U.S. patent application number 15/957608 was filed with the patent office on 2018-11-22 for optically-controlled cns dysfunction.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior Universit. Invention is credited to Karl Deisseroth, Lief Fenno, Kay Tye.
Application Number | 20180333456 15/957608 |
Document ID | / |
Family ID | 46025130 |
Filed Date | 2018-11-22 |
United States Patent
Application |
20180333456 |
Kind Code |
A1 |
Deisseroth; Karl ; et
al. |
November 22, 2018 |
OPTICALLY-CONTROLLED CNS DYSFUNCTION
Abstract
Provided herein are animals expressing light-responsive opsin
proteins in the basal lateral amygdala of the brain and methods for
producing the same wherein illumination of the light-responsive
opsin proteins causes anxiety in the animal. Also provided herein
are methods for alleviating and inducing anxiety in an animal as
well as methods for screening for a compound that alleviates
anxiety in an animal.
Inventors: |
Deisseroth; Karl; (Stanford,
CA) ; Tye; Kay; (Cambridge, MA) ; Fenno;
Lief; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
Universit |
Stanford |
CA |
US |
|
|
Family ID: |
46025130 |
Appl. No.: |
15/957608 |
Filed: |
April 19, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15194379 |
Jun 27, 2016 |
9968652 |
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15957608 |
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14555048 |
Nov 26, 2014 |
9421258 |
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15194379 |
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13882719 |
Jul 29, 2013 |
8932562 |
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PCT/US2011/059298 |
Nov 4, 2011 |
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14555048 |
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61464806 |
Mar 8, 2011 |
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61410748 |
Nov 5, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 41/00 20130101;
C07K 14/705 20130101; A61K 38/177 20130101; C12N 15/86 20130101;
A61P 25/22 20180101; C12N 2799/025 20130101; A01K 2267/0393
20130101; A61K 9/0019 20130101; A01K 67/0275 20130101; A61K 49/0004
20130101; A01K 2217/072 20130101; A61K 49/0008 20130101; A01K
2227/105 20130101; A61B 5/165 20130101; C12N 7/00 20130101; A01K
67/0278 20130101; A61N 5/062 20130101; C12N 15/8509 20130101; C12N
2015/859 20130101; A61K 48/0058 20130101; A01K 2267/0356 20130101;
G01N 33/5088 20130101; C12N 2750/14143 20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A01K 67/027 20060101 A01K067/027; G01N 33/50 20060101
G01N033/50; A61K 9/00 20060101 A61K009/00; A61K 49/00 20060101
A61K049/00; C07K 14/705 20060101 C07K014/705; A61K 41/00 20060101
A61K041/00; C12N 15/85 20060101 C12N015/85; C12N 15/86 20060101
C12N015/86; C12N 7/00 20060101 C12N007/00; A61K 48/00 20060101
A61K048/00; A61N 5/06 20060101 A61N005/06; A61B 5/16 20060101
A61B005/16 |
Claims
1.-31. (canceled)
32. A method for alleviating anxiety in an individual, the method
comprising: (a) administering directly into the brain of the
individual an effective amount of a recombinant expression vector
comprising a nucleic acid encoding a light-responsive opsin that
comprises an amino acid sequence having at least 90% amino acid
sequence identity to any one of SEQ ID NOs:6-11, wherein the
nucleic acid is operably linked to a promoter that controls the
specific expression of the opsin in the glutamatergic pyramidal
neurons of the basolateral amygdala (BLA), wherein the opsin is
expressed in the glutamatergic pyramidal neurons of the BLA,
wherein the opsin is an opsin that induces depolarization by light;
(b) implanting a beveled cannula over the centrolateral nucleus
(CeL) to prevent light delivery to the BLA somata; and (c)
delivering light from a light delivery device ensheathed in the
beveled cannula to selectively illuminate the opsin in the
glutamatergic pyramidal neurons in the central nucleus of the
amygdala (CeA) to alleviate anxiety.
33. The method of claim 32, comprising selectively illuminating the
centrolateral nuclei (CeL) to alleviate anxiety.
34. The method of claim 32, wherein the opsin comprises an amino
acid sequence having at least 95% amino acid sequence identity to
the amino acid sequence set forth in any one of SEQ ID
NOs:6-11.
35. The method of claim 32, wherein the opsin comprises an amino
acid sequence having at least 90% amino acid sequence identity to
the amino acid sequence set forth in SEQ ID NO:6.
36. The method of claim 32, wherein the opsin comprises an amino
acid sequence having at least 90% amino acid sequence identity to
the amino acid sequence set forth in SEQ ID NO:7.
37. The method of claim 32, wherein the opsin comprises an amino
acid sequence having at least 90% amino acid sequence identity to
the amino acid sequence set forth in SEQ ID NO:8.
38. The method of claim 32, wherein the opsin comprises an amino
acid sequence having at least 90% amino acid sequence identity to
the amino acid sequence set forth in SEQ ID NO:9.
39. The method of claim 32, wherein the opsin comprises an amino
acid sequence having at least 90% amino acid sequence identity to
the amino acid sequence set forth in SEQ ID NO:10.
40. The method of claim 32, wherein the opsin comprises an amino
acid sequence having at least 90% amino acid sequence identity to
the amino acid sequence set forth in SEQ ID NO:11.
41. The method of claim 32, wherein the recombinant vector is an
adeno-associated vector, a retroviral vector, an adenoviral vector,
or a lentiviral vector.
42. The method of claim 32, wherein the promoter is a CaMKIIa
promoter.
43. The method of claim 32, wherein said light delivery device
comprises an optical fiber.
44. The method of claim 32, wherein the beveled cannula is beveled
to form a 45-55-degree angle for the restriction of the
illumination to the CeA.
45. The method of claim 44, wherein the beveled cannula guide
comprises a long side that shields the posterior-lateral portion of
the light delivery device.
46. The method of claim 32, further comprising delivering one or
more of a pharmacological agent, an electrical stimulus, or a
magnetic stimulus to the individual.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/882,719, filed Jul. 29, 2013, now U.S. Pat.
No. 8,932,562, which is a national stage filing under 35 U.S.C.
.sctn. 371 of PCT/US2011/059298, filed Nov. 4, 2011, which claims
the priority benefit of U.S. provisional application Ser. No.
61/410,748 filed on Nov. 5, 2010, and 61/464,806 filed on Mar. 8,
2011, the contents of each of which are incorporated herein by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Anxiety is a sustained state of heightened apprehension in
the absence of immediate threat, which in disease states becomes
severely debilitating. Anxiety disorders represent the most common
of the psychiatric diseases (with 28% lifetime prevalence), and
have been linked to the etiology of major depression and substance
abuse. While the amygdala, a brain region important for emotional
processing, has long been hypothesized to play a role in anxiety,
the neural mechanisms which control and mediate anxiety have yet to
be identified. Despite the high prevalence and severity of anxiety
disorders, the corresponding neural circuit substrates are poorly
understood, impeding the development of safe and effective
treatments. Available treatments tend to be inconsistently
effective or, in the case of benzodiazepines, addictive and linked
to significant side effects including sedation and respiratory
suppression that can cause cognitive impairment and death. A deeper
understanding of anxiety control mechanisms in the mammalian brain
is necessary to develop more efficient treatments that have fewer
side-effects. Of particular interest and novelty would be the
possibility of recruiting native pathways for anxiolysis.
SUMMARY OF THE INVENTION
[0003] Provided herein is an animal comprising a light-responsive
opsin expressed in glutamatergic pyramidal neurons of the
basolateral amygdala (BLA), wherein the selective illumination of
the opsin in the BLA-CeL induces anxiety or alleviates anxiety of
the animal.
[0004] Provided herein is an animal comprising a light-responsive
opsin expressed in glutamatergic pyramidal neurons of the BLA,
wherein the opsin is an opsin which induces hyperpolarization by
light, and wherein the selective illumination of the opsin in the
BLA-CeL induces anxiety of the animal. In some embodiments, the
opsin is NpHR, BR, AR, or GtR3. In some embodiments, the NpHR
comprises the amino acid sequence of SEQ ID NO:1, 2, or 3. In some
embodiments, the animal further comprises a second light-responsive
opsin expressed in glutamatergic pyramidal neurons of the BLA,
wherein the second opsin is an opsin that induces depolarization by
light, and wherein the selective illumination of the second opsin
in the BLA-CeL reduces anxiety of the animal. In some embodiments,
the second opsin is ChR2, VChR1, or DChR. In some embodiments, the
second opsin is a C1V1 chimeric protein comprising the amino acid
sequence of SEQ ID NO:8, 9, 10, or 11. In some embodiments, the
second opsin comprises the amino acid sequence of SEQ ID NO:6 or
7.
[0005] Provided herein is an animal comprising a light-responsive
opsin expressed in the glutamatergic pyramidal neurons of the BLA,
wherein the opsin is an opsin that induces depolarization by light,
and wherein the selective illumination of the opsin in the BLA-CeL
reduces anxiety of the animal. In some embodiments, the opsin is
ChR2, VChR1, or DChR. In some embodiments, the opsin is a C1V1
chimeric protein comprising the amino acid sequence of SEQ ID NO:8,
9, 10, or 11. In some embodiments, the opsin comprises the amino
acid sequence of SEQ ID NO:6 or 7.
[0006] Also provided herein is a vector for delivering a nucleic
acid to glutamatergic pyramidal neurons of the BLA in an
individual, wherein the vector comprises the nucleic acid encoding
a light-responsive opsin and the nucleic acid is operably linked to
a promoter that controls the specific expression of the opsin in
the glutamatergic pyramidal neurons. In some embodiments, the
promoter is a CaMKIIa promoter. In some embodiments, the vector is
an AAV vector. In some embodiments, the opsin is an opsin that
induces depolarization by light, and wherein selective illumination
of the opsin in the BLA-CeL alleviates anxiety. In some
embodiments, the opsin that induces depolarization by light is
ChR2, VChR1, or DChR. In some embodiments, the opsin is a C1V1
chimeric protein comprising the amino acid sequence of SEQ ID NO:8,
9, 10, or 11. In some embodiments, the opsin comprises the amino
acid sequence of SEQ ID NO:6 or 7. In some embodiments, the opsin
is an opsin that induces hyperpolarization by light, and wherein
selective illumination of the opsin in the BLA-CeL and induces
anxiety. In some embodiments, the opsin that induces
hyperpolarization by light is NpHR, BR, AR, or GtR3. In some
embodiments, the NpHR comprises the amino acid sequence of SEQ ID
NO:1, 2, or 3. In some embodiments, the individual is a mouse or a
rat. In some embodiments, the individual is a human.
[0007] Also provided here is a method of delivering a nucleic acid
to glutamatergic pyramidal neurons of the BLA in an individual,
comprising administering to the individual an effective amount of a
vector comprising a nucleic acid encoding a light-responsive opsin
and the nucleic acid is operably linked to a promoter that controls
the specific expression of the opsin in the glutamatergic pyramidal
neurons. In some embodiments, the promoter is a CaMKII.alpha.
promoter. In some embodiments, the vector is an AAV vector. In some
embodiments, the opsin is an opsin that induces depolarization by
light, and wherein selective illumination of the opsin in the
BLA-CeL alleviates anxiety. In some embodiments, the opsin that
induces depolarization by light is ChR2, VChR1, or DChR. In some
embodiments, the opsin is a C1V1 chimeric protein comprising the
amino acid sequence of SEQ ID NO:8, 9, 10, or 11. In some
embodiments, the opsin comprises the amino acid sequence of SEQ ID
NO:6 or 7. In some embodiments, the opsin is an opsin that induces
hyperpolarization by light, and wherein selective illumination of
the opsin in the BLA-CeL and induces anxiety. In some embodiments,
the opsin that induces hyperpolarization by light is NpHR, BR, AR,
or GtR3. In some embodiments, the NpHR comprises the amino acid
sequence of SEQ ID NO:1, 2, or 3. In some embodiments, the
individual is a mouse or a rat. In some embodiments, the individual
is a human.
[0008] Also provided herein is a coronal brain tissue slice
comprising BLA, CeL, and CeM, wherein a light-responsive opsin is
expressed in the glutamatergic pyramidal neurons of the BLA. In
some embodiments, the opsin is an opsin that induces depolarization
by light. In some embodiments, the opsin that induces
depolarization by light is ChR2, VChR1, or DChR. In some
embodiments, the opsin is a C1V1 chimeric protein comprising the
amino acid sequence of SEQ ID NO:8, 9, 10, or 11. In some
embodiments, the opsin comprises the amino acid sequence of SEQ ID
NO:6 or 7. In some embodiments, the opsin is an opsin that induces
hyperpolarization by light. In some embodiments, the opsin that
induces hyperpolarization by light is NpHR, BR, AR, or GtR3. In
some embodiments, the NpHR comprises the amino acid sequence of SEQ
ID NO:1, 2, or 3. In some embodiments, the tissue is a mouse or a
rat tissue.
[0009] Also provided herein is a method for screening for a
compound that alleviates anxiety, comprising (a) administering a
compound to an animal having anxiety induced by selectively
illumination of an opsin expressed in the glutamatergic pyramidal
neurons of the BLA, wherein the animal comprises a light-responsive
opsin expressed in the glutamatergic pyramidal neurons of the BLA,
wherein the opsin is an opsin that induces hyperpolarization by
light; and (b) determining the anxiety level of the animal, wherein
a reduction of the anxiety level indicates that the compound may be
effective in treating anxiety. In some embodiments, the opsin is
NpHR, BR, AR, or GtR3. In some embodiments, the NpHR comprises the
amino acid sequence of SEQ ID NO:1, 2, or 3.
[0010] Also provided herein is a method for alleviating anxiety in
an individual, comprising: (a) administering to the individual an
effective amount of a vector comprising a nucleic acid encoding a
light-responsive opsin and the nucleic acid is operably linked to a
promoter that controls the specific expression of the opsin in the
glutamatergic pyramidal neurons of the BLA, wherein the opsin is
expressed in the glutamatergic pyramidal neurons of the BLA,
wherein the opsin is an opsin that induces depolarization by light;
and (b) selectively illuminating the opsin in the glutamatergic
pyramidal neurons in the BLA-CeL to alleviate anxiety. In some
embodiments, the promoter is a CaMKII.alpha. promoter. In some
embodiments, the vector is an AAV vector. In some embodiments, the
opsin is ChR2, VChR1, or DChR. In some embodiments, the opsin is a
C1V1 chimeric protein comprising the amino acid sequence of SEQ ID
NO:8, 9, 10, or 11. In some embodiments, the opsin comprises the
amino acid sequence of SEQ ID NO:6 or 7.
[0011] Also provided herein is a method for inducing anxiety in an
individual, comprising: (a) administering to the individual an
effective amount of a vector comprising a nucleic acid encoding an
opsin and the nucleic acid is operably linked to a promoter that
controls the specific expression of the opsin in the glutamatergic
pyramidal neurons of the BLA, wherein the opsin is expressed in the
glutamatergic pyramidal neurons, wherein the opsin is an opsin that
induces hyperpolarization by light; and (b) selectively
illuminating the opsin in the glutamatergic pyramidal neurons in
the BLA-CeL to induce anxiety. In some embodiments, the promoter is
a CaMKII.alpha. promoter. In some embodiments, the vector is an AAV
vector. In some embodiments, the opsin is NpHR, BR, AR, or GtR3. In
some embodiments, the NpHR comprises the amino acid sequence of SEQ
ID NO:1, 2, or 3.
[0012] It is to be understood that one, some, or all of the
properties of the various embodiments described herein may be
combined to form other embodiments of the present invention. These
and other aspects of the invention will become apparent to one of
skill in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Various example embodiments may be more completely
understood in consideration of the following description and the
accompanying drawings, in which:
[0014] FIG. 1 shows a system for providing optogenetic targeting of
specific projections of the brain, consistent with an embodiment of
the present disclosure; and
[0015] FIG. 2 shows a flow diagram for use of an anxiety-based
circuit model, consistent with an embodiment of the present
disclosure.
[0016] FIGS. 3A-3K show that projection-specific excitation of BLA
terminals in the CeA induced acute reversible anxiolysis. FIG. 3A:
All mice were singly-housed in a high-stress environment for at
least 1 week prior to behavioral manipulations and receive 5-ms
light pulses at 20 Hz for all light on conditions. Mice in the
ChR2:BLA-CeA group received viral transduction of ChR2 in BLA
neurons under the CaMKII promoter and were implanted with a beveled
cannula shielding light away from BLA somata to allow selective
illumination of BLA terminals in the CeA, while control groups
either received a virus including fluorophore only (EYFP:BLA-CeA
group) or a light fiber directed to illuminate BLA somata (ChR2:BLA
Somata group). FIG. 3B-3C: Mice in the ChR2:BLA-CeA group (n=8)
received selective illumination of BLA terminals in the CeA during
the light on epoch during the elevated plus maze, as seen in this
ChR2:BLA-CeA representative path FIG. 3B, which induced a 5-fold
increase in open arm time during the light on epoch relative to the
light off epochs and EYFP:BLA-CeA (n=9) and ChR2:BLA Somata (n=7)
controls FIG. 3C, as well as a significant increase in the
probability of entering the open arm (see inset). FIG. 3D-3F: Mice
in the ChR2:BLA-CeA group also showed an increase in the time spent
in the center of the open field chamber, as seen in this
representative trace (FIG. 3D), during light on epochs relative to
light off epochs and EYFP:BLA-CeA and ChR2:BLA Somata controls FIG.
3E, but did not show a significant change in locomotor activity
during light on epochs (FIG. 3F). FIG. 3G: Confocal image of a
coronal slice showing the CeA and BLA regions from a mouse in the
ChR2:BLA-CeA group wherein 125 .mu.mx125 .mu.m squares indicate
regions used for quantification. FIG. 3D-3H: Expanded regions are
arranged in rows by group and in columns by brain region. FIG.
3I-3K: Percent of EYFP-positive and c-fos-positive neurons of all
DAPI-identified cells for all groups, by region. Numbers of counted
per group and region are indicated in legends. None of the regions
examined showed detectable differences in the proportion of
EYFP-positive cells among groups. FIG. 31: Proportion of BLA
neurons that were EYFP-positive or c-fos-positive. The ChR2:BLA
Somata group had a significantly higher proportion of
c-fos-positive BLA neurons (F.sub.2,9=10.12, p<0.01) relative to
ChR2:BLA-CeA (p<0.01) or EYFP:BLA-CeA (p<0.05) groups. FIG.
3J: The ChR2:BLA-CeA group had a significantly higher proportion of
c-fos-positive cells in the CeL relative to the EYFP:BLA-CeA group
(p<0.05), but not the ChR2:BLA Somata group. FIG. 3K:Summary
data for CeM neurons show no detectable differences among
groups.
