U.S. patent application number 10/705755 was filed with the patent office on 2004-09-30 for cellular physiology workstations for automated data acquisition and perfusion control.
This patent application is currently assigned to Trustees of Boston University, a university in Massachusetts. Invention is credited to Farb, David H., Gibbs, Terrell T., Yaghoubi, Nader.
Application Number | 20040191853 10/705755 |
Document ID | / |
Family ID | 27358099 |
Filed Date | 2004-09-30 |
United States Patent
Application |
20040191853 |
Kind Code |
A1 |
Farb, David H. ; et
al. |
September 30, 2004 |
Cellular physiology workstations for automated data acquisition and
perfusion control
Abstract
Cellular physiology workstations for automated data acquisition
and perfusion control are described. The cellular physiology
workstation may be used for physiological and electrophysiological
experiments. Methods for employing such cellular physiology
workstations in physiological and electrophysiological experiments
are also disclosed. The cellular physiology workstations comprise
one or more recording chambers each for holding one or more cells
to be measured. One or more cells are place in each recording
chamber. Perfusions means, such as an automatic perfusion system is
connected to the recording chamber to perfuse the cells with a
plurality of solutions containing different concentration of one or
more agents to be tested. Biosensors, such as patch clamps,
electrodes, or microscopes are positioned to detect a response from
the cell. The cellular physiology workstation may optionally
comprise injecting means for introducing an injection solution into
the cell before and during analysis.
Inventors: |
Farb, David H.; (Chestnut
Hill, MA) ; Gibbs, Terrell T.; (Jamaica Plain,
MA) ; Yaghoubi, Nader; (Boston, MA) |
Correspondence
Address: |
EDWARDS & ANGELL, LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
Trustees of Boston University, a
university in Massachusetts
|
Family ID: |
27358099 |
Appl. No.: |
10/705755 |
Filed: |
November 10, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10705755 |
Nov 10, 2003 |
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09918370 |
Jul 30, 2001 |
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6762036 |
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09918370 |
Jul 30, 2001 |
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09519109 |
Mar 6, 2000 |
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6268168 |
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09519109 |
Mar 6, 2000 |
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08888691 |
Jul 7, 1997 |
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6048722 |
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08888691 |
Jul 7, 1997 |
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PCT/US96/18832 |
Nov 8, 1996 |
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60006326 |
Nov 8, 1995 |
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Current U.S.
Class: |
435/29 ;
455/15 |
Current CPC
Class: |
G01N 33/5044 20130101;
G01N 33/502 20130101; G01N 33/48728 20130101; G01N 33/5008
20130101; G01N 33/6872 20130101 |
Class at
Publication: |
435/029 ;
455/015 |
International
Class: |
C12Q 001/02 |
Goverment Interests
[0001] This invention was made, in part, with United States
Government support under grant number MH-49469, awarded by the
National Institute of Mental Health, and the United States
Government has certain rights in the invention.
Claims
1. An apparatus for detecting cellular response following exposure
to an agent, the apparatus comprising: a) a plurality of recording
chambers each adapted to receive and support a cell; b) a plurality
of video cameras, one video camera positioned for viewing a single
recording chamber; c) a video switching mechanism for controlling a
video camera input source selection to a video monitor; d) a
perfusion system adapted to perfuse a cell positioned in a
recording chamber with a predetermined set of perfusion solutions;
e) at least one invasive biosensor adapted for use with each
recording chamber, the at least one invasive biosensor being
controllable using computer-controlled electronic
micromanipulators; and f) a computer adapted to monitor recording
chambers, manipulate a biosensor, collect, analyze and display
responses detected using the biosensor.
2. The apparatus of claim 1 wherein the cell is a mammalian, insect
or amphibian cell.
3. The apparatus of claim 2 wherein the cell is a Xenopus
oocyte.
4. The apparatus of claim 1 wherein the biosensor is an
electrode.
