asp Ile Gly Thr Ile145 150 155 160 Val Trp Gly
Ala Thr Ser Ala Met Ala Thr Gly Tyr Val Lys Val Ile 165 170 175 Phe
Phe Cys Leu Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe His Ala 180 185
190 Ala Lys Ala Tyr Ile Glu Gly Tyr His Thr Val Pro Lys Gly Arg Cys
195 200 205 Arg Gln Val Val Thr Gly Met Ala Trp Leu Phe Phe Val Ser
Trp Gly 210 215 220 Met Phe Pro Ile Leu Phe Ile Leu Gly Pro Glu Gly
Phe Gly Val Leu225 230 235 240 Ser Val Tyr Gly Ser Thr Val Gly His
Thr Ile Ile Asp Leu Met Ser 245 250 255 Lys Asn Cys Trp Gly Leu Leu
Gly His Tyr Leu Arg Val Leu Ile His 260 265 270 Glu His Ile Leu Ile
His Gly Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280 285 Ile Gly Gly
Thr Glu Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala 290 295 300 Glu
Ala Gly Ala Val Pro305 310 2344PRTChlamydomonas reinhardtii 2Met
Ser Arg Arg Pro Trp Leu Leu Ala Leu Ala Leu Ala Val Ala Leu1 5 10
15 Ala Ala Gly Ser Ala Gly Ala Ser Thr Gly Ser Asp Ala Thr Val Pro
20 25 30 Val Ala Thr Gln Asp Gly Pro Asp Tyr Val Phe His Arg Ala
His Glu 35 40 45 Arg Met Leu Phe Gln Thr Ser Tyr Thr Leu Glu Asn
Asn Gly Ser Val 50 55 60 Ile Cys Ile Pro Asn Asn Gly Gln Cys Phe
Cys Leu Ala Trp Leu Lys65 70 75 80 Ser Asn Gly Thr Asn Ala Glu Lys
Leu Ala Ala Asn Ile Leu Gln Trp 85 90 95 Ile Thr Phe Ala Leu Ser
Ala Leu Cys Leu Met Phe Tyr Gly Tyr Gln 100 105 110 Thr Trp Lys Ser
Thr Cys Gly Trp Glu Glu Ile Tyr Val Ala Thr Ile 115 120 125 Glu Met
Ile Lys Phe Ile Ile Glu Tyr Phe His Glu Phe Asp Glu Pro 130 135 140
Ala Val Ile Tyr Ser Ser Asn Gly Asn Lys Thr Val Trp Leu Arg Tyr145
150 155 160 Ala Glu Trp Leu Leu Thr Cys Pro Val Ile Leu Ile His Leu
Ser Asn 165 170 175 Leu Thr Gly Leu Ala Asn Asp Tyr Asn Lys Arg Thr
Met Gly Leu Leu 180 185 190 Val Ser Asp Ile Gly Thr Ile Val Trp Gly
Thr Thr Ala Ala Leu Ser 195 200 205 Lys Gly Tyr Val Arg Val Ile Phe
Phe Leu Met Gly Leu Cys Tyr Gly 210 215 220 Ile Tyr Thr Phe Phe Asn
Ala Ala Lys Val Tyr Ile Glu Ala Tyr His225 230 235 240 Thr Val Pro
Lys Gly Ile Cys Arg Asp Leu Val Arg Tyr Leu Ala Trp 245 250 255 Leu
Tyr Phe Cys Ser Trp Ala Met Phe Pro Val Leu Phe Leu Leu Gly 260 265
270 Pro Glu Gly Phe Gly His Ile Asn Gln Phe Asn Ser Ala Ile Ala His
275 280 285 Ala Ile Leu Asp Leu Ala Ser Lys Asn Ala Trp Ser Met Met
Gly His 290 295 300 Phe Leu Arg Val Lys Ile His Glu His Ile Leu Leu
Tyr Gly Asp Ile305 310 315 320 Arg Lys Lys Gln Lys Val Asn Val Ala
Gly Gln Glu Met Glu Val Glu 325 330 335 Thr Met Val His Glu Glu Asp
Asp 340 3300PRTVolvox carteri 3Met Asp Tyr Pro Val Ala Arg Ser Leu
Ile Val Arg Tyr Pro Thr Asp1 5 10 15 Leu Gly Asn Gly Thr Val Cys
Met Pro Arg Gly Gln Cys Tyr Cys Glu 20 25 30 Gly Trp Leu Arg Ser
Arg Gly Thr Ser Ile Glu Lys Thr Ile Ala Ile 35 40 45 Thr Leu Gln