[0017] FIGS. 4A-4F show projection-specific excitation of BLA
terminals in the CeA activates CeL neurons and elicits feed-forward
inhibition of CeM neurons. FIG. 4A: Live two-photon images of
representative light-responsive BLA, CeL and CeM cells all imaged
from the same slice, overlaid on a brightfield image. FIG. 4B-4F:
Schematics of the recording and illumination sites for the
associated representative current-clamp traces (V.sub.m=.about.70
mV). FIG. 4B: Representative trace from a BLA pyramidal neuron
expressing ChR2, all BLA neurons expressing ChR2 in the BLA spiked
for every 5 ms pulse (n=4). FIG. 4C: Representative trace from a
CeL neuron in the terminal field of BLA projection neurons, showing
both sub-threshold and supra-threshold excitatory responses to
light-stimulation (n=16). Inset left, population summary of mean
probability of spiking for each pulse in a 40-pulse train at 20 Hz,
dotted lines indicate SEM. Inset right, frequency histogram showing
individual cell spiking fidelity for 5 ms light pulses delivered at
20 Hz, y-axis is the number of cells per each 5% bin. FIG. 4D: Six
sweeps from a CeM neuron spiking in response to a current step
(.about.60 pA; indicated in black) and inhibition of spiking upon
20 Hz illumination of BLA terminals in the CeL. Inset, spike
frequency was significantly reduced during light stimulation of CeL
neurons (n=4). FIG. 4E-4F: Upon broad illumination of the CeM,
voltage-clamp summaries show that the latency of EPSCs is
significantly shorter than the latency of IPSCs, while there was a
non-significant difference in the amplitude of EPSCs and IPSCs
(n=11; *p=0.04, see insets). The same CeM neurons (n=7) showed
either net excitation when receiving illumination of the CeM FIG.
4E: or net inhibition upon selective illumination of the CeL (FIG.
4F).
[0018] FIGS. 5A-5K show light-induced anxiolytic effects were
attributable to activation of BLA-CeL synapses alone. FIG. 5A-5B:
2-photon z-stack images of 18 dye-filled BLA neurons were
reconstructed, and their projections to the CeL and CeM are
summarized in FIG. 5A, with their images shown in FIG. 5B wherein
red indicates projections to CeL, blue indicates projections to CeM
and purple indicates projections to both CeL and CeM. FIG. 5C:
Schematic of the recording site and the light spot positions, as
whole-cell recordings were performed at each location of the light
spot, which was moved in 100 um-steps away from the cell soma both
over a visualized axon and in a direction that was not over an
axon. FIG. 5D: Normalized current-clamp summary of spike fidelity
to a 20 Hz train delivered at various distances from the soma,
showing that at .about.300um away from the cell soma, illumination
of an axon terminal results in low (<5%) spike fidelity. FIG.
5E: Normalized voltage-clamp summary of depolarizing current seen
at the cell soma upon illumination per distance from cell soma.
FIG. 5F-5I: Representative current-clamp traces upon illumination
with a .about.150 um-diameter light spot over various locations
within each slice preparation (n=7). Illumination of the cell soma
elicits high-fidelity spiking (FIG. 5F). Illumination of BLA
terminals in CeL elicits strong sub- and supra-threshold excitatory
responses in the postsynaptic CeL neuron (FIG. 5G), but does not
elicit reliable antidromic spiking in the BLA neuron itself (FIG.
5H), and light delivered off axon is shown for comparison as a
control for light scattering (FIG. 5I). FIG. 5J-5K: A separate
group of ChR2:BLA-CeL mice (n=8) were each run twice on the
elevated plus maze and the open field test, one session preceded
with intra-CeA infusions of saline (red) and the other session with
the glutamate receptor antagonists NBQX and AP5 (purple),
counterbalanced for order. FIG. 5K: Glutamate receptor blockade in
the CeA attenuated light-induced increases in both time spent in
open arms as well as the probability of open arm entry (inset) on
the elevated plus maze without impairing performance during light
off epochs. FIG. FIG. 5J: Local glutamate receptor antagonism
significantly attenuated light-induced increases in center time on
the open field test, inset shows pooled summary.
[0019] FIGS. 6A-6P show that selective inhibition of BLA terminals
in the CeA induced an acute and reversible increase in anxiety.
FIG. 6A: All mice were group-housed in a low-stress environment and
received bilateral constant 591 nm light during light on epochs.
Mice in the eNpHR3.0:BLA-CeA group (n=9) received bilateral viral
transduction of eNpHR3.0 in BLA neurons under the CaMKII promoter
and were implanted with a beveled cannula shielding light away from
BLA somata to allow selective illumination of BLA terminals in the
CeA, while control groups either received bilateral virus
transduction of a fluorophore only (EYFP:BLA-CeA bil group; n=8) or
a light fiber directed to illuminate BLA somata (eNpHR3.0:BLA
Somata group; n=6). FIG. 6B: Confocal image of the BLA and CeA of a
mouse treated with eNpHR3.0. FIG. 6C-6E: In the same animals used
in anxiety assays below, a significantly higher proportion of
neurons in the CeM (FIG. 6E) from the eNpHR3.0 group expressed
c-fos relative to the EYFP group (*p<0.05). FIG. 6F:
Representative path of a mouse in the eNpHR3.0:BLA-CeA group,
showing a decrease in open arm exploration on the elevated plus
maze during epochs of selective illumination of BLA terminals in
the CeA. FIG. 6G: eNpHR3.0 mice showed a reduction in the time
spent in open arms and probability of open arm entry (inset) during
light stimulation, relative to controls. FIG. 6H: Representative
path of a mouse from the eNpHR3.0:BLA-CeA group during pooled light
off and on epochs in the open field test. FIG. 61: Significant
reduction in center time in the open field chamber for the
eNpHR3.0:BLA-CeA group during light on, but not light off, epochs
as compared to controls, inset shows pooled data summary. FIGS.
6J-L: Selective illumination of eNpHR3.0-expressing BLA terminals
is sufficient to reduce spontaneous vesicle release in the presence
of carbachol. Representative trace of a CeL neuron (FIG. 6J) from
an acute slice preparation in which BLA neurons expressed eNpHR
3.0, shows that when BLA terminals .about.300 .mu.m away from the
BLA soma are illuminated, there is a reduction in the amplitude (k)
and frequency (l) of sEPSCs seen at the postsynaptic CeL neuron.
Cumulative distribution frequency of the amplitude (k) and
frequency (l) of sEPSCs recorded at CeL neurons (n=5) upon various
lengths of illumination 5-60 s, insets show respective mean+SEM in
the epochs of matched duration before, during and after
illumination (**p<0.01; ***p<0.001). FIG. 6M-6P: Selective
illumination of BLA terminals expressing eNpHR 3.0 suppresses
vesicle release evoked by electrical stimulation in the BLA. FIG.
6M: Schematic indicating the locations of the stimulating
electrode, the recording electrode and the .about.150 .mu.m
diameter light spot. FIG. 6N: Representative traces of EPSCs in a
CeL neuron before (OM), during (On) and after (Off.sub.2) selective
illumination of BLA terminals expressing eNpHR3.0. Normalized EPSC
amplitude summary data (FIG. 6O) and individual cell data (FIG. 6P)
from slice preparations containing BLA neurons expressing eNpHR 3.0
(n=7) and non-transduced controls (n=5) show that selectively
illuminating BLA terminals in the CeL significantly (*p=0.006)
reduces the amplitude of electrically-evoked EPSCs in postsynaptic
CeL neurons.
[0020] FIG. 7 is a diagram showing the histologically verified
placements of mice treated with 473 nm light. Unilateral placements
of the virus injection needle (circle) and the tip of beveled
cannula (x) are indicated, counter-balanced for hemisphere. Colors
indicate treatment group, see legend. Coronal sections containing
the BLA and the CeA are shown here, numbers indicate the
anteroposterior coordinates from bregma (Aravanis et al., J Neural
Eng, 4:S143-156, 2007).
[0021] FIGS. 8A-8E show the beveled cannula and illumination
profile design. FIG. 8A:Light cone from bare fiber emitting 473 nm
light over cuvette filled with fluorescein in water. The angle of
the light cone is approximately 12 degrees. FIG. 8B: Light cone
from the same fiber and light ensheathed in a beveled cannula. The
beveled cannula blocks light delivery to one side, without
detectably altering perpendicular light penetrance. FIG. 8C:
Diagram of light delivery via the optical fiber with the beveled
cannula over CeA. FIG. 8D: Chart indicating estimated light power
density seen at various distances from the fiber tip in mouse brain
tissue when the light power density seen at the fiber tip was 7 mW
(.about.99 mW/mm.sup.2). Inset, cartoon indicating the
configuration. Optical fiber is perpendicular and aimed at the
center of the power meter, through a block of mouse brain tissue.
FIG. 8E: Table showing light power (mW) as measured by a standard
power meter and the estimated light power density (mW/mm.sup.2)
seen at the tip, at the CeL (.about.0.5-0.7 mm depth in brain
tissue) and at the CeM (.about.1.1 mm depth in brain tissue).
[0022] FIGS. 9A-9D demonstrate that the beveled cannula prevented
light delivery to BLA and BLA spiking at light powers used for
behavioral assays. FIG. 9A: Schematic indicating the configuration
of light delivery by optical fiber to the CeA and recording
electrode (red) in the BLA. FIG. 9B: Scatterplot summary of
recordings in the BLA with various light powers delivered to the
CeA with and without the beveled cannula (n=4 sites). For each
site, repeated alternations of recordings were made with and
without the beveled cannula. The x-axis shows both the light power
density at the fiber tip (black) and the estimated light power
density at the CeL (grey). The blue vertical or shaded region
indicates the range of light power densities used for behavioral
assays (.about.7 mW; .about.99 mW/mm.sup.2 at the tip of the
fiber). Reliable responses from BLA neurons were not observed in
this light power density range. FIG. 9C: Representative traces of
BLA recordings with 20 Hz 5 ms pulse light stimulation at 7mW
(.about.99 mW/mm.sup.2 at fiber tip; .about.5.9 mW/mm.sup.2 at CeL)
at the same recording site in the CeA. FIG. 9D: Population spike
waveforms in response to single pulses of light reveal substantial
light restriction even at high 12 mV power (.about.170 mW/mm.sup.2
at the tip of the fiber; .about.10.1 mW/mm.sup.2 at CeL).
[0023] FIGS. 10A-10F demonstrate that viral transduction excluded
intercalated cell clusters. FIG. 10A: Schematic of the intercalated
cells displayed in subsequent confocal images. FIG. 10B-10D:
Representative images of intercalated cells from mice that received
EYFP (FIG. 10B) , eNpHR 3.0 (FIG. 10C) and ChR2 (FIG. 10D)
injections into the BLA that were used for behavioral
manipulations. Viral expression was not observed in intercalated
cell clusters. FIG. 10E-10F: There were very low (<2%) levels of
YFP expression in intercalated cell clusters for all 6 groups used
in behavioral assays. There were no statistically significant
differences among groups in c-fos expression.
[0024] FIG. 11 shows that unilateral intra-CeA administration of
glutamate antagonists did not alter locomotor activity.
Administration of NBQX and AP5 prior to the open field test did not
impair locomotor activity (as measured by mean velocity) relative
to saline infusion (F.sub.1,77=2.34, p=0.1239).
[0025] FIGS. 12A-12C demonstrate that bath application of glutamate
antagonists blocked optically-evoked synaptic transmission. 4-6
weeks following intra-BLA infusions of AAV5-CAMKII-ChR2-EYFP into
the BLA of wild-type mice, we examined the ability of the glutamate
receptor antagonists NBQX and AP5 to block glutamatergic
transmission. FIG. 12A: Representative current-clamp (top) and
voltage-clamp (bottom) traces of a representative CeL neuron upon a
20 Hz train of 473 nm light illumination of BLA terminals
expressing ChR2. FIG. 12B:The same cell's responses following bath
application of NBQX and AP5 show abolished spiking and EPSCs. FIG.
12C: Population summary (n=5) of the depolarizing current seen
before and after bath application of NBQX and AP5, normalized to
the pre-drug response.
[0026] FIG. 13 is a diagram depicting the histologically verified
placements of mice treated with 594 nm light. Bilateral placements
of virus injection needle (circle) and tip of beveled cannula (x)
are indicated. Colors indicate treatment group, see legend. Coronal
sections containing BLA and CeA are shown; numbers indicate AP
coordinates from bregma (Aravanis et el., J Neural Eng, 4:S143-156,
2007).
[0027] FIGS. 14A-14E show that light stimulation parameters used in
the eNpHR 3.0 terminal inhibition experiments does not block
spiking at the cell soma. FIG. 14A-14C: Schematics of the light
spot location and recording sites alongside corresponding
representative traces upon a current step lasting the duration of
the spike train, paired with yellow light illumination at each
location during the middle epoch (indicated by yellow horizontal
bar). FIG. 14A: Representative current-clamp trace from a BLA
neuron expressing eNpHR 3.0 upon direct illumination shows potent
inhibition of spiking during illumination of cell soma. FIG. 14B:
Representative current-clamp trace from a BLA neuron expressing
eNpHR 3.0 when a .about.125 um diameter light spot is presented
.about.300 um away from the cell soma without illuminating an axon.
FIG. 14C: Representative current-clamp trace from a BLA neuron
expressing eNpHR 3.0 when a .about.125 .mu.m diameter light spot is
presented .about.300 .mu.m away from the cell soma when
illuminating an axon. FIG. 14D:While direct illumination of the
cell soma induced complete inhibition of spiking that was
significant from all other conditions (F.sub.3,9=81.50,
p<0.0001; n=3 or more per condition), there was no significant
difference among the distal illumination .about.300 um away from
the soma of BLA neurons expressing eNpHR 3.0 conditions and the no
light condition (F.sub.2,7=0.79, p=0.49), indicating that distal
illumination did not significantly inhibit spiking at the cell
soma. FIG. 14E: Schematic indicating light spot locations relative
to recording site, regarding the population summary shown to the
right. Population summary shows the normalized hyperpolarizing
current recorded from the cell soma per distance of light spot from
cell soma, both on and off axon collaterals (n=5).
[0028] FIG. 15 demonstrates that selective illumination of BLA
terminals induced vesicle release onto CeL neurons without reliably
eliciting antidromic action potentials. Schematics and descriptions
refer to the traces below, and trace color indicates cell type.
Light illumination patterns are identical for both series of
traces. Left column, CeL traces for three overlaid sweeps of a
40-pulse light train per cell (n=8). Here, both time-locked EPSCs
indicate vesicle release from the presynaptic ChR2-expressing BLA
terminal, and for all postsynaptic CeL cells, there were excitatory
responses to 100% of light pulses. Right column, BLA traces for
three overlaid 40-pulse sweeps per cell (n=9), with the mean number
of light pulses delivered at the axon terminal resulting in a
supra-threshold antidromic action potential (5.4% .+-.2%,
mean.+-.SEM).
[0029] FIG. 16 is a graph demonstrating that light stimulation did
not alter locomotor activity in eNpHR 3.0 and control groups. There
were no detectable differences in locomotor activity among groups
nor light epochs (F.sub.1,20=0.023, p=0.3892; F.sub.1,100=3.08,
p=0.086).
DETAILED DESCRIPTION
[0030] The present disclosure relates to control over nervous
system disorders, such as disorders associated with anxiety and
anxiety symptoms, as described herein. While the present disclosure
is not necessarily limited in these contexts, various aspects of
the invention may be appreciated through a discussion of examples
using these and other contexts.
[0031] 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 anxiety. The optogenetic
system targets a neural circuit within a patient for selective
control thereof. The optogenetic system involves monitoring the
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.
[0032] 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.
[0033] Aspects of the present disclosure are directed to using an
artificially-induced anxiety state for the study of anxiety in
otherwise healthy animals. This can be particularly useful for
monitoring symptoms and aspects that are poorly understood and
otherwise difficult to accurately model in living animals For
instance, it can be difficult to test and/or study anxiety states
due to the lack of available animals exhibiting the anxiety state.
Moreover, certain embodiments allow for reversible anxiety 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 anxiety state and when not
exhibiting the anxiety state. The reversible anxiety states of
certain embodiments can also allow for a reset to baseline between
testing the effects of different treatments on the same animal
[0034] Certain aspects of the present disclosure are directed to a
method related to control over anxiety and/or anxiety symptoms in a
living animal In certain more specific embodiments, the monitoring
of the symptoms also includes assessing the efficacy of the
stimulus in mitigating the symptoms of anxiety. Various other
methods and applications exist, some of which are discussed in more
detail herein.
[0035] Light-responsive opsins that may be used in the present
invention includes opsins that induce hyperpolarization in neurons
by light and opsins that induce depolarization in neurons by light.
Examples of opsins are shown in Tables 1 and 2 below.
[0036] Table 1 shows identified opsins for inhibition of cellular
activity across the visible spectrum:
TABLE-US-00001 TABLE 1 Fast optogenetics: inhibition across the
visible spectrum Opsin Wavelength Type Biological Origin
Sensitivity Defined action NpHR Natronomonas 589 nm max Inhibition
pharaonis (hyperpolarization) BR Halobacterium 570 nm max
Inhibition helobium (hyperpolarization) AR Acetabulaira 518 nm max
Inhibition acetabulum (hyperpolarization) GtR3 Guillardia theta 472
nm max Inhibition (hyperpolarization) Mac Leptosphaeria 470-500 nm
max Inhibition maculans (hyperpolarization) NpHr3.0 Natronomonas
680 nm utility Inhibition pharaonis 589 nm max (hyperpolarization)
NpHR3.1 Natronomonas 680 nm utility Inhibition pharaonis 589 nm max
(hyperpolarization)
[0037] Table 2 shows identified opsins for excitation and
modulation across the visible spectrum:
TABLE-US-00002 TABLE 2 Fast optogenetics: excitation and modulation
across the visible spectrum Wavelength Opsin Type Biological Origin
Sensitivity Defined action VChR1 Volvox carteri 589 nm utility
Excitation 535 nm max (depolarization) DChR Dunaliella salina 500
nm max Excitation (depolarization) ChR2 Chlamydomonas 470 nm max
Excitation reinhardtii 380-405 nm utility (depolarization) ChETA
Chlamydomonas 470 nm max Excitation reinhardtii 380-405 nm utility
(depolarization) SFO Chlamydomonas 470 nm max Excitation
reinhardtii 530 nm max (depolarization) Inactivation SSFO
Chlamydomonas 445 nm max Step-like activation reinhardtii 590 nm;
390-400 nm (depolarization) Inactivation C1V1 Volvox carteri and
542 nm max Excitation Chlamydomonas (depolarization) reinhardtii
C1V1 E122 Volvox carteri and 546 nm max Excitation Chlamydomonas
(depolarization) reinhardtii C1V1 E162 Volvox carteri and 542 nm
max Excitation Chlamydomonas (depolarization) reinhardtii C1V1
E122/E162 Volvox carteri and 546 nm max Excitation Chlamydomonas
(depolarization) reinhardtii
[0038] As used herein, a light-responsive opsin (such as NpHR, BR,
AR, GtR3, Mac, ChR2, VChR1, DChR, and ChETA) includes naturally
occurring protein and functional variants, fragments, fusion
proteins comprising the fragments or the full length protein. For
example, the signal peptide may be deleted. A variant may have an
amino acid sequence at least about any of 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the naturally
occurring protein sequence. A functional variant may have the same
or similar hyperpolarization function or depolarization function as
the naturally occurring protein.