5. The apparatus of claim 4 wherein the electrode is a voltage
measuring or a current injecting electrode.
6. The apparatus of claim 1 wherein the perfusion system comprises
a plurality of reservoirs containing one or more different
perfusion solutions, and a valve in fluid communication with said
plurality of reservoirs for the delivery of perfusion solution to a
recording chamber.
7. The apparatus of claim 1 further comprising a means for
controlling the temperature of each recording chamber.
8. The apparatus of claim 1 further comprising means for
controlling the oxygen, nitrogen or carbon dioxide levels in a
recording chamber.
9. The apparatus of claim 1 wherein the response of the cell to the
agent is mediated by a cell surface receptor produced by
recombinant DNA technology.
Description
MICROFICHE APPENDIX
[0002] One microfiche appendix is filed with this application.
Microfiche appendix A contains a total of 1 microfiche and 18
frames.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to apparatuses for data
acquisition and perfusion control in the analysis of cellular
physiology and electrophysiology and to methods for automated
perfusion and membrane voltage and current measurement for
physiological and electrophysiological analysis.
[0005] 2. Description of the Background
[0006] Cell membranes communicate information from the
extracellular environment by means of receptor and channel proteins
located within the cell membrane. Receptor proteins are gated by
molecules which can bind to the receptor and signal that a binding
event has taken place, often by triggering the opening of ion
channels through which ions such as sodium and chloride ions can
flow. Ionic flux across a cell membrane generates electrical
current that can be measured with appropriate recording equipment.
Electrophysiological analysis is widely used today to study the
pharmacology and biophysics of membrane proteins.
[0007] An expression system utilizing unfertilized eggs, or
oocytes, taken from the South African clawed frog, Xenopus laevis,
is a preferred material for electrophysiological studies of
receptor and ion channel function. Xenopus oocytes have the ability
to synthesize functional proteins when microinjected with exogenous
mRNA or cDNA constructs.
[0008] In electrophysiological analysis, an oocyte is electrically
connected to intracellular voltage and current measuring and
clamping devices. Detection of an electrophysiological response may
comprise steps of applying appropriate receptor ligands and
adjusting the holding potential manually and measuring any changes
in membrane voltage or current.
[0009] Recently, electrophysiological analysis of Xenopus oocytes
has been actively applied to many fields. In particular,
electrophysiological analysis has been used for the study of
membrane protein function, such as the function and pharmacology of
membrane receptors, voltage-gated ion channels, molecular
transporters and ion pumps. Defined combinations of recombinant
subunits, chimeric proteins, or mutagenized constructs can be
efficiently reconstituted in the oocyte membrane for
electrophysiological analysis. For such analysis, the oocyte
response may be monitored using intracellular recording, patch
clamp and internal perfusion techniques.
[0010] It has been difficult to achieve a highly reproducible and
reliable assay or to achieve quantitative analysis of
electrophysiological response by conventional manual perfusion and
membrane potential measurement techniques. These techniques have
many shortcomings because of variabilities due to human errors,
operator fatigue and inconsistencies between operators, and less
than optimal reproducibility and reliability. Further, the
perfusion and detection steps typically require long and
complicated manual manipulations which create additional problems.
The cultured cell becomes less viable with time and it is difficult
to control the temperature and oxygen tension. The limited
dexterity of even the most experienced operator limits the number
of experiments may be performed on one cell. Reliance on human
operators has resulted in reaction times that are considerably
longer than theoretically possible.
[0011] Conventional systems for analysis of cells have attempted to
address some of the problems of automated cell analysis. These
systems have suffered generally from inability to individually
measure a physiological response of a cell. Examples of systems
that do not address individual physiological measurements include
Kearney, Engstrom, Frnzl et al. and Capco et al.