Trp Val Val Phe Ala Leu Ser Val Ala Cys Leu Gly Trp 50 55 60 Tyr
Ala Tyr Gln Ala Trp Arg Ala Thr Cys Gly Trp Glu Glu Val Tyr65 70 75
80 Val Ala Leu Ile Glu Met Met Lys Ser Ile Ile Glu Ala Phe His Glu
85 90 95 Phe Asp Ser Pro Ala Thr Leu Trp Leu Ser Ser Gly Asn Gly
Val Val 100 105 110 Trp Met Arg Tyr Gly Glu Trp Leu Leu Thr Cys Pro
Val Leu Leu Ile 115 120 125 His Leu Ser Asn Leu Thr Gly Leu Lys Asp
Asp Tyr Ser Lys Arg Thr 130 135 140 Met Gly Leu Leu Val Ser Asp Val
Gly Cys Ile Val Trp Gly Ala Thr145 150 155 160 Ser Ala Met Cys Thr
Gly Trp Thr Lys Ile Leu Phe Phe Leu Ile Ser 165 170 175 Leu Ser Tyr
Gly Met Tyr Thr Tyr Phe His Ala Ala Lys Val Tyr Ile 180 185 190 Glu
Ala Phe His Thr Val Pro Lys Gly Ile Cys Arg Glu Leu Val Arg 195 200
205 Val Met Ala Trp Thr Phe Phe Val Ala Trp Gly Met Phe Pro Val Leu
210 215 220 Phe Leu Leu Gly Thr Glu Gly Phe Gly His Ile Ser Pro Tyr
Gly Ser225 230 235 240 Ala Ile Gly His Ser Ile Leu Asp Leu Ile Ala
Lys Asn Met Trp Gly 245 250 255 Val Leu Gly Asn Tyr Leu Arg Val Lys
Ile His Glu His Ile Leu Leu 260 265 270 Tyr Gly Asp Ile Arg Lys Lys
Gln Lys Ile Thr Ile Ala Gly Gln Glu 275 280 285 Met Glu Val Glu Thr
Leu Val Ala Glu Glu Glu Asp 290 295 300 4747PRTVolvox carteri 4Met
Asp His Pro Val Ala Arg Ser Leu Ile Gly Ser Ser Tyr Thr Asn1 5 10
15 Leu Asn Asn Gly Ser Ile Val Ile Pro Ser Asp Ala Cys Phe Cys Met
20 25 30 Lys Trp Leu Lys Ser Lys Gly Ser Pro Val Ala Leu Lys Met
Ala Asn 35 40 45 Ala Leu Gln Trp Ala Ala Phe Ala Leu Ser Val Ile
Ile Leu Ile Tyr 50 55 60 Tyr Ala Tyr Ala Thr Trp Arg Thr Thr Cys
Gly Trp Glu Glu Val Tyr65 70 75 80 Val Cys Cys Val Glu Leu Thr Lys
Val Val Ile Glu Phe Phe His Glu 85 90 95 Phe Asp Glu Pro Gly Met
Leu Tyr Leu Ala Asn Gly Asn Arg Val Leu 100 105 110 Trp Leu Arg Tyr
Gly Glu Trp Leu Leu Thr Cys Pro Val Ile Leu Ile 115 120 125 His Leu
Ser Asn Leu Thr Gly Leu Lys Asp Asp Tyr Asn Lys Arg Thr 130 135 140
Met Arg Leu Leu Val Ser Asp Val Gly Thr Ile Val Trp Gly Ala Thr145
150 155 160 Ala Ala Met Ser Thr Gly Tyr Ile Lys Val Ile Phe Phe Leu
Leu Gly 165 170 175 Cys Met Tyr Gly Ala Asn Thr Phe Phe His Ala Ala
Lys Val Tyr Ile 180 185 190 Glu Ser Tyr His Thr Val Pro Lys Gly Leu
Cys Arg Gln Leu Val Arg 195 200 205 Ala Met Ala Trp Leu Phe Phe Val
Ser Trp Gly Met Phe Pro Val Leu 210 215 220 Phe Leu Leu Gly Pro Glu
Gly Phe Gly His Leu Ser Val Tyr Gly Ser225 230 235 240 Thr Ile Gly
His Thr Ile Ile Asp Leu Leu Ser Lys Asn Cys Trp Gly 245 250 255 Leu
Leu Gly His Phe Leu Arg Leu Lys Ile His Glu His Ile Leu Leu 260 265
270 Tyr Gly Asp Ile Arg Lys Val Gln Lys Ile Arg Val Ala Gly Glu Glu
275 280 285 Leu Glu Val Glu Thr Leu Met Thr Glu Glu Ala Pro Asp Thr