[0039] In some embodiments, the NpHR is eNpHR3.0 or eNpHR3.1 (See
www.stanford.edu/group/dlab/optogenetics/sequence_info.html). In
some embodiments, the light-responsive opsin is a C1V1 chimeric
protein or a C1V1-E162 (SEQ ID NO:10), C1V1-E122 (SEQ ID NO:9), or
C1V1-E122/E162 (SEQ ID NO:11) mutant chimeric protein (See, Yizhar
et al, Nature, 2011, 477(7363):171-78 and
www.stanford.edu/group/dlab/optogenetics/sequence_info.html). In
some embodiments, the light-responsive opsin is a SFO (SEQ ID NO:6)
or SSFO (SEQ ID NO:7) (See, Yizhar et al, Nature, 2011,
477(7363):171-78; Berndt et al., Nat. Neurosci., 12(2):229-34 and
www.stanford.edu/group/dlab/optogenetics/sequence_info.htme.
[0040] In some embodiments, the light-activated protein is a NpHR
opsin comprising an amino acid sequence at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% or 100% identical to the
sequence shown in SEQ ID NO:1. In some embodiments, the NpHR opsin
further comprises an endoplasmic reticulum (ER) export signal
and/or a membrane trafficking signal. For example, the NpHR opsin
comprises an amino acid sequence at least 95% identical to the
sequence shown in SEQ ID NO:1 and an endoplasmic reticulum (ER)
export signal. In some embodiments, the amino acid sequence at
least 95% identical to the sequence shown in SEQ ID NO:1 is linked
to the ER export signal through a linker. In some embodiments, the
ER export signal comprises the amino acid sequence FXYENE (SEQ ID
NO:12), where X can be any amino acid. In another embodiment, the
ER export signal comprises the amino acid sequence VXXSL (SEQ ID
NO:15), where X can be any amino acid. In some embodiments, the ER
export signal comprises the amino acid sequence FCYENEV (SEQ ID
NO:13). In some embodiments, the NpHR opsin comprises an amino acid
sequence at least 95% identical to the sequence shown in SEQ ID
NO:1, an ER export signal, and a membrane trafficking signal. In
other embodiments, the NpHR opsin comprises, from the N-terminus to
the C-terminus, the amino acid sequence at least 95% identical to
the sequence shown in SEQ ID NO:1, the ER export signal, and the
membrane trafficking signal. In other embodiments, the NpHR opsin
comprises, from the N-terminus to the C-terminus, the amino acid
sequence at least 95% identical to the sequence shown in SEQ ID
NO:1, the membrane trafficking signal, and the ER export signal. In
some embodiments, the membrane trafficking signal is derived from
the amino acid sequence of the human inward rectifier potassium
channel K.sub.ir2.1. In some embodiments, the membrane trafficking
signal comprises the amino acid sequence K S R I T S E G E Y I P L
D Q I D I N V (SEQ ID NO:14). In some embodiments, the membrane
trafficking signal is linked to the amino acid sequence at least
95% identical to the sequence shown in SEQ ID NO:1 by a linker. In
some embodiments, the membrane trafficking signal is linked to the
ER export signal through a linker. The linker may 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. The linker may further
comprise a fluorescent protein, for example, but not limited to, a
yellow fluorescent protein, a red fluorescent protein, a green
fluorescent protein, or a cyan fluorescent protein. In some
embodiments, the light-activated opsin further comprises an
N-terminal signal peptide. In some embodiments, the light-activated
opsin comprises the amino acid sequence of SEQ ID NO:2. In some
embodiments, the light-activated protein comprises the amino acid
sequence of SEQ ID NO:3.
[0041] In some embodiments, the light-activated opsin is a chimeric
protein derived from VChR1 from Volvox carteri and ChR1 from
Chlamydomonas reinhardti. In some embodiments, the chimeric protein
comprises the amino acid sequence of VChR1 having at least the
first and second transmembrane helices replaced by the
corresponding first and second transmembrane helices of ChR1. In
other embodiments, the chimeric protein comprises the amino acid
sequence of VChR1 having the first and second transmembrane helices
replaced by the corresponding first and second transmembrane
helices of ChR1 and further comprises at least a portion of the
intracellular loop domain located between the second and third
transmembrane helices replaced by the corresponding portion from
ChR1. In some embodiments, the entire intracellular loop domain
between the second and third transmembrane helices of the chimeric
light-activated protein can be replaced with the corresponding
intracellular loop domain from ChR1. In some embodiments, the
light-activated chimeric protein comprises 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:8 without the signal
peptide sequence. In some embodiments, the light-activated chimeric
protein comprises 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:8. C1V1 chimeric light-activated opsins
that may have specific amino acid substitutions at key positions
throughout the retinal binding pocket of the VChR1 portion of the
chimeric polypeptide. In some embodiments, the C1V1 protein has a
mutation at amino acid residue E122 of SEQ ID NO:8. In some
embodiments, the C1V1 protein has a mutation at amino acid residue
E162 of SEQ ID NO:8. In other embodiments, the C1V1 protein has a
mutation at both amino acid residues E162 and E122 of SEQ ID NO:8.
In some embodiments, each of the disclosed mutant C1V1 chimeric
proteins can have specific properties and characteristics for use
in depolarizing the membrane of an animal cell in response to
light.
[0042] As used herein, a vector comprises a nucleic acid encoding a
light-responsive opsin described herein and the nucleic acid is
operably linked to a promoter that controls the specific expression
of the opsin in the glutamatergic pyramidal neurons. Any vectors
that are useful for delivering a nucleic acid to glutamatergic
pyramidal neurons may be used. Vectors include viral vectors, such
as AAV vectors, retroviral vectors, adenoviral vectors, HSV
vectors, and lentiviral vectors. Examples of AAV vectors are AAV1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,
AAV12, AAV13, AAV14, AAV15, and AAV16. A CaMKII.alpha. promoter and
any other promoters that can control the expression of the opsin in
the glutamatergic pyramidal neurons may be used.
[0043] An "individual" is a mammal, such as a human. Mammals also
include, but are not limited to, farm animals, sport animals, pets
(such as cats, dogs, horses), primates, mice and rats. An "animal"
is a non-human mammal
[0044] As used herein, "treatment" or "treating" or "alleviation"
is an approach for obtaining beneficial or desired results
including and preferably clinical results. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, one or more of the following: showing observable
and/or measurable reduction in one or more signs of the disease
(such as anxiety), decreasing symptoms resulting from the disease,
increasing the quality of life of those suffering from the disease,
decreasing the dose of other medications required to treat the
disease, and/or delaying the progression of the disease.
[0045] As used herein, an "effective dosage" or "effective amount"
of a drug, compound, or pharmaceutical composition is an amount
sufficient to effect beneficial or desired results. For therapeutic
use, beneficial or desired results include clinical results such as
decreasing one or more symptoms resulting from the disease,
increasing the quality of life of those suffering from the disease,
decreasing the dose of other medications required to treat the
disease, enhancing effect of another medication such as via
targeting, and/or delaying the progression of the disease. As is
understood in the clinical context, an effective dosage of a drug,
compound, or pharmaceutical composition may or may not be achieved
in conjunction with another drug, compound, pharmaceutical
composition, or another treatment. Thus, an "effective dosage" may
be considered in the context of administering one or more
therapeutic agents or treatments, and a single agent may be
considered to be given in an effective amount if, in conjunction
with one or more other agents or treatments, a desirable result may
be or is achieved.
[0046] The above overview is not intended to describe each
illustrated embodiment or every implementation of the present
disclosure.
DETAILED DESCRIPTION AND EXAMPLE EXPERIMENTAL EMBODIMENTS
[0047] The present disclosure is believed to be useful for
controlling anxiety states and/or symptoms of anxiety. 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 associated with anxiety states and/or
symptoms thereof. 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 Examples. 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.
[0048] Anxiety refers to a sustained state of heightened
apprehension in the absence of an immediate threat, which in
disease states becomes severely debilitating. Embodiments of the
present disclosure are directed toward the use of one or more of
cell type-specific optogenetic tools with two-photon microscopy,
electrophysiology, and anxiety assays to study and develop
treatments relating to neural circuits underlying anxiety-related
behaviors.
[0049] Aspects of the present disclosure are related to the
optogenetic targeting of specific projections of the brain, rather
than cell types, in the study of neural circuit function relevant
to psychiatric disease.
[0050] Consistent with particular embodiments of the present
disclosure, temporally-precise optogenetic stimulation of
basolateral amygdala (BLA) terminals in the central nucleus of the
amygdala (CeA) are used to produce a reversible anxiolytic effect.
The optogenetic stimulation can be implemented by viral
transduction of BLA with a light-responsive opsin, such as ChR2,
followed by restricted illumination in downstream CeA.
[0051] Consistent with other embodiments of the present disclosure,
optogenetic inhibition of the basolateral amygdala (BLA) terminals
in the central nucleus of the amygdala (CeA) are used to increase
anxiety-related behaviors. The optogenetic stimulation can be
implemented by viral transduction of BLA with a light-responsive
opsin, such as eNpHR3.0, followed by restricted illumination in
downstream CeA.
[0052] Embodiments of the present disclosure are directed towards
the specific targeting of neural cell populations, as anxiety-based
effects were not observed with direct optogenetic control of BLA
somata. For instance, targeting of specific BLA-CeA projections as
circuit elements have been experimentally shown to be sufficient
for endogenous anxiety control in the mammalian brain.
[0053] Consistent with embodiments of the present disclosure, the
targeting of the specific BLA-CeA projections as circuit elements
is based upon a number of factors discussed in more detail
hereafter. The amygdala is composed of functionally and
morphologically heterogeneous subnuclei with complex
interconnectivity. A primary subdivision of the amygdala is the
basolateral amygdala complex (BLA), which encompasses the lateral
(LA), basolateral (BL) and basomedial (BM) amygdala nuclei
(.about.90% of BLA neurons are glutamatergic). In contrast, the
central nucleus of the amygdala (CeA), which is composed of the
centrolateral (CeL) and centromedial (CeM) nuclei, is predominantly
(.about.95%) comprised of GABAergic medium spiny neurons. The BLA
is ensheathed in dense clusters of GABAergic intercalated cells
(ITCs), which are functionally distinct from both local
interneurons and the medium spiny neurons of the CeA. The primary
output nucleus of the amygdala is the CeM, which, when chemically
or electrically excited, is believed to mediate autonomic and
behavioral responses that are associated with fear and anxiety via
projections to the brainstem. While the CeM is not directly
controlled by the primary amygdala site of converging environmental
and cognitive information (LA), LA and BLA neurons excite GABAergic
CeL neurons, which can provide feed-forward inhibition onto CeM
"output" neurons and reduce amygdala output. The BLA-CeL-CeM is a
less-characterized pathway suggested to be involved not in fear
extinction but in conditioned inhibition. The suppression of fear
expression, possibly due to explicit unpairing of the tone and
shock, suggested to be related to the potentiation of BLA-CeL
synapses.
[0054] BLA cells have promiscuous projections throughout the brain,
including to the bed nucleus of the stria terminalis (BNST),
nucleus accumbens, hippocampus and cortex. Aspects of the present
disclosure relate to methods for selective control of BLA terminals
in the CeL, without little or no direct affect/control of other BLA
projections. Preferential targeting of BLA-CeL synapses can be
facilitated by restricting opsin gene expression to BLA
glutamatergic projection neurons and by restricting light delivery
to the CeA.
[0055] For instance, control of BLA glutamatergic projection
neurons can be achieved with an adeno-associated virus (AAVS)
vector carrying light-activated optogenetic control genes under the
control of a CaMKIIa promoter. Within the BLA, CaMKII.alpha. is
only expressed in glutamatergic pyramidal neurons, not in local
interneurons or intercalated cells.
[0056] FIG. 1 shows a system for providing optogenetic targeting of
specific projections of thebrain, consistent with an embodiment of
the present disclosure. For instance, a beveled guide cannula can
be used to direct light, e.g., prevent light delivery to the BLA
and allow selective illumination of the CeA. This preferential
delivery of light to the CeA projection can be accomplished using
stereotaxic guidance along with implantation over the CeL.
Geometric and functional properties of the resulting light
distribution can be quantified both in vitro and in vivo, e.g.,
using in vivo electrophysiological recordings to determine light
power parameters for selective control of BLA terminals but not BLA
cell bodies. Experimental results, such as those described in the
Examples, support that such selective excitation or inhibition
result in significant, immediate and reversible anxiety-based
effects.
[0057] Embodiments of the present disclosure are directed toward
the above realization being applied to various ones of the
anatomical, functional, structural, and circuit targets identified
herein. For instance, the circuit targets can be studied to develop
treatments for the psychiatric disease of anxiety. These treatments
can include, as non-limiting examples, pharmacological, electrical,
magnetic, surgical and optogenetic, or other treatment means.
[0058] FIG. 2 shows a flow diagram for use of an anxiety-based
circuit model, consistent with an embodiment of the present
disclosure. An optogenetic delivery device, such as a. viral
delivery device, is generated 202. This delivery device can be
configured to introduce optically responsive opsins to the target
cells and may include targeted promoters for specific cell types.
The delivery device can then be stereotaxically (or otherwise)
injected 204 into the BLA. A light delivery device can then be
surgical implanted 206. This light delivery device can be
configured to provide targeted illumination (e.g., using a
directional optical element). The target area is then illuminated
208. The target area can be, for example, the BLA-CeA. The effects
thereof can then be monitored and/or assessed 210. This can also be
used in connection with treatments or drug screening.
[0059] Various embodiments of the present disclosure relate to the
use of the identified model for screening new treatments for
anxiety. For instance, anxiety can be artificially induced or
repressed using the methods discussed herein, while
pharmacological, electrical, magnetic, surgical, or optogenetic
treatments are then applied and assessed. In other embodiments of
the present disclosure, the model can be used to develop an in
vitro approximation or simulation of the identified circuit, which
can then be used in the screening of devices, reagents, tools,
technologies, methods and approaches and for studying and probing
anxiety and related disorders. This study can be directed towards,
but not necessarily limited to, identifying phenotypes,
endophenotypes, and treatment targets.
[0060] Embodiments of the present disclosure are directed toward
modeling the BLA-CeL pathway as an endogenous neural substrate for
bidirectionally modulating the unconditioned expression of anxiety.
Certain embodiments are directed toward other downstream circuits,
such as CeA projections to the BNST, for their role in the
expression of anxiety or anxiety-related behaviors. For instance,
it is believed that corticotropin releasing hormone (CRH) networks
in the BNST may be critically involved in modulating
anxiety-related behaviors, as the CeL is a primary source of CRH
for the BNST. Other neurotransmitters and neuromodulators may
modulate or gate effects on distributed neural circuits, including
serotonin, dopamine, acetylcholine, glycine, GABA and CRH. Still
other embodiments are directed toward control of the neural
circuitry converging to and diverging from this pathway, as
parallel or downstream circuits of the BLA-CeL synapse are believed
to contribute to the modulation or expression of anxiety
phenotypes. Moreover, upstream of the amygdala, this microcircuit
is well-positioned to be recruited by top-down cortical control
from regions important for processing fear and anxiety, including
the prelimbic, infralimbic and insular cortices that provide robust
innervation to the BLA and CeL.
[0061] Experimental results based upon the BLA anatomy suggest that
the populations of BLA neurons projecting to CeL and CeM neurons
are largely non-overlapping. In natural states, the CeL-projecting
BLA neurons may excite CeM-projecting BLA neurons in a microcircuit
homeostatic mechanism, which can then be used to study underlying
anxiety disorders when there are synaptic changes that skew the
balance of the circuit to allow uninhibited CeM activation.
[0062] The embodiments and specific applications discussed herein
(including the Examples) may be implemented in connection with one
or more of the above-described aspects, embodiments and
implementations, as well as with those shown in the figures and
described below. Reference may be made to the following Example,
which is fully incorporated herein by reference. For further
details on light-responsive molecules and/or opsins, including
methodology, devices and substances, reference may also be made to
the following background publications: U.S. Patent Publication No.
2010/0190229, entitled "System for Optical Stimulation of Target
Cells" to Zhang et al.; U.S. Patent Publication No. 2010/0145418,
also entitled "System for Optical Stimulation of Target Cells" to
Zhang et al.; U.S. Patent Publication No. 2007/0261127, entitled
"System for Optical Stimulation of Target Cells" to Boyden et al.;
and PCT WO 2011/116238, Entitled "Light Sensitive Ion Passing
Molecules". These applications form part of the patent document and
are fully incorporated herein by reference. Consistent with these
publications, numerous opsins can be used in mammalian cells in
vivo and in vitro to provide optical stimulation and control of
target cells. For example, when ChR2 is introduced into an
electrically-excitable cell, such as a neuron, light activation of
the ChR2 channelrhodopsin can result in excitation and/or firing of
the cell. In instances when NpHR is introduced into an
electrically-excitable cell, such as a neuron, light activation of
the NpHR opsin can result in inhibition of firing of the cell.
These and other aspects of the disclosures of the above-referenced
patent applications may be useful in implementing various aspects
of the present disclosure.
[0063] While the present disclosure is amenable to various
modifications and alternative forms, specifics thereof have been
shown by way of example in the drawings and will be described in
further detail. It should be understood that the intention is not
to limit the disclosure to the particular embodiments and/or
applications described. On the contrary, the intention is to cover
all modifications, equivalents, and alternatives falling within the
spirit and scope of the present disclosure.