[0012] Kearney (U.S. Pat. No. 5,424,209), discloses a system for
culturing and testing of cells. This culturing and testing system
was designed for the culturing and testing of cell populations and
not individual cells. Engstrom (U.S. Pat. No. 5,312,731) discloses
a method and apparatus for studying a reaction pattern of a cell or
cell aggregate during perfusion with different media. The system is
limited to analysis of cell response of a through transmission
microscopy. Franzl et al., (U.S. Pat. No. 5,432,086) discloses an
apparatus for the automatic monitoring of microorganism culture.
The system is limited to the monitoring of microorganism growth and
multiplication by an impedance measuring process. Capco et al.,
(U.S. Pat. No. 4,983,527) discloses a method for detection of tumor
promoting compounds. Amphibian oocytes are contacted to a tumor
promoting compound and the oocytes are examined visually to detect
a change in the size of the light/dark hemisphere of the oocyte.
Capco's disclosed method is limited to contacting the oocytes to
one solution comprising a candidate tumor promoting compound.
SUMMARY OF THE INVENTION
[0013] The present invention overcomes the problems and
disadvantages associated with current strategies and designs and
provides novel apparatus and methods for the study of membrane
physiology.
[0014] One embodiment of the invention is directed to cellular
physiology workstations that enable automated execution of
experimental protocols for electrophysiological experiments and for
the development of more complex protocols based on extended
recording sessions. As currently developed for oocyte
electrophysiology, the apparatus comprises one or more custom-built
recording chambers, a perfusion control system designed for rapid
application of about 2 to about 16 or more solutions under
automated control, software-based virtual instrumentation developed
to automate the execution of experimental protocols, and a data
acquisition and control platform which integrates the entire
system. The system is fully customizable through a sophisticated
object-oriented programming language and can be easily adapted to
applications such as patch clamp electrophysiology, calcium imaging
studies, confocal microscopy and other applications where perfusion
control and data acquisition need to be tightly integrated.
[0015] Another embodiment of the invention is directed to apparatus
for reproducibly detecting the electrical response of a cell to an
agent. The apparatus comprises a plurality of recording chambers.
Each chamber is designed to contain one or more cells such as, for
example, one or more Xenopus oocytes. Means are provided to perfuse
each recording chamber with a plurality of perfusion solutions.
Each perfusion solution may contain a different concentration of
one or more agent. A plurality of electrodes such as, for example,
a voltage measuring electrode, a current injecting electrode or a
glass patch electrode, may be connected to each cell to measure the
electrical response of the cell to the presence, absence or change
in concentration of the agent. The electrical response may also be
measured at various holding potentials.
[0016] Another embodiment of the invention is directed to automated
apparatuses for electrophysiological measurement which comprises
injecting means, such as a needle, for delivering an injection
solution into the cell. The injection solution may comprise a
second agent, a protein, a nucleic acid or a combination thereof.
The nucleic acid may be, for example, DNA, RNA or PNA. PNAs,
peptide nucleic acids or protein nucleic acids, are synthetic
polymers capable of hybridizing in a sequence specific manner with
natural nucleic acids.
[0017] Another embodiment of the invention is directed to methods
for reproducibly detecting a physiological response of a cell to a
agent. A cell such as, for example, a Xenopus oocyte, is perfused
using an automated perfusion system with a plurality of solutions,
which may comprise different concentrations of one or more agents,
and the electrophysiological response of the cell measured. The
automated perfusion control system may be, for example, a gravity
fed flow through perfusion system. The automated perfusion control
system may have an optimized lag time of less than about 100
milliseconds and a rise time of less than about 140 milliseconds
such as less than about 70 milliseconds.
[0018] Another embodiment of the invention is directed to assays
for detecting a substance which affects cellular physiology. A cell
is injected with a nucleic acid such as, for example, DNA or RNA
encoding a membrane receptor. The cell is perfused with a plurality
of solutions comprising different concentration of said substance
using an automated perfusion system. A change in cellular
electrophysiology of the cell is detected to determine the effect
of the substance. The period of time between the injecting step and
the perfusing and measuring steps may be between about one hour to
about 15 days.