Val Lys 290 295 300 Lys Ser Thr Ala Gln Tyr Ala Asn Arg Glu Ser Phe
Leu Thr Met Arg305 310 315 320 Asp Lys Leu Lys Glu Lys Gly Phe Glu
Val Arg Ala Ser Leu Asp Asn 325 330 335 Ser Gly Ile Asp Ala Val Ile
Asn His Asn Asn Asn Tyr Asn Asn Ala 340 345 350 Leu Ala Asn Ala Ala
Ala Ala Val Gly Lys Pro Gly Met Glu Leu Ser 355 360 365 Lys Leu Asp
His Val Ala Ala Asn Ala Ala Gly Met Gly Gly Ile Ala 370 375 380 Asp
His Val Ala Thr Thr Ser Gly Ala Ile Ser Pro Gly Arg Val Ile385 390
395 400 Leu Ala Val Pro Asp Ile Ser Met Val Asp Tyr Phe Arg Glu Gln
Phe 405 410 415 Ala Gln Leu Pro Val Gln Tyr Glu Val Val Pro Ala Leu
Gly Ala Asp 420 425 430 Asn Ala Val Gln Leu Val Val Gln Ala Ala Gly
Leu Gly Gly Cys Asp 435 440 445 Phe Val Leu Leu His Pro Glu Phe Leu
Arg Asp Lys Ser Ser Thr Ser 450 455 460 Leu Pro Ala Arg Leu Arg Ser
Ile Gly Gln Arg Val Ala Ala Phe Gly465 470 475 480 Trp Ser Pro Val
Gly Pro Val Arg Asp Leu Ile Glu Ser Ala Gly Leu 485 490 495 Asp Gly
Trp Leu Glu Gly Pro Ser Phe Gly Leu Gly Ile Ser Leu Pro 500 505 510
Asn Leu Ala Ser Leu Val Leu Arg Met Gln His Ala Arg Lys Met Ala 515
520 525 Ala Met Leu Gly Gly Met Gly Gly Met Leu Gly Ser Asn Leu Met
Ser 530 535 540 Gly Ser Gly Gly Val Gly Leu Met Gly Ala Gly Ser Pro
Gly Gly Gly545 550 555 560 Gly Gly Ala Met Gly Val Gly Met Thr Gly
Met Gly Met Val Gly Thr 565 570 575 Asn Ala Met Gly Arg Gly Ala Val
Gly Asn Ser Val Ala Asn Ala Ser 580 585 590 Met Gly Gly Gly Ser Ala
Gly Met Gly Met Gly Met Met Gly Met Val 595 600 605 Gly Ala Gly Val
Gly Gly Gln Gln Gln Met Gly Ala Asn Gly Met Gly 610 615 620 Pro Thr
Ser Phe Gln Leu Gly Ser Asn Pro Leu Tyr Asn Thr Ala Pro625 630 635
640 Ser Pro Leu Ser Ser Gln Pro Gly Gly Asp Ala Ser Ala Ala Ala Ala
645 650 655 Ala Ala Ala Ala Ala Ala Ala Thr Gly Ala Ala Ser Asn Ser
Met Asn 660 665 670 Ala Met Gln Ala Gly Gly Ser Val Arg Asn Ser Gly
Ile Leu Ala Gly 675 680 685 Gly Leu Gly Ser Met Met Gly Pro Pro Gly
Ala Pro Ala Ala Pro Thr 690 695 700 Ala Ala Ala Thr Ala Ala Pro Ala
Val Thr Met Gly Ala Pro Gly Gly705 710 715 720 Gly Gly Ala Ala Ala
Ser Glu Ala Glu Met Leu Gln Gln Leu Met Ala 725 730 735 Glu Ile Asn
Arg Leu Lys Ser Glu Leu Gly Glu 740 745 520PRTArtificial
SequenceSynthetic peptide 5Lys Ser Arg Ile Thr Ser Glu Gly Glu Tyr
Ile Pro Leu Asp Gln Ile1 5 10 15 Asp Ile Asn Val 20
647PRTArtificial SequenceSynthetic peptide 6Met Asp Tyr Gly Gly Ala
Leu Ser Ala Val Gly Arg Glu Leu Leu Phe1 5 10 15 Val Thr Asn Pro
Val Val Val Asn Gly Ser Val Leu Val Pro Glu Asp 20 25 30 Gln Cys
Tyr Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn Gly 35 40 45
76PRTArtificial SequenceSynthetic peptide 7Phe Xaa Tyr Glu Asn Glu1
5 87PRTArtificial SequenceSynthetic peptide 8Phe Cys Tyr Glu Asn
Glu Val1 5
* * * * *