EXAMPLES
[0064] Introduction
[0065] Anxiety is a sustained state of heightened apprehension in
the absence of immediate threat, which in disease states becomes
severely debilitating'. Anxiety disorders represent the most common
of the psychiatric diseases (with 28% lifetime prevalence).sup.2,
and have been linked to the etiology of major depression and
substance abuse.sup.3-5. While the amygdala, a brain region
important for emotional processing.sup.9-17, has long been
hypothesized to play a role in anxiety.sup.18-23, the neural
mechanisms which control and mediate anxiety have yet to be
identified. Here, we combine cell type-specific optogenetic tools
with two-photon microscopy, electrophysiology, and anxiety assays
in freely-moving mice to identify neural circuits underlying
anxiety-related behaviors. Capitalizing on the unique capability of
optogenetics.sup.24-26 to control not only cell types, but also
specific connections between cells, we observed that
temporally-precise optogenetic stimulation of basolateral amygdala
(BLA) terminals in the central nucleus of the amygdala (CeA),
resolved by viral transduction of BLA with ChR2 followed by
restricted illumination in downstream CeA, exerted a profound,
immediate, and reversible anxiolytic effect. Conversely, selective
optogenetic inhibition of the same defined projection with
eNpHR3.0.sup.25 potently, swiftly, and reversibly increased
anxiety-related behaviors. Importantly, these effects were not
observed with direct optogenetic control of BLA somata themselves.
Together, these results implicate specific BLA-CeA projections as
circuit elements both necessary and sufficient for endogenous
anxiety control in the mammalian brain, and demonstrate the
importance of optogenetically targeting specific projections,
rather than cell types, in the study of neural circuit function
relevant to psychiatric disease.
[0066] Despite the high prevalence and severity.sup.1 of anxiety
disorders, the corresponding neural circuit substrates are poorly
understood, impeding the development of safe and effective
treatments. Available treatments tend to be inconsistently
effective or, in the case of benzodiazepines, addictive and linked
to significant side effects including sedation and respiratory
suppression that can cause cognitive impairment and death.sup.27,
28. A deeper understanding of anxiety control mechanisms in the
mammalian brain.sup.29, 30 is necessary to develop more efficient
treatments that have fewer side-effects. Of particular interest and
novelty would be the possibility of recruiting native pathways for
anxiolysis.
[0067] The amygdala is critically involved in processing
associations between neutral stimuli and positive or negative
outcomes, and has also been implicated in processing unconditioned
emotional states. While the amygdala microcircuit has been
functionally dissected in the context of fear conditioning,
amygdalar involvement has been implicated in a multitude of other
functions and emotional states, including unconditioned anxiety.
The amygdala is composed of functionally and morphologically
heterogeneous subnuclei with complex interconnectivity. A primary
subdivision of the amygdala is the basolateral amygdala complex
(BLA), which encompasses the lateral (LA), basolateral (BL) and
basomedial (BM) amygdala nuclei (.about.90% of BLA neurons are
glutamatergic).sup.33, 34. In contrast, the central nucleus of the
amygdala (CeA), which is composed of the centrolateral (CeL) and
centromedial (CeM) nuclei, is predominantly (.about.95%) comprised
of GABAergic medium spiny neurons.sup.35. The BLA is ensheathed in
dense clusters of GABAergic intercalated cells (ITCs), which are
functionally distinct from both local interneurons and the medium
spiny neurons of the CeA.sup.36, 37. The primary output nucleus of
the amygdala is the CeM,.sup.32, 35, 38-40 which when chemically or
electrically excited mediates autonomic and behavioral responses
associated with fear and anxiety via projections to the brainstem6,
12, 32, 35. While the CeM is not directly controlled by the primary
amygdala site of converging environmental and cognitive information
(LA).sup.12, 38, 41, LA and BLA neurons excite GABAergic CeL
neurons.sup.42 which can provide feed-forward inhibition onto
CeM.sup.40, 46 "output" neurons and reduce amygdala output. The
BLA-CeL-CeM is a less-characterized pathway suggested to be
involved not in fear extinction but in conditioned inhibition, the
suppression of fear expression due to explicit unpairing of the
tone and shock, due to the potentiation of BLA-CeL synapses.sup.47.
Although fear is characterized to be a phasic state triggered by an
external cue, while anxiety is a sustained state that may occur in
the absence of an external trigger, we wondered if circuits
modulating conditioned inhibition of fear might also be involved in
modulating unconditioned inhibition of anxiety.
[0068] Materials and Methods
[0069] Subjects: Male C57BL/6 mice, aged 4-6 weeks at the start of
experimental procedures, were maintained with a reverse 12-hr
light/dark cycle and given food and water ad libitum. Animals shown
in FIGS. 3A-3K, 4A-4F and 5A-5J (mice in the ChR2 Terminals, EYFP
Terminals and ChR2 Cell Bodies groups) were all single-housed in a
typical high-traffic mouse facility to increase baseline anxiety
levels. Each mouse belonged to a single treatment group. Animals
shown in FIG. 6A-6P (Bilateral EYFP and eNpHR 3.0 groups) were
group-housed in a special low-traffic facility to decrease baseline
anxiety levels. Animal husbandry and all aspects of experimental
manipulation of our animals were in accordance with the guidelines
from the National Institute of Health and have been approved by
members of the Stanford Institutional Animal Care and Use
Committee.
[0070] Optical Intensity Measurements: Light transmission
measurements were conducted with blocks of brain tissue from
acutely sacrificed mice. The tissue was then placed over the
photodetector of a power meter (ThorLabs, Newton, N.J.) to measure
the light power of the laser penetrated the tissue. The tip of a
300 .mu.m diameter optical fiber was coupled to a 473 nm blue laser
(OEM Laser Systems, East Lansing, Mich.). To characterize the light
transmission to the opposite side of the bevel, the photodetector
of the power meter was placed parallel to the beveled cannula. For
visualization of the light cone, we used Fluorescein
isothiocyanate-dextran (FD150s; Sigma, Saint Louis, Mo.) at
approximately 5 mg/ml placed in a cuvette with the optical fibers
either with or without beveled cannula shielding aimed
perpendicularly over the fluorescein solution. Power density at
specific depths were calculated considering both fractional
decrease in intensity due to the conical output of light from the
optical fiber and the loss of light due to scattering in tissue
(Aravanis et al., J Neural Eng, 4:S143-156, 2007) (Gradinaru et
al., Neurosci, 27:14231-14238, 2007). The half-angle of divergence
.theta..sub.div for a multimode optical fiber, which determines the
angular spread of the output light, is
.theta. div = sin - 1 ( NA fib n tiz ) ##EQU00001##
where n.sub.tis is the index of refraction of gray matter (1.36,
Vo-Dinh T 2003, Biomedical Photonics Handbook (Boca Raton, Fla.:
CRC Press)) and NA.sub.fib(0.37) is the numerical aperture of the
optical fiber. The fractional change in intensity due to the
conical spread of the light with distance (z) from the fiber end
was calculated using trigonometry
I ( z ) I ( Z = 0 ) = .rho. 2 ( z + .rho. ) 2 , where .rho. = r ( n
NA ) 2 - 1 ##EQU00002##
and r is the radius of the optical fiber (100 pm).
[0071] The fractional transmission of light after loss due to
scattering was modeled as a hyperbolic function using empirical
measurements and the Kubelka-Munk model .sup.1, 2, and the combined
product of the power density at the tip of the fiber and the
fractional changes due to the conical spread and light scattering,
produces the value of the power density at a specific depth below
the fiber.
[0072] Virus construction and packaging: The recombinant AAV
vectors were serotyped with AAV5 coat proteins and packaged by the
viral vector core at the University of North Carolina. Viral titers
were 2.times.10e.sup.12 particles/mL, 3.times.10e.sup.12
particles/mL, 4.times.10e.sup.12 particles/mL respectively for
AAV-CaMKII.alpha.-hChR2(H134R)-EYFP, AAV-CaMKII.alpha.-EYFP, and
AAV-CaMKII.alpha.-eNpHR 3.0-EYFP. The
pAAV-CaMKII.alpha.-eNpHR3.0-EYFP plasmid was constructed by cloning
CaMKII.alpha.-eNpHR3.0-EYFP into an AAV backbone using MluI and
EcoRI restriction sites. Similarly, The pAAV-CaMKII.alpha.-EYFP
plasmid was constructed by cloning CaMKII.alpha.-EYFP into an AAV
backbone using MluI and EcoRI restriction sites. The maps are
available online at www.optogenetics.org, which are incorporated
herein by reference.
[0073] Stereotactic injection and optical fiber placement: All
surgeries were performed under aseptic conditions under stereotaxic
guidance. Mice were anaesthetized using 1.5-3.0% isoflourane. All
coordinates are relative to bregma in mm.sup.3. In all experiments,
both in vivo and in vitro, virus was delivered to the BLA only, and
any viral expression in the CeA rendered exclusion from all
experiments. Cannula guides were beveled to form a 45-55 degree
angle for the restriction of the illumination to the CeA. The short
side of the beveled cannula guide was placed antero-medially, the
long side of the beveled cannula shielded the posterior-lateral
portion of the light cone, facing the opposite direction of the
viral injection needle. To preferentially target BLA-CeL synapses,
we restricted opsin gene expression to BLA glutamatergic projection
neurons and restricted light delivery to the CeA. Control of BLA
glutamatergic projection neurons was achieved using an
adeno-associated virus (AAV5) vector carrying light-activated
optogenetic control genes under the control of a CaMKII.alpha.
promoter. Within the BLA, CaMKII.alpha. is only expressed in
glutamatergic pyramidal neurons, not in local interneurons.sup.4.
Mice in the ChR2 Terminals and EYFP Terminals groups received
unilateral implantations of beveled cannulae for the optical fiber
(counter-balanced for hemisphere), while mice in the eNpHR 3.0 or
respective EYFP group received bilateral implantations of the
beveled cannulae over the CeA (-1.06 mm anteroposterior (AP);
.+-.2.25 mm mediolateral (ML); and -4.4 mm dorsoventral (DV);
PlasticsOne, Roanoke, Va.).sup.3. Mice in the ChR2 Cell Bodies
groups received unilateral implantation of a Doric patchcord
chronically implantable fiber (NA=0.22; Doric lenses, Quebec,
Canada) over the BLA at (-1.6 mm AP; .+-.3.1 mm ML; -4.5 mm
DV).sup.3. For all mice, 0.5 .mu.l of purified AAV.sub.5 was
injected unilaterally or bilaterally in the BLA (.+-.3.1 mm AP, 1.6
mm ML, -4.9 mm DV).sup.3 using beveled 33 or 35 gauge metal needle
facing postero-lateral side to restrict the viral infusion to the
BLA. 10 .mu.l Hamilton microsyringe (nanofil; WPI, Sarasota, Fla.)
were used to deliver concentrated AAV solution using a microsyringe
pump (UMP3; WPI, Sarasota, Fla.) and its controller (Micro4; WPI,
Sarasota, Fla.). Then, 0.5 .mu.l of virus solution was injected at
each site at a rate of 0.1 .mu.l per min. After injection
completion, the needle was lifted 0.1 mm and stayed for 10
additional minutes and then slowly withdrawn. One layer of adhesive
cement (C&B metabond; Parkell, Edgewood, N.Y.) followed by
cranioplastic cement (Dental cement; Stoelting, Wood Dale, Ill.)
was used to secure the fiber guide system to the skull. After 20
min, the incision was closed using tissue adhesive (Vetbond;
Fisher, Pittsburgh, Pa.). The animal was kept on a heating pad
until it recovered from anesthesia. A dummy cap (rat: C312G, mouse:
C313G) was inserted to keep the cannula guide patent. Behavioral
and electrophysiological experiments were conducted 4-6 weeks later
to allow for viral expression.
[0074] In vivo recordings: Simultaneous optical stimulation of
central amygdala (CeA) and electrical recording of basolateral
amygdala (BLA) of adult male mice previously (4-6 weeks prior)
transduced in BLA with AAV-CaMKIIa-ChR2-eYFP viral construct was
carried out as described previously (Gradinaru et al., J Neurosci,
27:14231-14238, 2007). Animals were deeply anesthetized with
isoflurane prior to craniotomy and had negative toe pinch. After
aligning mouse stereotaxically and surgically removing
approximately 3 mm.sup.2 skull dorsal to amygdala. Coordinates were
adjusted to allow for developmental growth of the skull and brain,
as mice received surgery when they were 4-6 weeks old and
experiments were performed when the mice were 8-10 weeks old
(centered at -1.5 mm AP, .+-.2.75 mm ML).sup.3, a 1Mohm 0.005-in
extracellular tungsten electrode (A-M systems) was stereotactically
inserted into the craniotomized brain region above the BLA (in mm
-1.65 AP, .+-.3.35 ML, -4.9 DV).sup.3. Separately, a 0.2 N.A. 200
.mu.m core diameter fiber optic cable (Thor Labs) was
stereotactically inserted into the brain dorsal to CeA (-1.1 AP,
.+-.2.25 ML, -4.2 DV).sup.3. After acquiring a light evoked
response, voltage ramps were used to vary light intensity during
stimulation epochs (20 Hz, 5 ms pulse width) 2 s in length. After
acquiring optically evoked signal, the exact position of the fiber
was recorded, the fiber removed from the brain, inserted into a
custom beveled cannula, reinserted to the same position, and the
same protocol was repeated. In most trials, the fiber/cannula was
then extracted from the brain, the cannula removed, and the bare
fiber reinserted to ensure the fidelity of the population of
neurons emitting the evoked signal. Recorded signals were bandpass
filtered between 300 Hz and 20 kHz, AC amplified either 1000.times.
or 10000.times. (A-M Systems 1800), and digitized (Molecular
Devices Digidata 1322A) before being recorded using Clampex
software (Molecular Devices). Clampex software was used for both
recording field signals and controlling a 473 nm (OEM Laser
Systems) solidstate laser diode source coupled to the optrode.
Light power was titrated between <1 mW (.about.14 mW/mm.sup.2)
and 28 mW (.about.396 mW/mm.sup.2) from the fiber tip and measured
using a standard light power meter (ThorLabs). Electrophysiological
recordings were initiated approximately 1 mm dorsal to BLA after
lowering isoflurane anesthesia to a constant level of 1%. Optrode
was lowered ventrally in .about.0.1 mm steps until localization of
optically evoked signal.
[0075] Behavioral assays: All animals used for behavior received
viral transduction of BLA neurons and the implantation enabling
unilateral (for ChR2 groups and controls) or bilateral (for
eNpHR3.0 groups and controls) light delivery. For behavior,
multimode optical fibers (NA 0.37; 300 pm core, BFL37-300;
ThorLabs, Newton, N.J.) were precisely cut to the optimal length
for restricting the light to the CeA, which was shorter than the
long edge of the beveled cannula, but longer than the shortest edge
of the beveled cannula. For optical stimulation, the fiber was
connected to a 473 nm or 594 nm laser diode (OEM Laser Systems,
East Lansing, Mich.) through an FC/PC adapter. Laser output was
controlled using a Master-8 pulse stimulator (A.M.P.I., Jerusalem,
Israel) to deliver light trains at 20 Hz, 5 ms pulse-width for 473
nm light, and constant light for 594 nm light experiments. All
included animals had the center of the viral injection located in
the BLA, though there was sometimes leak to neighboring regions or
along the needle tract. Any case in which there was any detectable
viral expression in the CeA, the animals were excluded. All
statistically significant effects of light were discussed, and
undiscussed comparisons did not show detectable differences.
[0076] 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) at 90 degrees in the form of a plus. The
maze was placed 30 cm above the floor. Mice were individually
placed in the center. 1-5 minutes were allowed for recovery from
handling before the session was initiated. Video tracking software
(BiObserve, Fort Lee, N.J.) was used to track mouse location,
velocity and movement of head, body and tail. All measurements
displayed were relative to the mouse body. Light stimulation
protocols are specified by group. ChR2:BLA-CeA mice and
corresponding controls groups (EYFP:BLA-CeA and ChR2:BLA Somata)
were singly-housed in a high-stress environment for at least 1 week
prior to anxiety assays: unilateral illumination of BLA terminals
in the CeA at 7-8 mW (.about.106 mW/mm.sup.2 at the tip of the
fiber, .about.6.3 mW/mm2 at CeL and .about.2.4 mW/mm.sup.2 at the
CeM) of 473 nm light pulse trains (5 ms pulses at 20 Hz). For the
ChR2 Cell Bodies group BLA neurons were directly illuminated with a
lower light power because illumination with 7-8 mW induced seizure
activity, so we unilaterally illuminated BLA neurons at 3-5 mW
(.about.57 mW/mm.sup.2) of 473 nm light pulse trains (5 ms pulses
at 20 Hz). For the eNpHR 3.0 and corresponding EYFP group, all mice
were group-housed and received bilateral viral injections and
bilateral illumination of BLA terminals in the CeA at 4-6 mW
(.about.71 mW/mm.sup.2 at the tip of the fiber, .about.4.7
mW/mm.sup.2 at the CeL and .about.1.9 mW/mm.sup.2 at the CeM) of
594 nm light with constant illumination throughout the 5-min light
on epoch. The 15-min session was divided into 3 5-min epochs, the
first epoch there was no light stimulation (off), the second epoch
light was delivered as specified above (on), and the third epoch
there was no light stimulation (off).
[0077] The open-field chamber (50.times.50 cm) and the open field
was divided into a central field (center, 23 x 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 by using the same
video-tracking software, Viewer.sup.2 (BiObserve, Fort Lee, N.J.).
The open field assessment was made immediately after the
elevated-plus maze test. The open field test consisted of an 18-min
session in which there were six 3-min epochs. The epochs alternated
between no light and light stimulation periods, beginning with a
light off epoch. For all analyses and charts where only "off" and
"on" conditions are displayed, the 3 "off" epochs were pooled and
the 3 "on" epochs were pooled.
[0078] For the glutamate receptor antagonist manipulation, a
glutamate antagonist solution consisting of 22.0 mM of NBQX and
38.0 mM of D-APV (Tocris, Ellisville, Mo.) dissolved in saline
(0.9% NaCl). 5-15 min before the anxiety assays, 0.3 .mu.l of the
glutamate antagonist solution was infused into the CeA via an
internal infusion needle, inserted into the same guide cannulae
used for light delivery via optical fiber, that was connected to a
10-.mu.l Hamilton syringe (nanofil; WPI, Sarasota, Fla.). The flow
rate (0.1 .mu.l per min) was regulated by a syringe pump (Harvard
Apparatus, Mass.). Placements of the viral injection, guide cannula
and chronically-implanted fiber were histologically verified as
indicated in FIGS. 7 and 10A-10F.