[0019] Another embodiment of the invention is directed to a
substance detected by the assay. A candidate substance is used and
the assay is performed to detect a desirable effect. A substance
capable of inducing a desirable effect is identified by the
assay.
[0020] Another embodiment of the invention is directed to a kit for
performing the assay. The kit may comprise reagents and biosensors
for the performance of the assay.
[0021] Other embodiments and advantages of the invention are set
forth, in part, in the description which follows and, in part, will
be obvious from this description and may be learned from practice
of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 depicts an automated oocyte perfusion control system
according to one embodiment of the present invention.
[0023] FIG. 2 depicts a voltage clamp and recording chamber
connection according to one embodiment of the present
invention.
[0024] FIG. 3 depicts an internal perfusion control system
according to one embodiment of the present invention.
[0025] FIG. 4 depicts a block diagram showing information transfer
between the system components.
[0026] FIG. 5 depicts components of the perfusion system.
[0027] FIG. 6 depicts an instrumentation graphical user interface
according to one embodiment of the present invention.
[0028] FIG. 7 depicts responses for several ligand-gated ion
channels expressed after injection of the oocytes with rat brain
mRNA.
[0029] FIG. 8 depicts averaged kainate dose-response data from four
oocytes injected with GluR6 cDNA.
[0030] FIG. 9 depicts the effect of pregnenolone sulfate on kainate
dose-response curve in oocytes injected with rat brain poly A.sup.+
mRNA.
[0031] FIG. 10 depicts the measurement of pregnenolone sulfate IC50
in oocytes injected with rat brain poly A.sup.+ mRNA.
[0032] FIG. 11 depicts the effects of steroids on recombinant GluR6
kainate receptors.
[0033] FIG. 12 depicts steroid IC50 determinations for recombinant
GluR6 receptors.
[0034] FIG. 13 depicts the kainate concentration dependence of
pregnenolone sulfate inhibition.
[0035] FIG. 14 depicts inhibition of kainate responses by
stimulation of metabotropic glutamate receptors.
[0036] FIG. 15 depicts .gamma.-aminobutyric acid (GABA)
dose-response curves revealing reproducibility of GABA
EC.sub.50.
[0037] FIG. 16 depicts a high-resolution dose-response curve
generated by the cellular physiology workstation.
[0038] FIG. 17 depicts current responses to 30 consecutive
applications of 100 .mu.M GABA.
[0039] FIG. 18 depicts determination of reversal potential
generated automatically by the cellular physiology workstation.
[0040] FIG. 19 depicts an examination of the endogenous
calcium-dependent chloride current (I.sub.C1-.sub.(Ca)) present in
native oocytes.
[0041] FIG. 20 depicts an alternate embodiment of the perfusion
control system of the present invention.
[0042] FIG. 21 depicts a font panel which aids in the creation and
editing of recipe files.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0043] Performing multiple electrophysiological measurement on a
plurality of cells while maintaining consistency between individual
experiments is problematic. Difficulties associated with
reproducibility and reliability render any more than two or three
measurements suspect. The present invention overcomes these
problems using automated perfusion systems and methods that are
capable of controlled and consistent perfusions on a plurality of
recording chambers.
[0044] The cellular physiology workstation of the invention, when
applied to electrophysiology, has several advantages. Automation
allows experimenters to maximize the amount of data that can be
obtained from a given cell during the limited viability of
microelectrode-impaled cells. Automation also increases the speed
and throughput of electrophysiological experiments while reducing
errors associated with manual manipulations and waveform analysis.
Manipulations required during a recording trial such as, toggling
switches, applying drugs, measuring the response and timing the
experiment are minimized or eliminated. Automation also enables the
development of more complex protocols based on repetitive
recordings that permit averaging of data before and after a given
manipulation. Finally, automation of experimental protocols makes
it feasible to utilize technician-level operators for the
collection of electrophysiological data. This is advantageous for
drug screening and receptor characterization. By minimizing timing,
perfusion and instrument control manipulations, automation reduces
experimenter fatigue during extended recording sessions. Protocols
which are too complex, fast, or repetitive for manual performance
may be preprogrammed and executed consistently and reliably with a
single keypress. Further, the experimenter may focus on the
hypothesis being tested rather than the mechanics of
electrophysiological technique. Automation enables the precise
timing of agent application, such as drug application, and improves
the quality of experimental data by reducing inadvertent errors,
idiosyncratic variations in protocol between different
investigators, and introduction of noise through manual
manipulations. Automated waveform analysis reduces measurement
errors as well as the post-processing time necessary for the
analysis of experimental data, while enabling real-time evaluation
of results. Additionally, the efficiency and speed of data
generation are increased, thereby allowing mass, parallel
screenings of large chemical libraries. This added throughput also
allows the researcher the opportunity to test secondary hypotheses
that might otherwise have been neglected due to recording time
limitations or the tedious nature of the task. The physiology
workstation is especially useful and advantageous in
pharmaceutical, chemical, and biotechnical research and
development. The methods and apparatus of the invention is
especially suited for repetitive or complex protocols, such as a
drug dose response analysis.
[0045] Accordingly, the present invention provides an
application-specific integrated workstation, particularly one for
cellular electrophysiology such as oocyte electrophysiology, which
results in a tremendous reduction in time expenditure in assembly.
Additionally, the invention provides an automated workstation which
provides greater efficiency and higher productivity. The
workstation provides a tightly integrated system comprising
recording chambers, perfusion system, data acquisition platform and
instrumentation software that enables immediate experimentation
without additional set up. At the same time, system flexibility is
preserved by allowing selection of amplifiers, microscopes,
micromanipulators and other such devices that are most appropriate
for the experimenter's requirements.
[0046] The apparatuses and methods of the present invention may
also be used for techniques such as, internal perfusion of oocytes,
patch clamp electrophysiology, brain slice recording,
receptor-ligand interactions on cell surfaces, calcium imaging
studies, confocal microscopy, and in vivo microdialysis, for
example. The system of the present invention may also be used to
examine the function of ligand-gated ion channels, voltage-gated
ion channels, G-protein coupled receptors, activities across the
synapse, molecular transporters, cell-cell interactions and ion
pumps. The system may also be useful for screening compound
libraries to search for novel classes of compounds, screening
members of a given class of compounds for effects on specific
receptors, detailed pharmacological characterizations of compounds
having receptor effects, rapid evaluation of EC.sub.50 (potency)
and E.sub.max (efficiency), investigation of interactions between
receptors and rapid characterization of a series of receptor
mutants. The invention provides repetitive application,
dose-response data generation, evaluation of receptors expressed
from poly A.sup.+ mRNA, and evaluation of recombinant receptors
such as, for example, .gamma.-aminobutyric acid (GABA) receptors,
kainate receptors, and N-Methyl-D-aspartic acid (NMDA)
receptors.
[0047] Examples of agents that may be used for the apparatus and
methods of the invention include drugs, receptor agonists, receptor
antagonists, neurotransmitter, neurotransmitter analogues, enzyme
inhibitors, ion channel modulators, G-protein coupled receptor
modulators, transport inhibitors, hormones, peptides, toxins,
antibodies, pharmaceutical agents, chemicals and combinations of
these agents. Specific agents which may be used for the apparatus
and methods of the invention include purinergics, cholinergics,
serotonergics, dopaminergics, anesthetics, benzodiazepines,
barbiturates, steroids, alcohols, metal cations, cannabinoids,
cholecystokinins, cytokines, excitatory amino acids, GABAergics,
gangliosides, histaminergics, melatonins, neuropeptides,
neurotoxins, endothelins, NO compounds, opioids, sigma receptor
ligands, somatostatins, tachykinins, angiotensins, bombesins,
bradykinins, prostaglandins and combinations of these agents.