[0079] Two-photon optogenetic circuit mapping and ex vivo
electrophysiological recording: Mice were injected with
AAV5-CaMKII.alpha.-ChR2-EYFP at 4 weeks of age, and were sacrificed
for acute slice preparation 4-6 weeks to allow for viral
expression. Coronal slices containing the BLA and CeA were prepared
to examine the functional connectivity between the BLA and the CeA.
Two-photon images and electrophysiological recordings were made
under the constant perfusion of aCSF, which contained (in mM): 126
NaCl, 26 NaHCO.sub.3, 2.5 KCl, 1.25 NaH.sub.2PO.sub.4, 1
MgCl.sub.2, 2 CaCl.sub.2, and 10 glucose. All recordings were at
32.degree. C. Patch electrodes (4-6 MOhms) were filled (in min): 10
HEPES, 4 Mg-ATP, 0.5 MgCl.sub.2, 0.4 Na.sub.3-GTP, 10 NaCl, 140
potassium gluconate, and 80 Alexa-Fluor 594 hydrazide (Molecular
Probes, Eugene Oreg.). Whole-cell patch-clamp recordings were
performed in BLA, CeL and CeM neurons, and cells were allowed to
fill for approximately 30 minutes before imaging on a modified
two-photon microscope (Prairie Microscopes, Madison Wis.) where
two-photon imaging, whole-cell recording and optogenetic
stimulation could be done simultaneously. Series resistance of the
pipettes was usually 10-20 MOhms. Blue light pulses were elicited
using a 473 nm LED at .about.7 mW/mm.sup.2 (Thorlabs, Newton N.J.)
unless otherwise noted. A Coherent Ti-Saphire laser was used to
image both ChR2-YFP (940 nm) and Alexa-Fluor 594 (800 nm). A FF560
dichroic with filters 630/69 and 542/27 (Semrock, Rochester N.Y.)
was also used to separate both molecules' emission. All images were
taken using a 40X/.8 NA LUMPlanFL/IR Objective (Olympus, Center
Valley Pa.). In order to isolate fibers projecting to CeL from the
BLA and examine responses in the CeM, slices were prepared as
described above with the BLA excluded from illumination. Whole-cell
recordings were performed in the CeM with illumination from the
objective aimed over the CeL. To further ensure activation of
terminals from the BLA to CeL was selective, illumination was
restricted to a .about.125 .mu.m diameter around the center of the
CeL. Here, blue light pulses were elicited using an XCite halogen
light source (EXPO, Mississauga, Ontario) with a 470/3 filter at
6.5 mW/mm.sup.2 coupled to a shutter (Uniblitz, Rochester N.Y.).
For functional mapping, we first recorded from a BLA neuron
expressing ChR2 and simultaneously collected electrophysiological
recordings and filled the cell with Alexa-Fluor 594 hydrazide dye
to allow for two-photon imaging. Two-photon z-stacks were collected
at multiple locations along the axon of the filled BLA neuron. We
then followed the axon of the BLA neuron projecting to the CeL
nucleus and recorded from a CeL neuron in the BLA terminal field.
We then simultaneously recorded from a CeL neuron, filled the cell
with dye and performed two-photon live imaging before following the
CeL neuronal axons to the CeM. We then repeated this procedure in a
CeM neuron, but moved the light back to the terminal field in the
CeL to mimic the preferential illumination of BLA-CeL synapses with
the same stimulation parameters as performed in vivo. Voltage-clamp
recordings were made at both -70 mV, to isolate EPSCs, and at 0 mV,
to isolate IPSCs. EPSCs were confirmed to be EPSCs via bath
application of the glutamate receptor antagonists (n=5), NBQX (22
.mu.M) and AP5 (38 .mu.M), IPSCs were confirmed to be IPSCs via
bath application of bicuculline (10 .mu.M; n=2), which abolished
them, respectively. We also performed current-clamp recordings when
the cell was resting at approximately -70 mV.
[0080] For the characterization of optogenetically-driven
antidromic stimulation in BLA axon terminals, animals were injected
with AAV5-CaMKII.alpha.-ChR2-EYFP at 4 weeks of age, and were
sacrificed for acute slice preparation 4-6 weeks to allow for viral
expression. Slice preparation was the same as above. To the aCSF we
added 0.1 mM picrotoxin, 10 .mu.M CNQX and 25 .mu.M AP5 (Sigma, St.
Louis, Mo.). Whole-cell patch-clamp recordings were performed in
BLA neurons and were allowed to fill for approximately 30 minutes
before two-photon imaging. Series resistance of the pipettes was
usually 10-20 MOhms. All images were taken using a 40X/.8 NA
LUMPlanFL/IR Objective (Olympus, Center Valley Pa.). Blue light
pulses were elicited using an XCite halogen light source (EXPO,
Mississauga, Ontario) with a 470/30 filter at 6.5 mW/mm.sup.2
coupled to a shutter (Uniblitz, Rochester N.Y.). Two-photon
z-stacks were collected at multiple locations along the axon of the
filled BLA neuron. Only neurons whose axons could be visualized for
over .about.300 .mu.m diameter towards the CeL nucleus were
included for the experiment, and neurons that had processes going
in all directions were also excluded. Stimulation on/off axon was
accomplished by moving the slice relative to a .about.125 .mu.m
diameter blue light spot. In order to calibrate the slice for
correct expression, whole-cell patch-clamp was performed on a CeL
cell and a .about.125 .mu.m diameter spot blue pulse was used to
ensure that synaptic release from the BLA terminals on to the CeL
neuron was reliable.
[0081] For the dissection of direct and indirect projections to
CeM, animals were injected with AAV.sub.5-CaMKII.alpha.-ChR2-EYFP
at 4 weeks of age, and were sacrificed for acute slice preparation
4-6 weeks to allow for viral expression. Slice preparation was the
same as above. Light was delivered through a 40X/.8 NA LUMPlanFL/IR
Objective (Olympus, Center Valley Pa.). Prior to whole cell patch
clamping in the CeM nucleus, the location of the CeL nucleus was
noted in order to revisit it with the light spot restricted to this
region. Whole-cell patch-clamp recordings were performed in CeM
neurons. Series resistance of the pipettes was usually 10-20 MOhms.
Blue light pulses were elicited using a XCite halogen light source
(EXPO, Mississauga, Ontario) with a 470/30 filter at 6.5
mW/mm.sup.2 coupled to a shutter (Uniblitz, Rochester N.Y.). During
CeM recordings, broad illumination (.about.425-450 .mu.m in
diameter) of BLA terminals in the CeA and 20 Hz, 5 ms light train
for 2 s was applied. Voltage-clamp recordings were made at 70 mV
and 0 mV to isolate EPSCs and IPSCs respectively. Current-clamp
recordings were also made. Then, illumination was moved to the CeL
using a restricted light spot .about.125 .mu.m in diameter. We
again performed voltage clamp recordings at -70 mV and 0 mV and
used 20 Hz, 5 ms light train for 2 s. For the CeM neuron spiking
inhibition experiments, in current-clamp, we applied the minimal
current step required to induce spiking (.about.60 pA) and
simultaneously applied preferential illumination of ChR2-expressing
BLA terminals in the CeL with a 20 Hz, 5 ms light train for 2s
(mean over 6 sweeps per cell). For the experiments comparing the
broad illumination of the BLA terminal field centered in the CeM to
selective illumination of BLA-CeL terminals, these conditions were
performed in repeated alternation in the same CeM cells (n=7).
[0082] To verify that terminal inhibition did not alter somatic
spiking, animals were injected with
AAV5-CaMKII.alpha.-eNpHR3.0-EYFP at 4 weeks of age, and were
sacrificed for acute slice preparation 4-6 weeks to allow for viral
expression. Slice preparation was the same as above. Whole-cell
patch-clamp recordings were performed in BLA neurons and were
allowed to fill for approximately 30 minutes. Light was delivered
through a 40X/.8 NA LUMPlanFL/IR Objective (Olympus, Center Valley
Pa.). Whole-cell patch-clamp recordings were performed on BLA
neurons. Series resistance of the pipettes was usually 10-20 MOhms.
Yellow light pulses were elicited using a XCite halogen light
source (EXPO, Mississauga, Ontario) with a 589/24 filter at 6.5
mW/mm.sup.2 coupled to a shutter (Uniblitz, Rochester N.Y.). After
patching, an unrestricted light spot (.about.425-450 microns in
diameter) was placed over the BLA soma and a is pulse was applied.
Cells were excluded if the current recorded was under 600 pA of
hyperpolarizing current and the axon did not travel over .about.300
.mu.m towards the CeL nucleus. The light spot was then restricted
to .about.125 pm in diameter. On and off axon voltage clamp
recordings were taken with a is pulse of light. For the current
clamp recordings, action potentials were generated by applying 250
pA of current to the cell soma through the patch pipette.
[0083] To demonstrate that selective illumination of
eNpHR3.0-expressing BLA terminals reduced the probability of
spontaneous vesicle release, animals were injected with
AAV5-CaMKII.alpha.-eNpHR3.0-EYFP at 4 weeks of age, and were
sacrificed for acute slice preparation 4-6 weeks to allow for viral
expression. Slice preparation was the same as above. Whole-cell
patch-clamp recordings were performed in central lateral neurons.
Light was delivered through a 40X/.8 NA LUMPlanFL/IR Objective
(Olympus, Center Valley Pa.). Series resistance of the pipettes was
usually 10-20 MOhms. Yellow light pulses were elicited using a
XCite halogen light source (EXPO, Mississauga, Ontario) with a
589/24 filter at 6.5 mW/mm.sup.2 coupled to a shutter (Uniblitz,
Rochester N.Y.). The light spot was restricted to .about.125 .mu.m
in diameter. Carbachol was added to the bath at a concentration of
20 .about.M. After sEPSC activity increased in the CeL neuron,
light pulses were applied ranging in times from 5s to 30 s.
[0084] To demonstrate that selective illumination of
eNpHR3.0-expressing BLA terminals could reduce the probability of
vesicle release evoked by electrical stimulation, animals were
injected with AAVS-CaMKII.alpha.-eNpHR3.0-EYFP at 4 weeks of age,
and were sacrificed for acute slice preparation 4-6 weeks to allow
for viral expression. Slice preparation was the same as above. A
bipolar concentric stimulation probe (FHC, Bowdoin Me.) was placed
in the BLA. Whole-cell patch-clamp recordings were performed in CeL
neurons. Light was delivered through a 40X/.8 NA LUMPlanFL/IR
Objective (Olympus, Center Valley Pa.). Series resistance of the
pipettes was usually 10-20 MOhms. Amber light pulses over the
central lateral cell were elicited using a XCite halogen light
source (EXPO, Mississauga, Ontario) with a 589/24 filter at 6.5
mW/mm.sup.2 coupled to a shutter (Uniblitz, Rochester N.Y.). The
light spot was restricted to .about.125 .mu.m in diameter.
Electrical pulses were delivered for 40 seconds and light was
delivered starting at 10 seconds and shut off at 30 seconds in the
middle.
[0085] For the anatomical tracing experiments, neurons were
excluded when the traced axons were observed to be severed and all
BLA neurons included in the anatomical assay (FIGS. 5A-5I) showed
spiking patterns typical of BLA pyramidal neurons.sup.18 upon a
current step.
[0086] Slice immunohistochemistry: Anesthetized mice were
transcardially perfused with ice-cold 4% paraformaldehyde (PFA) in
PBS (pH 7.4) 100-110 min after termination of in vivo light
stimulation. Brains were fixed overnight in 4% PFA and then
equilibrated in 30% sucrose in PBS. 40 .mu.m-thick coronal sections
were cut on a freezing microtome and stored in cryoprotectant at
4.degree. C. until processed for immunohistochemistry.
Free-floating sections were washed in PBS and then incubated for 30
min in 0.3% Tx100 and 3% normal donkey serum (NDS). Primary
antibody incubations were performed overnight at 4.degree. C. in 3%
NDS/PBS (rabbit anti-c-fos 1:500, Calbiochem, La Jolla, Calif.;
mouse anti-CaMKII 1:500, Abcam, Cambridge, Mass.). Sections were
then washed and incubated with secondary antibodies (1:1000)
conjugated to Cy3 or Cy5 (Jackson Laboratories, West Grove, Pa.)
for 3 hrs at room temperature. Following a 20 min incubation with
DAPI (1:50,000) sections were washed and mounted on microscope
slides with PVD-DABCO.
[0087] Confocal microscopy and analysis: Confocal fluorescence
images were acquired on a Leica TCS SP5 scanning laser microscope
using a 20X/0.70NA or a 40X/1.25NA oil immersion objective. Serial
stack images covering a depth of 10 .mu.m through multiple sections
were acquired using equivalent settings. The Volocity image
analysis software (Improvision/PerkinElmer, Waltham, Mass.)
calculated the number of c-fos positive cells per field by
thresholding c-fos immunoreactivity above background levels and
using the DAPI staining to delineate nuclei. All imaging and
analysis was performed blind to the experimental conditions.
[0088] Statistics: For behavioral experiments and the ex vivo
electrophysiology data, binary comparisons were tested using
nonparametric bootstrapped t-tests (paired or unpaired where
appropriate).sup.5, while hypotheses involving more than two group
means were tested using linear contrasts (using the "boot" and
"lme4" packages in R.sup.6, respectively); the latter were
formulated as contrasts between coefficients of a linear
mixed-effects model (a "two-way repeated-measures ANOVA") with the
fixed effects being the genetic or pharmacological manipulation and
the light treatment (on or off). All hypothesis tests were
specified a priori. Subjects were modeled as a random effects. For
c-fos quantification comparisons, we used a one-way ANOVA followed
by Tukey's multiple comparisons test.
[0089] Plots of the data clearly show a relationship between
observation mean and observation variance (that is, they are
heteroskedastic; see for example, FIG. 3E and FIG. 5J). We found
that a standard square-root transformation corrected this well.
Additionally, eNpHR3.0 elevated plus maze (EPM) data required
detrending by a linear fit over time to account for a decrease in
exploration behavior over time. As is standard for a two-way linear
mixed effects model (also known as a two-way repeated-measures
ANOVA), we model (the square-root corrected value of) the kth
observation in the ijth cell (y.sub.ijk) as
{square root over
(y.sub.ijk)}=.mu.+c.sub.j+t.sub.j+(c:t).sub.ij+b.sub.j+c.sub.ijk
(1)
[0090] where [0091] .mu. is the grand mean across all cells (where
the ijth "cell" in the collection of observations corresponding to
the ith condition and jth treatment) [0092] c.sub.i is a fixed
effect due to the ith animal condition across treatments (for
example, a genetic manipulation) [0093] t.sub.j is a fixed effect
due to the jth treatment across conditions (for example, light on
or light off) [0094] (c : t).sub.ij is a fixed effect due to the
interaction of the ith condition and jth treatment in the ijth cell
[0095] b.sub.j is a random effect corresponding to animals being
used across treatments, and [0096] e.sub.ijk is an independent and
identically distributed (i.i.d.) random normal disturbance in the
ijkth observation with mean 0 and variance .sigma..sup.2, and
independent of b.sub.j for all j
[0097] Collecting the fixed effects into a 2-way analysis of
variance (ANOVA) design matrix X .di-elect cons.R.sup.nxp, dummy
coding the random effects in a sparse matrix Z .di-elect
cons.R.sup.nxq, and letting .mu.= {square root over (u)} we can
express the model in matrix form as
where ij .di-elect cons. , b .di-elect cons. .sup.q, and
.epsilon..di-elect cons. are observations of random y, and
.epsilon. respectively and our model assumes
.about.(0, .sigma..sup.2.SIGMA.)
.about.(0, .sigma..sup.2I), .di-elect cons..perp.
(y)|=b) .about.(X .beta.+Zb, .sigma..sup.2I)
where N (.mu., .SIGMA.) denotes the multivariate Gaussian
distribution with mean vector .mu. and variance-covariance matrix
.SIGMA., and .perp. indicates that two variables are independent.
To estimate the coefficient vectors .beta. .di-elect cons. R.sup.p,
b .di-elect cons. R.sup.q, and the variance parameter .sigma. and
sparse (block-diagonal) relative variance-covariance matrix
.SIGMA..di-elect cons. R.sup.qxq, we use the lme4 package in R
written by Douglas Bates and Martin Maechler, which first finds a
linear change of coordinates that "spheres" the random effects and
then finds the maximum likelihood estimates for .beta., .sigma.,
and .SIGMA. using penalized iteratively reweighted least-squares,
exploiting the sparsity of the random effects matrix to speed
computation. For more details see the documentation accompanying
the package in the lme4 repository at
http://www.r-project.org/.
[0098] To solve for the maximum likelihood estimates, the design
matrix X in equation 2 must be of full column rank. It is well
known that this is not the case for a full factorial design matrix
with an intercept (as in equation 1), and thus linear combinations
("contrasts") must be used to define the columns of X in order for
the fixed-effect coefficients to be estimable. As our designs are
balanced (or nearly balanced), we used orthogonal (or nearly
orthogonal) Helmert contrasts between the coefficients associated
with light on as compared to light off conditions, terminal
stimulation as compared to control conditions, and so on, as
reported in the main text. Such contrasts allowed us to compare
pooled data (e.g., from several sequential light on vs. light off
conditions) against each other within a repeated-measures
design--yielding improved parameter estimation and test power while
accounting for within-animal correlations.
[0099] Results
[0100] BLA cells have promiscuous projections throughout the brain,
including to the bed nucleus of the stria terminalis (BNST),
nucleus accumbens, hippocampus and cortex.sup.38, 43. To test
whether BLA-CeL synapses could be causally involved in anxiety, it
was therefore necessary to develop a method to selectively control
BLA terminals in the CeL, without directly affecting other BLA
projections. To preferentially target BLA-CeL synapses, we
restricted opsin gene expression to BLA glutamatergic projection
neurons and restricted light delivery to the CeA. Control of BLA
glutamatergic projection neurons was achieved with an
adeno-associated virus (AAV5) vector carrying light-activated
optogenetic control genes under the control of a CaMKII.alpha.
promoter; within the BLA, CaMKII.alpha. is only expressed in
glutamatergic pyramidal neurons, not in local interneurons or
intercalated cells.sup.48. To preferentially deliver light to the
CeA projection, virus was delivered unilaterally into the BLA under
stereotaxic guidance (FIGS. 7 and 8A-8E) along with implantation of
a beveled guide cannula over the CeL to prevent light delivery to
the BLA and allow selective illumination of the CeA. Geometric and
functional properties of the resulting light distribution were
quantified both in vitro and in vivo, with in vivo
electrophysiological recordings to determine light power parameters
for selective control of BLA terminals but not BLA cell bodies
(FIGS. 9A-9D).
[0101] To test the hypothesis that the BLA-CeA pathway could
implement an endogenous mechanism for anxiolysis, we probed
freely-moving mice under projection-specific optogenetic control in
two distinct and well-validated anxiety assays: the elevated plus
maze and the open field test (FIGS. 3A-3F). Mice display
anxiety-related behaviors when exposed to open or exposed spaces,
therefore increased time spent in the exposed arms of the elevated
plus maze or in the center of the open field chamber indicates
reduced anxiety.sup.49, 50. To test for both induction and reversal
of relevant behaviors, we first exposed mice to the elevated plus
maze for three 5-min epochs, in which light was delivered during
the second epoch only.
[0102] To determine whether the anxiolytic effect we observed would
be specific to activation of BLA terminals in the CeA, and not BLA
cells in general, we compared mice receiving projection-specific
control (in the ChR2:BLA-CeA group; FIG. 3A to both a negative
control group receiving transduction with a control virus given the
same pattern of illumination (EYFP:BLA-CeA) and a positive control
group transduced with the AAV-CaMKII.alpha.-ChR2-EYFP virus in the
BLA with a fiber implanted directly over the BLA (ChR2:BLA Somata).
For this group (ChR2:BLA Somata), light stimulation did not elicit
the anxiolysis observed in the ChR2:BLA-CeA group (FIG. 3B-3C;
indeed, the ChR2:BLA-CeA group spent significantly more time in
open arms (t(42)=8.312; p<0.00001; FIG. 3b,c) during
light-induced activation of BLA terminals in the CeA , in
comparison to controls (EYFP:BLA-CeA and ChR2:BLA Somata groups).
The ChR2:BLA-CeA mice also showed an increase in the probability of
entering an open arm rather than a closed arm, from the choice
point of the center of the maze (FIG. 3C inset), indicating an
increased probability of selecting the normally anxiogenic
environment.
[0103] We also probed mice on the open field arena for six 3-minute
epochs, again testing for reversibility by alternating between no
light (off) and light stimulation (on) conditions. Experimental
(ChR2:BLA-CeA) mice displayed an immediate, robust, and reversible
light-induced anxiolytic response as measured by the time in center
of the open field chamber (FIG. 3D-3E), while mice in the
EYFP:BLA-CeA and ChR2:BLA Somata groups did not (FIG. 3E). Light
stimulation did not significantly alter locomotor activity (FIG.
3F). While there was no detectable difference among groups in the
off conditions, there was a significant increase in center time of
the open field spent by mice in the ChR2:BLA-CeA group relative to
the EYFP:BLA-CeA or ChR2:BLA Somata groups during the on conditions
(t(105)=4.96178; p<0.0001 for each contrast). We concluded that
selective stimulation of BLA projections to the CeA, but not BLA
somata, produces an acute, rapidly reversible anxiolytic effect,
supporting the hypothesis that the BLA-CeL-CeM pathway could
represent a native microcircuit for anxiety control.
[0104] We next investigated the physiological basis of this
light-induced anxiolytic effect. Glutamatergic neurons in the BLA
send robust excitatory projections to CeL neurons as well as to CeM
neurons.sup.38; however, not only are the CeM synapses distant from
the light source (FIG. 8A-8E), but also any residual direct
excitation of these CeM neurons would be expected to result in an
anxiogenic, rather than an anxiolytic, effect.sup.12. However, CeL
neurons exert strong inhibition onto these brainstem-projecting CeM
output neurons.sup.32, 35, 40, and we therefore hypothesized that
illumination of BLA terminals in the CeA could activate BLA-CeL
neurons and thereby elicit feed-forward inhibition onto CeM neurons
and implement the observed anxiolytic phenomenon.
[0105] To confirm the operation of this optogenetically-defined
projection, we undertook in vivo experiments, with light delivery
protocols matched to those delivered in the behavioral experiments,
and activity-dependent immediate early gene (c-fos) expression
analysis as the readout to verify the pattern of neuronal
activation (FIG. 3G-3K). Under blinded conditions, we quantified
the proportion of neurons in the BLA, CeL and CeM (FIG. 3I-3K) for
ChR2:BLA-CeA, EYFP:BLA-CeA and ChR2:BLA Somata groups that
expressed EYFP or showed c-fos immunoreactivity. Virus expression
under the CaMKII.alpha. promoter in the BLA targeted glutamatergic
neurons.sup.47, and we did not observe EYFP expression in local
interneurons nor intercalated cells (FIG. 10A-10F). No significant
differences among groups were detected in the proportion of
EYFP-positive cells within each region (FIG. 3G-3K), but we found a
significantly higher proportion of c-fos positive BLA cells in the
ChR2:BLA Somata group, relative to ChR2:BLA-CeA or EYFP:BLA-CeA
groups (FIG.3I;p<0.01 and p<0.05, respectively). There was no
detectable difference in c-fos between the ChR2:BLA-CeA and
EYFP:BLA-CeA groups, indicating that the beveled cannula shielding
effectively prevented direct illumination to BLA cell bodies. A
significantly higher proportion of CeL neurons expressed c-fos in
the ChR2:BLA-CeA group relative to the EYFP:BLA-CeA group
(p<0.05), but not the ChR2:BLA Somata group (FIG. 3J). Thus,
selective illumination of BLA terminals expressing ChR2 in the CeA
led to preferential activation of CeL neurons, without activating
BLA somata. In the CeM, we found twice as many c-fos positive
neurons (relative to total neurons) in the ChR2:BLA Somata group
than in the ChR2:BLA-CeA (FIG. 3K), consistent with anatomical
projections, as LA neurons selectively innervate CeL neurons, while
neurons in the BL and BM nuclei of the amygdala have monosynaptic
projections to both the CeL and the CeM.sup.38, 43, 51. Together,
these data reveal that the in vivo illumination that triggers an
acute anxiolytic behavioral phenotype implements selective
illumination of BLA-CeL synapses without activating BLA cell
bodies.
[0106] To test the hypothesis that selective illumination of BLA
terminals in the CeL induces feed-forward inhibition of CeM output
neurons, we combined whole-cell patch-clamp recording with live
two-photon imaging to visualize the microcircuit while
simultaneously probing the functional relationships among these
cells during projection-specific optogenetic control (FIG. 4A-4F).
While the light-stimulation parameters used in vivo were delivered
via a fiber optic and the parameters used in our ex vivo
experiments were delivered onto acute slices, we matched the light
power density at our target location .about.6 mW/mm.sup.2. A
two-photon image of the BLA-CeL-CeM circuit is shown in FIG. 4A,
with all three cells imaged from the same slice (FIG.4A). The BLA
neuron expressing ChR2-EYFP showed robust, high-fidelity spiking to
direct illumination with 20 Hz, 5 ms pulses of 473 nm light (FIG.
4B). A representative trace from a CeL neuron, recorded during
illumination of the terminal field of BLA neurons expressing
ChR2-EYFP, demonstrates the typical excitatory responses seen in
CeL (FIG. 4C), with population summaries revealing that spiking
fidelity was steady throughout the 40-pulse light train and that
responding cells include both weakly and strongly-excited CeL cells
(n=16; FIG. 4C). To test whether illumination of BLA-CeL synapses
would be functionally significant at the level of blocking spiking
in CeM cells due to the robust feed-forward inhibition from CeL
neurons, we recorded from CeM neurons while selectively
illuminating BLA-CeL synapses (FIG. 4D). Indeed, we observed potent
spiking inhibition (F.sub.2,11=15.35, p=0.0044) in the CeM due to
light stimulation of BLA terminals in the CeL (FIG.4d; spikes per
second before (49.+-.9.0), during (1.5.+-.0.87), and after
(33.+-.8.4) illumination; mean.+-.s.e.m). Next, FIG. 4E shows CeM
responses recorded during illumination of the terminal field of BLA
neurons in the CeM expressing ChR2-EYFP, and the combined
excitatory and inhibitory input. Population summaries from
voltage-clamp recordings indicated that latencies of EPSCs were
shorter than those of the disynaptic IPSCs, as expected, and that
the mean IPSC amplitude was greater than mean EPSC amplitude
(recorded at 0 and -70 mV, respectively; FIG. 4E). Importantly, the
very same CeM neurons (n=7) yielded net excitation with broad
illumination of BLA inputs to the CeM (FIG. 4E), but displayed net
inhibition with selective illumination of BLA inputs to the CeL
(FIG. 4F) in a repeatable fashion with alternation between sites.
This demonstrates that the balance of direct and indirect inputs
from the BLA to the CeM can modulate CeM output. Together, these
data reveal a structurally- and functionally-identified
physiological microcircuit, whereby selective illumination of BLA
terminals in the CeA activates BLA-CeL synapses, thus increasing
feed-forward inhibition from CeL neurons onto the
brainstem-projecting CeM neurons.
[0107] To further elucidate the amygdalar microcircuits underlying
this anxiolytic effect, we carefully dissected the anatomical and
functional properties governing this phenomenon. While some efforts
to map the projections of BLA collaterals in the CeA have been made
in the rat, we empirically tested whether overlapping or distinct
populations of BLA neurons projected to the CeL and CeM (FIG.
5A-5B). A noteworthy caveat is that we visualized these neurons in
.about.350 um thick coronal sections and while every attempt was
made to exclude neurons in which the axons were severed, we cannot
exclude the possibility that this occurred nor can we deny that
this induced some sampling bias for BLA neurons closer to the CeA.
FIG. 5A summarizes the anatomical projections of the BLA neurons
sampled (n=18) and shows that the 44% of neurons projected to the
CeL alone and 17% projected to the CeM alone. However, a minority
of BLA cells (n=1;6%), projected to both the CeL and the CeM, one
of which sent separate collaterals to the CeL and CeM and one of
which sent a collateral that sent branches to the CeL and CeM. FIG.
5B shows the 2-photon image of each cell sampled, all of which
showed spiking patterns typical of BLA pyramidal neurons upon a
current step.
[0108] Next, as our c-fos assays suggested that illumination of BLA
terminals in the CeL were sufficient to excite CeL neurons, but not
BLA neurons themselves, we sought to confirm this hypothesis with
whole-cell recordings. With electrical stimulation, depolarization
of axon terminals leads to antidromic spiking at the cell soma.
However, there has been evidence that optogenetically-induced
depolarization functions via a distinct mechanism. To evaluate the
properties of optogenetically-induced terminal stimulation in this
amygdalar microcircuit, we recorded from BLA pyramidal neurons
expressing ChR2 and moved a light spot (.about.120 .mu.m in
diameter) in 100 .mu.m steps from the cell soma, both in a
direction over a visually-identified axon collateral and in a
direction where there was no axon (FIG. 5C). The spike fidelity of
the BLA neuron given a 20 Hz train of light at each distance from
the soma is summarized in FIG. 5D, while the depolarizing current
is summarized in FIG.5e. In all preparations, we confirmed that the
light stimulation parameters used were sufficient to elicit
high-fidelity spiking at the BLA cell soma (FIG.Sf) and reliable
vesicle release at BLA terminals as shown by recordings from a
postsynaptic CeL neuron (FIG. 5G; FIG. 15). In contrast, when
recording from the same BLA neurons with the light spot 300 um away
from the cell soma we did not observe reliable action potential
induction, regardless of whether we were over an axon (FIG. 5H) or
not (FIG. 5I). This absence of antidromic spiking was observed even
upon bath application of GABA and AMPA receptor antagonists (n=7),
thus excluding the possible contribution of local inhibitory
constraints. While we demonstrate that optogenetically-induced
vesicle release can occur in the absence of antidromic stimulation
in BLA pyramidal neurons, it is possible that at antidromic
stimulation could be achieved with greater light power density than
we used here (.about.6 mW/mm.sup.2). Thusfar, we have demonstrated
that the populations of BLA neurons projecting to the CeL and the
CeM are largely distinct and that illumination of BLA-CeL synapses
induces vesicle release and CeL excitation without strong
activation of BLA somata themselves.
[0109] Finally, we further explored the mechanism with in vivo
pharmacological analysis in the setting of projection-specific
optogenetic control. To determine whether the anxiolytic effect we
observed could be due to the selective activation of BLA-CeL
synapses alone, and not BLA fibers passing through the CeA, nor
back-propagation of action potentials to BLA cell bodies which then
would innervate all BLA projection target regions, we tested
whether local glutamate receptor antagonism would attenuate
light-induced anxiolytic effects. This question is of substantial
interest since lesions in the CeA that alter anxiety are confounded
by the likelihood of ablation of BLA projections to the BNST which
pass through CeA.sup.6. We unilaterally transduced BLA neurons with
AAV-CaMKII.alpha.-ChR2-EYFP and implanted beveled cannulae to
implement selective illumination of BLA terminals in the CeA as
before (n=8; FIGS. 8A-8E), and tested mice on the elevated plus
maze and open field test. In this case, however, we infused either
the glutamate antagonists NBQX and AP5 using the optical fiber
guide cannula, or saline control on different trials in the same
animals, with trials counter-balanced for order. Confirming a local
synaptic mechanism rather than control of fibers of passage, for
the same mice and light stimulation parameters, local glutamate
receptor antagonism in the CeA abolished light-induced reductions
in anxiety on both the elevated plus maze (FIG. 5K) and the open
field test (FIG. 5J). Importantly, in control experiments, drug
treatment did not impair locomotor activity (FIG. 11), and in acute
slices time-locked light-evoked excitatory responses were abolished
upon bath application of NBQX and AP5 (FIG. 12A-12C). Together
these data indicate that the light-induced anxiolytic effects we
observed were caused by the activation of BLA-CeL synapses, and not
attributable to BLA projections to distal targets passing through
the CeA.
[0110] In a final series of experiments, to determine if endogenous
anxiety-reducing processes could be blocked by selectively
inhibiting this pathway, we tested whether the selective inhibition
of these optogenetically defined synapses could reversibly increase
anxiety. We performed bilateral viral transductions of either
eNpHR3.0, a light-activated chloride pump which hyperpolarizes
neuronal membranes upon illumination with amber light.sup.25, or
EYFP alone, both under the CaMKIIa promoter in the BLA, and
implanted bilateral beveled guide cannulae to allow selective
illumination of BLA terminals in the CeA (FIG.6a; FIG. 13).
eNpHR3.0 expression was restricted to glutamatergic
CaMKII.alpha.-positive neurons in the BLA (FIG. 6B). The
eNpHR3.0:BLA-CeA group only showed significantly elevated levels of
c-fos expression, relative to the EYFP:BLA-CeA bil and eNpHR
3.0:Soma groups, in the CeM (p<0.05; FIGS.6c-e), consistent with
the hypothesis that selective inhibition of BLA terminals in the
CeA suppresses feed-forward inhibition from CeL neurons to CeM
neurons, thus increasing CeM excitability and the downstream
processes leading to increased anxiety phenotypes. Importantly,
inhibition of BLA somata did not induce an anxiogenic response,
likely due to the simultaneous decrease in direct BLA-CeM
excitatory input. We also found that the eNpHR3.0:BLA-CeA group
showed a significant reduction in open arm time and probability of
open arm entry on the elevated plus maze during light-on epochs,
but not light-off epochs, relative to the EYFP and Soma groups
(FIGS. 6F-6G), without altering locomotor activity (FIG. 16). The
eNpHR3.0:BLA-CeA group also showed a significant reduction in
center time upon illumination with 594 nm light, relative to the
EYFP and Soma groups (statistics, p=0.002; FIGS. 6H-6I). Finally,
we also demonstrate that selective illumination of
eNpHR3.0-expressing axon terminals can reduce the probability of
both spontaneously occurring (FIGS. 6J-6L) and evoked (FIGS. 6M-6P)
vesicle release, without preventing spiking at the cell soma (FIGS.
14A-14E). These data demonstrate that selective inhibition of BLA
terminals in the CeA induces an acute increase in anxiety-like
behaviors.
[0111] Conclusions: In these experiments, we have identified the
BLA-CeL pathway as an endogenous neural substrate for
bidirectionally modulating the unconditioned expression of anxiety.
While we identify the BLA-CeL pathway as the critical substrate
rather than BLA fibers passing through the CeL, it is likely that
other downstream circuits, such as CeA projections to the BNST play
an important role in the expression of anxiety or anxiety-related
behaviors.sup.4, 6, 13. Indeed, our findings may support the notion
that corticotrophin releasing hormone (CRH) networks in the BNST
can be critically involved in modulating anxiety-related
behaviors.sup.6, 52, as the CeL is a primary source of CRH for the
BNST.sup.53.
[0112] Other neurotransmitters and neuromodulators may modulate or
gate effects on distributed neural circuits, including
serotonin.sup.54, 55, dopamine.sup.56, acetylcholine.sup.57,
glycine.sup.58, GABA.sup.13 and CRH.sup.59. The neural circuitry
converging to and diverging from this pathway will provide many
opportunities for modulatory control, as parallel or downstream
circuits of the BLA-CeL synapse likely contribute to modulate the
expression of anxiety phenotypes.sup.6, 56. Moreover, upstream of
the amygdala, this microcircuit is well-positioned to be recruited
by top-down cortical control from regions important for processing
fear and anxiety, including the prelimbic, infralimbic and insular
cortices that provide robust innervation to the BLA and CeL..sup.4,
13, 23, 60.
[0113] Our examination of the BLA anatomy suggests that the
populations of BLA neurons projecting to CeL and CeM neurons are
largely non-overlapping. In natural states, the CeL-projecting BLA
neurons may excite CeM-projecting BLA neurons in a microcircuit
homeostatic mechanism. This may also represent a potential
mechanism underlying anxiety disorders, when there are synaptic
changes that skew the balance of the circuit to allow uninhibited
CeM activation.
[0114] Together, the data presented here support identification of
the BLA-CeL synapse as a critical circuit element both necessary
and sufficient for the expression of endogenous anxiolysis in the
mammalian brain, providing a novel source of insight into anxiety
as well as a new kind of treatment target, and demonstrate the
importance of resolving specific projections in the study of neural
circuit function relevant to psychiatric disease.
[0115] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention.
[0116] All references, publications, and patent applications
disclosed herein are hereby incorporated by reference in their
entirety.
REFERENCES
[0117] 1. Lieb, R. Anxiety disorders: clinical presentation and
epidemiology. Handb Exp Pharmacol, 405-432 (2005). [0118] 2.
Kessler, R. C., et al. Lifetime prevalence and age-of-onset
distributions of DSM-IV disorders in the National Comorbidity
Survey Replication. Arch Gen Psychiatry 62, 593-602 (2005). [0119]
3. Koob, G. F. Brain stress systems in the amygdala and addiction.
Brain Res 1293, 61-75 (2009). [0120] 4. Ressler, K. J. &
Mayberg, H. S. Targeting abnormal neural circuits in mood and
anxiety disorders: from the laboratory to the clinic. Nat Neurosci
10, 1116-1124 (2007). [0121] 5. Vanderschuren, L. J. & Everitt,
B. J. Behavioral and neural mechanisms of compulsive drug seeking.
Eur J Pharmacol 526, 77-88 (2005). [0122] 6. Davis, M., Walker, D.