[0048] Another embodiment of the invention is directed to an
automated workstation for data acquisition and perfusion control to
facilitate electrophysiological measurement. A preferred apparatus
and method are described in relation to as follows, but it is clear
to one of ordinary skill in the art that the described apparatus
and methods are broadly applicable to many cell types.
[0049] Automated Perfusion Control System
[0050] One embodiment of the present invention, depicted in FIG. 1,
is an automated perfusion control system for electrophysiological
studies of. The system comprises computer 10, operating
instrumentation software, recording chamber 12 designed to receive
a cell such as for example, oocyte 32, and perfusion control system
14. Perfusion control system 14, depicted in FIG. 1 and FIG. 5,
comprises valve controller 16, connected to and under the control
of computer 10. Valve controller 16 is connected by a plurality of
tubes 44 to a plurality of constant flow reservoirs 18 to
multi-barrel manifold 20. Each reservoir comprises ventilation
means 46 which allow gravity flow of solution from the reservoir
18, through tube 44 when valve controller 16 is open. Valve
controller 16 comprises a plurality of solenoid valves. The system
may also comprise voltage clamp amplifier 22 connected to and under
control of computer 10 via BNC box 24. Voltage clamp amplifier 22
is connected to oscilloscope 26 and to two impaling electrodes 28
and 30 in recording chamber 12.
[0051] According to one embodiment of the present invention, the
automated perfusion control system of the cellular physiology
workstation may comprise a plurality of reservoirs 18 containing
one or more different perfusion solutions. The reA valve 16 may be
used to control delivery of the fluid to the one or more recording
chambers 12. The fluid valve may be manual or machine operated. A
machine operated valve may be controlled directly by computer means
within the cellular physiology workstation. The automated perfusion
control system may comprise between 2 to about 100 reservoirs,
preferably between about 4 and about 20 reservoirs. The automated
perfusion control system may optionally comprise a mixing means,
such as a mixing chamber, between the fluid valve and the recording
chamber.
[0052] Turning to FIG. 20, an alternate embodiment of the present
invention is shown as perfusion control system 14'. This system 14'
is in most respects substantially identical to perfusion control
system 14, while including a plurality of recording chambers 12 and
a plurality of manifolds 20.
[0053] Oocyte Preparation
[0054] In a preferred embodiment cells monitored by the cellular
physiology workstation are Xenopus oocytes. Numerous methods for
preparing oocytes and poly A.sup.+ RNA are known to those of skill
in the art. One method is described as follows.
[0055] Donor animals, female oocyte positive Xenopus laevis frogs,
are anesthetized in a solution of about 0.15% Tricaine for about 30
minutes. Ovarian sections are removed through a lateral abdominal
incision, after which the incision is sutured with about 4 to about
5 stitches and the frog is allowed to recover in isolation for
about 3 to about 4 hours. Ovarian lobules containing the follicular
oocytes are immediately rinsed with calcium-free ND96 solution (96
mM NaCl, 1 mM MgCl.sub.2, 2 mM KCl, 50 mM Hepes, 2.5 mM pyruvate)
and cut into clumps of about 10 to about 20 oocytes. Following 2
mg/ml collagenase treatment (Sigma) at room temperature for about 2
hours, individual oocytes are obtained free of their follicular
layer. Selected oocytes (Dumont stage V and VI) are then
transferred to 60.times.15 mm glass petri dishes containing ND96
(96 mM NaCl, 1 mM MgCl.sub.2, 2 mM KCl, 50 mM Hepes, 2.5 mM
pyruvate) and maintained in an incubator at a temperature of about
18.degree. C. to about 19.degree. C.