L., Miles, L. & Grillon, C. Phasic vs sustained fear in rats
and humans: role of the extended amygdala in fear vs anxiety.
Neuropsychopharmacology 35, 105-135. [0123] 7. Ehrlich, I., et al.
Amygdala inhibitory circuits and the control of fear memory. Neuron
62, 757-771 (2009). [0124] 8. Han, J. H., et al. Selective erasure
of a fear memory. Science 323, 1492-1496 (2009). [0125] 9. Herry,
C., et al. Switching on and off fear by distinct neuronal circuits.
Nature 454, 600-606 (2008). [0126] 10. LeDoux, J. The emotional
brain, fear, and the amygdala. Cell Mol Neurobiol 23, 727-738
(2003). [0127] 11. Maren, S. & Quirk, G. J. Neuronal signaling
of fear memory. Nat Rev Neurosci 5, 844-852 (2004). [0128] 12.
Pare, D., Quirk, G. J. & Ledoux, J. E. New vistas on amygdala
networks in conditioned fear. J Neurophysiol 92, 1-9 (2004). [0129]
13. Shin, L. M. & Liberzon, I. The neurocircuitry of fear,
stress, and anxiety disorders. Neuropsychopharmacology 35, 169-191.
[0130] 14. Davis, M. The role of the amygdala in conditioned and
unconditioned fear and anxiety. in The Amygdala (ed. A. JP) p.
213-288 (Oxford University Press, Oxford, UK, 2000). [0131] 15.
Killcross, S., Robbins, T. W. & Everitt, B. J. Different types
of fear-conditioned behaviour mediated by separate nuclei within
amygdala. Nature 388, 377-380 (1997). [0132] 16. Tye, K. M. &
Janak, P. H. Amygdala neurons differentially encode motivation and
reinforcement. J Neurosci 27, 3937-3945 (2007). [0133] 17. Tye, K.
M., Stuber, G. D., de Ridder, B., Bonci, A. & Janak, P. H.
Rapid strengthening of thalamo-amygdala synapses mediates
cue-reward learning. Nature 453, 1253-1257 (2008). [0134] 18. Bahi,
A., Mineur, Y. S. & Picciotto, M. R. Blockade of protein
phosphatase 2B activity in the amygdala increases anxiety- and
depression-like behaviors in mice. Biol Psychiatry 66, 1139-1146
(2009). [0135] 19. Davis, M. Are different parts of the extended
amygdala involved in fear versus anxiety? Biol Psychiatry 44,
1239-1247 (1998). [0136] 20. Etkin, A., et al. Individual
differences in trait anxiety predict the response of the
basolateral amygdala to unconsciously processed fearful faces.
Neuron 44, 1043-1055 (2004). [0137] 21. Kahn, N. H., Shelton, S. E.
& Davidson, R. J. The role of the central nucleus of the
amygdala in mediating fear and anxiety in the primate. J Neurosci
24, 5506-5515 (2004). [0138] 22. Roozendaal, B., McEwen, B. S.
& Chattarji, S. Stress, memory and the amygdala. Nat Rev
Neurosci (2009). [0139] 23. Stein, M. B., Simmons, A. N.,
Feinstein, J. S. & Paulus, M. P. Increased amygdala and insula
activation during emotion processing in anxiety-prone subjects. Am
J Psychiatry 164, 318-327 (2007). [0140] 24. Boyden, E. S., Zhang,
F., Bamberg, E, Nagel, G. & Deisseroth, K.
Millisecond-timescale, genetically targeted optical control of
neural activity. Nat Neurosci 8, 1263-1268 (2005). [0141] 25.
Gradinaru, V., at aL Molecular and cellular approaches for
diversifying and extending optogenetics. Ce//141, 154-165. [0142]
26. Nagel, G., at al. Channelrhodopsin-2, a directly light-gated
cation-selective membrane channel. Proc Nat/Acad Sci U S A 100,
13940-13945 (2003). [0143] 27. Fraser, A. D. Use and abuse of the
benzodiazepines. Ther Drug Monit 20, 481-489 (1998). [0144] 28.
Woods, J. H., Katz, J. L. & Winger, G. Benzodiazepines: use,
abuse, and consequences. Pharmacol Rev 44, 151-347 (1992). [0145]
29. Hovatta, I. & Barlow, C. Molecular genetics of anxiety in
mice and men. Anti Med 40, 92-109 (2008). [0146] 30. Hovatta, I.,
et al. Glyoxalase 1 and glutathione reductase 1 regulate anxiety in
mice. Nature 438, 662-666 (2005). [0147] 31. Blanchard, R. J.,
Yudko, E. B., Rodgers, R. J. & Blanchard, D. C. Defense system
psychopharmacology: an ethological approach to the pharmacology of
fear and anxiety. Behav Brain Res 58, 155-165 (1993). [0148] 32.
LeDoux, J. E., Iwata, J., Cicchetti, P. & Reis, D. J. Different
projections of the central amygdaloid nucleus mediate autonomic and
behavioral correlates of conditioned fear. J Neurosci 8, 2517-2529
(1988). [0149] 33. Carlson, J. lrnmunocytochemical localization of
glutamate decarboxylase in the rat basolateral amygdaloid nucleus,
with special reference to GABAergic innervation of amygdalostriatal
projection neurons. J Comp Neurol 273, 513-526 (1988). [0150] 34.
Smith, Y. & Pare, D. Intra-amygdaloid projections of the
lateral nucleus in the cat: PHA-L anterograde labeling combined
with postembedding GABA and glutamate immunocytochemistry. J Comp
Neurol 342, 232-248 (1994). [0151] 35. McDonald, A. J.
Cytoarchitecture of the central amygdaloid nucleus of the rat. J
Comp Neurol 208, 401-418 (1982). [0152] 36. Bissiere, S., Humeau,
Y. & Luthi, A. Dopamine gates LTP induction in lateral amygdala
by suppressing feedforward inhibition. Nat Neurosci 6, 587-592
(2003). [0153] 37. Marowsky, A., Yanagawa, Y., Obata, K. &
Vogt, K. E. A specialized subclass of interneurons mediates
dopaminergic facilitation of amygdala function. Neuron 48,
1025-1037 (2005). [0154] 38. Pitkanen, A. Connectivity of the rat
amygdaloid complex. in The Amygdala (ed. A. J P) p. 31-99 (Oxford
University Press, Oxford, UK, 2000). [0155] 39. Krettek, J. E.
& Price, J. L. Amygdaloid projections to subcortical structures
within the basal forebrain and brainstem in the rat and cat. J Comp
Neurol 178, 225-254 (1978). [0156] 40. Petrovich, G. D. &
Swanson, L. W. Projections from the lateral part of the central
amygdalar nucleus to the postulated fear conditioning circuit.
Brain Res 763, 247-254 (1997). [0157] 41. LeDoux, J. E., Cicchetti,
P., Xagoraris, A. & Romanski, L. M. The lateral amygdaloid
nucleus: sensory interface of the amygdala in fear conditioning. J
Neurosci 10, 1062-1069 (1990). [0158] 42. Krettek, J. E. &
Price, J. L. A description of the amygdaloid complex in the rat and
cat with observations on intra-amygdaloid axonal connections. J
Comp Neurol 178, 255-280 (1978). [0159] 43. Petrovich, G. D.,
Risold, P. Y. & Swanson, L. W. Organization of projections from
the basomedial nucleus of the amygdala: a PHAL study in the rat. J
Comp Neural 374, 387-420 (1996). [0160] 44. Pare, D. & Smith,
Y. The intercalated cell masses project to the central and medial
nuclei of the amygdala in cats. Neuroscience 57, 1077-1090 (1993).
[0161] 45. Likhtik, E., Popa, D., Apergis-Schoute, J., Fidacaro,
G.A. & Pare, D. Amygdala intercalated neurons are required for
expression of fear extinction. Nature 454, 642-645 (2008). [0162]
46. Jolkkonen, E. & Pitkanen, A. Intrinsic connections of the
rat amygdaloid complex:
[0163] projections originating in the central nucleus. J Comp
Neurol 395, 53-72 (1998). [0164] 47. Amano, T., Unal, C. T. &
Pare, D. Synaptic correlates of fear extinction in the amygdala.
Nat Neurosci 13, 489-494. [0165] 48. McDonald, A. J., Muller, J. F.
& Mascagni, F. GABAergic innervation of alpha type II
calcium/calmodulin-dependent protein kinase immunoreactive
pyramidal neurons in the rat basolateral amygdala. J Comp Neurol
446, 199-218 (2002). [0166] 49. Choleris, E., Thomas, A. W.,
Kavaliers, M. & Prato, F. S. A detailed ethological analysis of
the mouse open field test: effects of diazepam, chlordiazepoxide
and an extremely low frequency pulsed magnetic field. Neurosci
Biobehav Rev 25, 235-260 (2001). [0167] 50. Pellow, S., Chopin, P.,
File, S. E. & Briley, M. Validation of open:closed arm entries
in an elevated plus-maze as a measure of anxiety in the rat. J
Neurosci Methods 14, 149-167 (1985). [0168] 51. Sah, P. & Lopez
De Armentia, M. Excitatory synaptic transmission in the lateral and
central amygdala. Ann N YAcad Sci 985, 67-77 (2003). [0169] 52.
Davis, M. & Shi, C. The extended amygdala: are the central
nucleus of the amygdala and the bed nucleus of the stria terminalis
differentially involved in fear versus anxiety? Ann N YAcad Sci
877, 281291 (1999). [0170] 53. Sakanaka, M., Shibasaki, T. &
Lederis, K. Distribution and efferent projections of
corticotropin-releasing factor-like immunoreactivity in the rat
amygdaloid complex. Brain Res 382, 213-238 (1986). [0171] 54.
Holmes, A., Yang, R. J., Lesch, K. P., Crawley, J. N. & Murphy,
D. L. Mice lacking the serotonin transporter exhibit 5-HT(1A)
receptor-mediated abnormalities in tests for anxiety-like behavior.
Neuropsychopharmacology 28, 2077-2088 (2003). [0172] 55. Lesch, K.
P., et al. Association of anxiety-related traits with a
polymorphism in the serotonin transporter gene regulatory region.
Science 274, 1527-1531 (1996). [0173] 56. Graybiel, A. M. &
Rauch, S. L. Toward a neurobiology of obsessive-compulsive
disorder. Neuron 28, 343-347 (2000). [0174] 57. Picciotto, M. R.,
Brunzell, D. H. & Caldarone, B. J. Effect of nicotine and
nicotinic receptors on anxiety and depression. Neuroreport 13,
1097-1106 (2002). [0175] 58. Snyder, S. H. & Enna, S. J. The
role of central glycine receptors in the pharmacologic actions of
benzodiazepines. Adv Biochem Psychopharmacol, 81-91 (1975). [0176]
59. Lesscher, H. M., et al. Amygdala protein kinase C epsilon
regulates corticotropin-releasing factor and anxiety-like behavior.
Genes Brain Behav 7, 323-333 (2008). [0177] 60. Milad, M. R.,
Rauch, S. L., Pitman, R. K. & Quirk, G. J. Fear extinction in
rats: implications for human brain imaging and anxiety disorders.
Biol Psycho! 73, 61-71 (2006).
TABLE-US-00003 [0177] SEQUENCES (NpHR amino acid sequence without
the signal peptide): SEQ ID NO: 1
VTQRELFEFVLNDPLLASSLYINIALAGLSILLFVFMTRGLDDPRAKLIA
VSTILVPVVSIASYTGLASGLTISVLEMPAGHFAEGSSVMLGGEEVDGVV
TMWGRYLTWALSTPMILLALGLLAGSNATKLFTAITFDIAMCVTGLAAAL
TTSSHLMRWFWYAISCACFLVVLYILLVEWAQDAKAAGTADMFNTLKLLT
VVMWLGYPIVWALGVEGIAVLPVGVTSWGYSFLDIVAKYIFAFLLLNYLT
SNESVVSGSILDVPSASGTPADD (eYFP-NpHR3.0 amino acid sequence): SEQ ID
NO: 2 MTETLPPVTESAVALQAEVTQRELFEFVLNDPLLASSLYINIALAGLSIL
LFVFMTRGLDDPRAKLIAVSTILVPVVSIASYTGLASGLTISVLEMPAGH
FAEGSSVMLGGEEVDGVVTMWGRYLTWALSTPMILLALGLLAGSNATKLF
TAITFDIAMCVTGLAAALTTSSHLMRWFWYAISCACFLVVLYILLVEWAQ
DAKAAGTADMFNTLKLLTVVMWLGYPIVWALGVEGIAVLPVGVTSWGYSF
LDIVAKYIFAFLLLNYLTSNESVVSGSILDVPSASGTPADDAAAKSRITS
EGEYIPLDQIDINVVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGD
ATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKS
AMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNI
LGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNT
PIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDEL YKFCYENEV
(eYFP-NpHR3.1 amino acid sequence): SEQ ID NO: 3
MVTQRELFEFVLNDPLLASSLYINIALAGLSILLFVFMTRGLDDPRAKLI
AVSTILVPVVSIASYTGLASGLTISVLEMPAGHFAEGSSVMLGGEEVDGV
VTMWGRYLTWALSTPMILLALGLLAGSNATKLFTAITFDIAMCVTGLAAA
LTTSSHLMRWFWYAISCACFLVVLYILLVEWAQDAKAAGTADMFNTLKLL
TVVMWLGYPIVWALGVEGIAVLPVGVTSWGYSFLDIVAKYIFAFLLLNYL
TSNESVVSGSILDVPSASGTPADDAAAKSRITSEGEYIPLDQIDINVVSK
GEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL
PVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIM
ADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSY
QSASKDPNEKRDHMVLLEFVTAAGITLGMDELYKFCYENEV (GtR3 amino acid
sequence): SEQ ID NO: 4
ASSFGKALLEFVFIVFACITLLLGINAAKSKAASRVLFPATFVTGIASIA
YFSMASGGGWVIAPDCRQLFVARYLDWLITTPLLLIDLGLVAGVSRWDIM
ALCLSDVLMIATGAFGSLTVGNVKWVWWFFGMCWFLHIIFALGKSWAEAA
KAKGGDSASVYSKIAGITVITWFCYPVVWVFAEGFGNFSVTFEVLIYGVL
DVISKAVFGLILMSGAATGYESI (ChR2 amino acid sequence): SEQ ID NO: 5
MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQT
ASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFF
EFKNPSMLYLATGHRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSRRTM
GLLVSDIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGY
HTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHT
IIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLV EDEAEAGAVP (SFO
amino acid sequence): SEQ ID NO: 6
MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQT
ASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFF
EFKNPSMLYLATGHRVQWLRYAEWLLTSPVILIHLSNLTGLSNDYSRRTM
GLLVSDIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGY
HTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHT
IIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLV EDEAEAGAVP (SSFO
amino acid sequence): SEQ ID NO: 7
MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQT
ASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFF
EFKNPSMLYLATGHRVQWLRYAEWLLTSPVILIHLSNLTGLSNDYSRRTM
GLLVSAIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGY
HTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHT
IIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLV EDEAEAGAVP (C1V1
amino acid sequence): SEQ ID NO: 8
MSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERM
LFQTSYTLENNGSVICIPNNGQCFCLAWLKSNGTNAEKLAANILQWITFA
LSALCLMFYGYQTWKSTCGWEEIYVATIEMIKFIIEYFHEFDEPAVIYSS
NGNKTVWLRYAEWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGCIV
WGATSAMCTGWTKILFFLISLSYGMYTYFHAAKVYIEAFHTVPKGICREL
VRVMAWTFFVAWGMFPVLFLLGTEGFGHISPYGSAIGHSILDLIAKNMWG
VLGNYLRVKIHEHILLYGDIRKKQKITIAGQEMEVETLVAEEED (C1V1-E122T amino acid
sequence): SEQ ID NO: 9
MSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERM
LFQTSYTLENNGSVICIPNNGQCFCLAWLKSNGTNAEKLAANILQWITFA
LSALCLMFYGYQTWKSTCGWETIYVATIEMIKFIIEYFHEFDEPAVIYSS
NGNKTVWLRYAEWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGCIV
WGATSAMCTGWTKILFFLISLSYGMYTYFHAAKVYIEAFHTVPKGICREL
VRVMAWTFFVAWGMFPVLFLLGTEGFGHISPYGSAIGHSILDLIAKNMWG
VLGNYLRVKIHEHILLYGDIRKKQKITIAGQEMEVETLVAEEED (C1V1-E162T amino acid
sequence): SEQ ID NO: 10
MSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERM
LFQTSYTLENNGSVICIPNNGQCFCLAWLKSNGTNAEKLAANILQWITFA
LSALCLMFYGYQTWKSTCGWEEIYVATIEMIKFIIEYFHEFDEPAVIYSS
NGNKTVWLRYATWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGCIV
WGATSAMCTGWTKILFFLISLSYGMYTYFHAAKVYIEAFHTVPKGICREL
VRVMAWTFFVAWGMFPVLFLLGTEGFGHISPYGSAIGHSILDLIAKNMWG
VLGNYLRVKIHEHILLYGDIRKKQKITIAGQEMEVETLVAEEED (C1V1-E122T/E162T
amino acid sequence): SEQ ID NO: 11
MSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERM
LFQTSYTLENNGSVICIPNNGQCFCLAWLKSNGTNAEKLAANILQWITFA
LSALCLMFYGYQTWKSTCGWETIYVATIEMIKFIIEYFHEFDEPAVIYSS
NGNKTVWLRYATWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGCIV
WGATSAMCTGWTKILFFLISLSYGMYTYFHAAKVYIEAFHTVPKGICREL
VRVMAWTFFVAWGMFPVLFLLGTEGFGHISPYGSAIGHSILDLIAKNMWG
VLGNYLRVKIHEHILLYGDIRKKQKITIAGQEMEVETLVAEEED
Sequence CWU 1
1
151273PRTNatronomonas pharaonis 1Val Thr Gln Arg Glu Leu Phe Glu
Phe Val Leu Asn Asp Pro Leu Leu1 5 10 15 Ala Ser Ser Leu Tyr Ile
Asn Ile Ala Leu Ala Gly Leu Ser Ile Leu 20 25 30 Leu Phe Val Phe
Met Thr Arg Gly Leu Asp Asp Pro Arg Ala Lys Leu 35 40 45 Ile Ala
Val Ser Thr Ile Leu Val Pro Val Val Ser Ile Ala Ser Tyr 50 55 60
Thr Gly Leu Ala Ser Gly Leu Thr Ile Ser Val Leu Glu Met Pro Ala65
70 75 80 Gly His Phe Ala Glu Gly Ser Ser Val Met Leu Gly Gly Glu
Glu Val 85 90 95 Asp Gly Val Val Thr Met Trp Gly Arg Tyr Leu Thr
Trp Ala Leu Ser 100 105 110 Thr Pro Met Ile Leu Leu Ala Leu Gly Leu
Leu Ala Gly Ser Asn Ala 115 120 125 Thr Lys Leu Phe Thr Ala Ile Thr
Phe Asp Ile Ala Met Cys Val Thr 130 135 140 Gly Leu Ala Ala Ala Leu
Thr Thr Ser Ser His Leu Met Arg Trp Phe145 150 155 160 Trp Tyr Ala
Ile Ser Cys Ala Cys Phe Leu Val Val Leu Tyr Ile Leu 165 170 175 Leu
Val Glu Trp Ala Gln Asp Ala Lys Ala Ala Gly Thr Ala Asp Met 180 185
190 Phe Asn Thr Leu Lys Leu Leu Thr Val Val Met Trp Leu Gly Tyr Pro
195 200 205 Ile Val Trp Ala Leu Gly Val Glu Gly Ile Ala Val Leu Pro
Val Gly 210 215 220 Val Thr Ser Trp Gly Tyr Ser Phe Leu Asp Ile Val
Ala Lys Tyr Ile225 230 235 240 Phe Ala Phe Leu Leu Leu Asn Tyr Leu
Thr Ser Asn Glu Ser Val Val 245 250 255 Ser Gly Ser Ile Leu Asp Val
Pro Ser Ala Ser Gly Thr Pro Ala Asp 260 265 270
Asp2559PRTArtificial SequenceSynthetic polypeptide 2Met Thr Glu Thr
Leu Pro Pro Val Thr Glu Ser Ala Val Ala Leu Gln1 5 10 15 Ala Glu
Val Thr Gln Arg Glu Leu Phe Glu Phe Val Leu Asn Asp Pro 20 25 30
Leu Leu Ala Ser Ser Leu Tyr Ile Asn Ile Ala Leu Ala Gly Leu Ser 35
40 45 Ile Leu Leu Phe Val Phe Met Thr Arg Gly Leu Asp Asp Pro Arg
Ala 50 55 60 Lys Leu Ile Ala Val Ser Thr Ile Leu Val Pro Val Val
Ser Ile Ala65 70 75 80 Ser Tyr Thr Gly Leu Ala Ser Gly Leu Thr Ile
Ser Val Leu Glu Met 85 90 95 Pro Ala Gly His Phe Ala Glu Gly Ser
Ser Val Met Leu Gly Gly Glu 100 105 110 Glu Val Asp Gly Val Val Thr
Met Trp Gly Arg Tyr Leu Thr Trp Ala 115 120 125 Leu Ser Thr Pro Met
Ile Leu Leu Ala Leu Gly Leu Leu Ala Gly Ser 130 135 140 Asn Ala Thr
Lys Leu Phe Thr Ala Ile Thr Phe Asp Ile Ala Met Cys145 150 155 160
Val Thr Gly Leu Ala Ala Ala Leu Thr Thr Ser Ser His Leu Met Arg 165
170 175 Trp Phe Trp Tyr Ala Ile Ser Cys Ala Cys Phe Leu Val Val Leu
Tyr 180 185 190 Ile Leu Leu Val Glu Trp Ala Gln Asp Ala Lys Ala Ala
Gly Thr Ala 195 200 205 Asp Met Phe Asn Thr Leu Lys Leu Leu Thr Val
Val Met Trp Leu Gly 210 215 220 Tyr Pro Ile Val Trp Ala Leu Gly Val
Glu Gly Ile Ala Val Leu Pro225 230 235 240 Val Gly Val Thr Ser Trp
Gly Tyr Ser Phe Leu Asp Ile Val Ala Lys 245 250 255 Tyr Ile Phe Ala
Phe Leu Leu Leu Asn Tyr Leu Thr Ser Asn Glu Ser 260 265 270 Val Val
Ser Gly Ser Ile Leu Asp Val Pro Ser Ala Ser Gly Thr Pro 275 280 285
Ala Asp Asp Ala Ala Ala Lys Ser Arg Ile Thr Ser Glu Gly Glu Tyr 290
295 300 Ile Pro Leu Asp Gln Ile Asp Ile Asn Val Val Ser Lys Gly Glu
Glu305 310 315 320 Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
Asp Gly Asp Val 325 330 335 Asn Gly His Lys Phe Ser Val Ser Gly Glu
Gly Glu Gly Asp Ala Thr 340 345 350 Tyr Gly Lys Leu Thr Leu Lys Phe
Ile Cys Thr Thr Gly Lys Leu Pro 355 360 365 Val Pro Trp Pro Thr Leu
Val Thr Thr Phe Gly Tyr Gly Leu Gln Cys 370 375 380 Phe Ala Arg Tyr
Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser385 390 395 400 Ala
Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp 405 410
415 Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr
420 425 430 Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu
Asp Gly 435 440 445 Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn
Ser His Asn Val 450 455 460 Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly
Ile Lys Val Asn Phe Lys465 470 475 480 Ile Arg His Asn Ile Glu Asp
Gly Ser Val Gln Leu Ala Asp His Tyr 485 490 495 Gln Gln Asn Thr Pro
Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn 500 505 510 His Tyr Leu
Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys 515 520 525 Arg
Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr 530 535
540 Leu Gly Met Asp Glu Leu Tyr Lys Phe Cys Tyr Glu Asn Glu Val545
550 555 3542PRTArtificial SequenceSynthetic polypeptide 3Met Val
Thr Gln Arg Glu Leu Phe Glu Phe Val Leu Asn Asp Pro Leu1 5 10 15
Leu Ala Ser Ser Leu Tyr Ile Asn Ile Ala Leu Ala Gly Leu Ser Ile 20
25 30 Leu Leu Phe Val Phe Met Thr Arg Gly Leu Asp Asp Pro Arg Ala
Lys 35 40 45 Leu Ile Ala Val Ser Thr Ile Leu Val Pro Val Val Ser
Ile Ala Ser 50 55 60 Tyr Thr Gly Leu Ala Ser Gly Leu Thr Ile Ser
Val Leu Glu Met Pro65 70 75 80 Ala Gly His Phe Ala Glu Gly Ser Ser
Val Met Leu Gly Gly Glu Glu 85 90 95 Val Asp Gly Val Val Thr Met
Trp Gly Arg Tyr Leu Thr Trp Ala Leu 100 105 110 Ser Thr Pro Met Ile
Leu Leu Ala Leu Gly Leu Leu Ala Gly Ser Asn 115 120 125 Ala Thr Lys
Leu Phe Thr Ala Ile Thr Phe Asp Ile Ala Met Cys Val 130 135 140 Thr
Gly Leu Ala Ala Ala Leu Thr Thr Ser Ser His Leu Met Arg Trp145 150
155 160 Phe Trp Tyr Ala Ile Ser Cys Ala Cys Phe Leu Val Val Leu Tyr
Ile 165 170 175 Leu Leu Val Glu Trp Ala Gln Asp Ala Lys Ala Ala Gly
Thr Ala Asp 180 185 190 Met Phe Asn Thr Leu Lys Leu Leu Thr Val Val
Met Trp Leu Gly Tyr 195 200 205 Pro Ile Val Trp Ala Leu Gly Val Glu
Gly Ile Ala Val Leu Pro Val 210 215 220 Gly Val Thr Ser Trp Gly Tyr
Ser Phe Leu Asp Ile Val Ala Lys Tyr225 230 235 240 Ile Phe Ala Phe
Leu Leu Leu Asn Tyr Leu Thr Ser Asn Glu Ser Val 245 250 255 Val Ser
Gly Ser Ile Leu Asp Val Pro Ser Ala Ser Gly Thr Pro Ala 260 265 270
Asp Asp Ala Ala Ala Lys Ser Arg Ile Thr Ser Glu Gly Glu Tyr Ile 275
280 285 Pro Leu Asp Gln Ile Asp Ile Asn Val Val Ser Lys Gly Glu Glu
Leu 290 295 300 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly
Asp Val Asn305 310 315 320 Gly His Lys Phe Ser Val Ser Gly Glu Gly
Glu Gly Asp Ala Thr Tyr 325 330 335 Gly Lys Leu Thr Leu Lys Phe Ile
Cys Thr Thr Gly Lys Leu Pro Val 340 345 350 Pro Trp Pro Thr Leu Val
Thr Thr Phe Gly Tyr Gly Leu Gln Cys Phe 355 360 365 Ala Arg Tyr Pro
Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala 370 375 380 Met Pro
Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp385 390 395
400 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu
405 410 415 Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp
Gly Asn 420 425 430 Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser
His Asn Val Tyr 435 440 445 Ile Met Ala Asp Lys Gln Lys Asn Gly Ile
Lys Val Asn Phe Lys Ile 450 455 460 Arg His Asn Ile Glu Asp Gly Ser
Val Gln Leu Ala Asp His Tyr Gln465 470 475 480 Gln Asn Thr Pro Ile
Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 485 490 495 Tyr Leu Ser
Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg 500 505 510 Asp
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu 515 520
525 Gly Met Asp Glu Leu Tyr Lys Phe Cys Tyr Glu Asn Glu Val 530 535
540 4223PRTGuillardia theta 4Ala Ser Ser Phe Gly Lys Ala Leu Leu
Glu Phe Val Phe Ile Val Phe1 5 10 15 Ala Cys Ile Thr Leu Leu Leu
Gly Ile Asn Ala Ala Lys Ser Lys Ala 20 25 30 Ala Ser Arg Val Leu
Phe Pro Ala Thr Phe Val Thr Gly Ile Ala Ser 35 40 45 Ile Ala Tyr
Phe Ser Met Ala Ser Gly Gly Gly Trp Val Ile Ala Pro 50 55 60 Asp
Cys Arg Gln Leu Phe Val Ala Arg Tyr Leu Asp Trp Leu Ile Thr65 70 75
80 Thr Pro Leu Leu Leu Ile Asp Leu Gly Leu Val Ala Gly Val Ser Arg
85 90 95 Trp Asp Ile Met Ala Leu Cys Leu Ser Asp Val Leu Met Ile
Ala Thr 100 105 110 Gly Ala Phe Gly Ser Leu Thr Val Gly Asn Val Lys
Trp Val Trp Trp 115 120 125 Phe Phe Gly Met Cys Trp Phe Leu His Ile
Ile Phe Ala Leu Gly Lys 130 135 140 Ser Trp Ala Glu Ala Ala Lys Ala
Lys Gly Gly Asp Ser Ala Ser Val145 150 155 160 Tyr Ser Lys Ile Ala
Gly Ile Thr Val Ile Thr Trp Phe Cys Tyr Pro 165 170 175 Val Val Trp
Val Phe Ala Glu Gly Phe Gly Asn Phe Ser Val Thr Phe 180 185 190 Glu
Val Leu Ile Tyr Gly Val Leu Asp Val Ile Ser Lys Ala Val Phe 195 200
205 Gly Leu Ile Leu Met Ser Gly Ala Ala Thr Gly Tyr Glu Ser Ile 210
215 220 5310PRTArtificial SequenceSynthetic polypeptide 5Met 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 6310PRTArtificial
SequenceSynthetic polypeptide 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 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 Ser 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 7310PRTArtificial SequenceSynthetic polypeptide
7Met 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 Ser 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 Ala 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 8344PRTArtificial SequenceSynthetic
polypeptide 8Met 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
Leu Leu Ile His Leu Ser Asn 165 170 175 Leu Thr Gly Leu Lys Asp Asp
Tyr Ser Lys Arg Thr Met Gly Leu Leu 180 185 190 Val Ser Asp Val Gly
Cys Ile Val Trp Gly Ala Thr Ser Ala Met Cys 195 200 205 Thr Gly Trp
Thr Lys Ile Leu Phe Phe Leu Ile Ser Leu Ser Tyr Gly 210 215 220 Met
Tyr Thr Tyr Phe His Ala Ala Lys Val Tyr Ile Glu Ala Phe His225 230
235 240 Thr Val Pro Lys Gly Ile Cys Arg Glu Leu Val Arg Val Met Ala
Trp 245 250 255 Thr Phe Phe Val Ala Trp Gly Met Phe Pro Val Leu Phe
Leu Leu Gly 260 265 270 Thr Glu Gly Phe Gly His Ile Ser Pro Tyr Gly
Ser Ala Ile Gly His 275 280 285 Ser Ile Leu Asp Leu Ile Ala Lys Asn
Met Trp Gly Val Leu Gly Asn 290 295 300 Tyr Leu Arg Val Lys Ile His
Glu His Ile Leu Leu Tyr Gly Asp Ile305 310 315 320 Arg Lys Lys Gln
Lys Ile Thr Ile Ala Gly Gln Glu Met Glu Val Glu 325 330 335 Thr Leu
Val Ala Glu Glu Glu Asp 340 9344PRTArtificial SequenceSynthetic
polypeptide 9Met 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 Thr 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
Leu Leu Ile His Leu Ser Asn 165 170 175 Leu Thr Gly Leu Lys Asp Asp
Tyr Ser Lys Arg Thr Met Gly Leu Leu 180 185 190 Val Ser Asp Val Gly
Cys Ile Val Trp Gly Ala Thr Ser Ala Met Cys 195 200 205 Thr Gly Trp
Thr Lys Ile Leu Phe Phe Leu Ile Ser Leu Ser Tyr Gly 210 215 220 Met
Tyr Thr Tyr Phe His Ala Ala Lys Val Tyr Ile Glu Ala Phe His225 230
235 240 Thr Val Pro Lys Gly Ile Cys Arg Glu Leu Val Arg Val Met Ala
Trp 245 250 255 Thr Phe Phe Val Ala Trp Gly Met Phe Pro Val Leu Phe
Leu Leu Gly 260 265 270 Thr Glu Gly Phe Gly His Ile Ser Pro Tyr Gly
Ser Ala Ile Gly His 275 280 285 Ser Ile Leu Asp Leu Ile Ala Lys Asn
Met Trp Gly Val Leu Gly Asn 290 295 300 Tyr Leu Arg Val Lys Ile His
Glu His Ile Leu Leu Tyr Gly Asp Ile305 310 315 320 Arg Lys Lys Gln
Lys Ile Thr Ile Ala Gly Gln Glu Met Glu Val Glu 325 330 335 Thr Leu
Val Ala Glu Glu Glu Asp 340 10344PRTArtificial SequenceSynthetic
polypeptide 10Met 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 Thr Trp Leu Leu Thr Cys Pro Val
Leu Leu Ile His Leu Ser Asn 165 170 175 Leu Thr Gly Leu Lys Asp Asp
Tyr Ser Lys Arg Thr Met Gly Leu Leu 180 185 190 Val Ser Asp Val Gly
Cys Ile Val Trp Gly Ala Thr Ser Ala Met Cys 195 200 205 Thr Gly Trp
Thr Lys Ile Leu Phe Phe Leu Ile Ser Leu Ser Tyr Gly 210 215 220 Met
Tyr Thr Tyr Phe His Ala Ala Lys Val Tyr Ile Glu Ala Phe His225 230
235 240 Thr Val Pro Lys Gly Ile Cys Arg Glu Leu Val Arg Val Met Ala
Trp 245 250 255 Thr Phe Phe Val Ala Trp Gly Met Phe Pro Val Leu Phe
Leu Leu Gly 260 265 270 Thr Glu Gly Phe Gly His Ile Ser Pro Tyr Gly
Ser Ala Ile Gly His 275 280 285 Ser Ile Leu Asp Leu Ile Ala Lys Asn
Met Trp Gly Val Leu Gly Asn 290 295 300 Tyr Leu Arg Val Lys Ile His
Glu His Ile Leu Leu Tyr Gly Asp Ile305 310 315 320 Arg Lys Lys Gln
Lys Ile Thr Ile Ala Gly Gln Glu Met Glu Val Glu 325 330 335 Thr Leu
Val Ala Glu Glu Glu Asp 340 11344PRTArtificial SequenceSynthetic
polypeptide 11Met 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 Thr 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 Thr Trp Leu Leu Thr Cys Pro Val
Leu Leu Ile His Leu Ser Asn 165 170 175 Leu Thr Gly Leu Lys Asp Asp
Tyr Ser Lys Arg Thr Met Gly Leu Leu 180 185 190 Val Ser Asp Val Gly
Cys Ile Val Trp Gly Ala Thr Ser Ala Met Cys 195 200 205 Thr Gly Trp
Thr Lys Ile Leu Phe Phe Leu Ile Ser Leu Ser Tyr Gly 210 215 220 Met
Tyr Thr Tyr Phe His Ala Ala Lys Val Tyr Ile Glu Ala Phe His225 230
235 240 Thr Val Pro Lys Gly Ile Cys Arg Glu Leu Val Arg Val Met Ala
Trp 245 250 255 Thr Phe Phe Val Ala Trp Gly Met Phe Pro Val Leu Phe
Leu Leu Gly 260 265 270 Thr Glu Gly Phe Gly His Ile Ser Pro Tyr Gly
Ser Ala Ile Gly His 275 280 285 Ser Ile Leu Asp Leu Ile Ala Lys Asn
Met Trp Gly Val Leu Gly Asn 290 295 300 Tyr Leu Arg Val Lys Ile His
Glu His Ile Leu Leu Tyr Gly Asp Ile305 310 315 320 Arg Lys Lys Gln
Lys Ile Thr Ile Ala Gly Gln Glu Met Glu Val Glu 325 330 335 Thr Leu
Val Ala Glu Glu Glu Asp 340 126PRTArtificial SequenceSynthetic
peptideMisc_feature(2)...(2)X is any amino acid 12Phe Xaa Tyr Glu
Asn Glu1 5 137PRTArtificial SequenceSynthetic peptide 13Phe Cys Tyr
Glu Asn Glu Val1 5 1420PRTArtificial SequenceSynthetic peptide
14Lys Ser Arg Ile Thr Ser Glu Gly Glu Tyr Ile Pro Leu Asp Gln Ile1
5 10 15 Asp Ile Asn Val 20 155PRTArtificial SequenceSynthetic
peptideMisc_feature(2)...(2)Xaa is any amino
acidMisc_feature(3)...(3)Xaa is any amino acid 15Val Xaa Xaa Ser
Leu1 5
* * * * *
References