[0056] Poly A.sup.+ mRNA are extracted from brain tissue and
neuronal cell culture using a magnetic separation protocol based on
the Dynabeads Oligo (dT)25 kit (Dynal, Olso, Norway). Briefly, the
protocol utilized magnetic beads that have an attached poly-T
moiety which can bind poly A.sup.+ mRNA for separation. Tissue is
homogenized, cells are disrupted, and the lysate is added to an
aliquot of Dynabeads. Magnetic separation efficiently yields mRNA
that is suitable for direct injection into oocytes. Batches of
about 20 to about 30 select oocytes are injected with about 50 to
about 100 ng of neuronal poly A.sup.+ mRNA. Alternatively, oocytes
may be injected with about 30 .mu.l to about 80 .mu.l of RNA
prepared from in vitro transcription of cDNA clones. In either
case, injection may be performed using a Drummond electronic
microinjector. Oocytes are then maintained at about 18.degree. C.
to about 19.degree. C. for about 2 to about 4 days to allow protein
expression prior to electrophysiological recordings. After
incubation, electrophysiological analysis of expressed receptors
and ion channels may be performed using a system according to the
present invention.
[0057] Recording Chamber
[0058] A novel intracellular recording chamber for Xenopus oocyte
electrophysiology has been developed to enable rapid agent
application and automated control over perfusion protocols.
Recording chamber 12 features a flow-through design in which
gravity-feed eliminates the need for pressurization of solution
containers and drop-wise removal of perfusate eliminates the need
for a vacuum line. As depicted in FIGS. 1 and FIG. 2, oocyte 32
sits in V-shaped groove 34 in fluid chamber 36 with a capacity of
about 100 .mu.l and is stabilized against solution flow by
microelectrode impalement. The recording chamber may be adapted to
accept more than one cell such as an oocyte. For example, the
recording chamber may be adapted to receive about 3, about 10,
about 15, or about 100 cells. Optionally, if it is desired to
provide more stability against solution flow, additional devices
such as needles or electrodes may be employed. This has resulted in
rise times (5%-95%) of about 70 msec to about 140 msec for 100
.mu.M GABA responses from oocytes expressing GABA.sub.A receptors,
which represents the fastest rise times reported to date for
Xenopus oocyte electrophysiology. The chamber is preferably made of
non-conductive plastic and is clamped onto a microscope stage for
stability. In one embodiment the entire chamber is plastic and
electrodes 28 and 30 impale oocyte 32. The physiology workstation
may comprise more than one recording chamber such as about 3, about
10, about 15, or about 100 recording chambers.
[0059] Electrodes 28 and 30 are connected to operational amplifiers
A2 and A1 respectively of a voltage clamp amplifier. Operational
amplifier A1 measures the voltage difference between the voltage
recording electrode 30 and reference electrode 38 which is disposed
in KCl well 40. KCl well 40 preferably comprises about 3M KCl and
is in fluid communication with fluid chamber 36 by way of agar
bridge 42. Operational amplifier A1 feeds the difference between
electrode 30 and electrode 38 to operational amplifier A2.
Operational amplifier A2 compares the voltage difference with the
desired voltage difference, Vcom, and outputs current to injecting
electrode 28 to maintain the oocyte membrane at a desired
potential. In a preferred embodiment, the desired potential may be
any value between about 200 mV to about -200 mV such as about -60
mV and is stepped up to any value between about 200 mV to about
-200 mV such as about -100 mV during agent application to increase
ionic driving forces.
[0060] The electrical response detected by the cellular physiology
workstation may be a membrane potential or a membrane current.
After detection the electrical response may be recorded by a
recording means such as, for example, a digital recorder, a
computer, volatile memory, involatile memory, a chart recorder or a
combination of recording devices. The apparatus may further
comprise means for controlling the temperature and oxygen level of
the recording chamber.
[0061] Fluid chamber 36 has a port to which multi-barrel manifold
20 may be attached for delivery of solutions (FIG. 1). Perfusate
drips through an aperture into a plastic perfusate collection
chamber which feeds into a disposal bottle. Flow rates of about 1.5
ml to about 3.0 ml per minute can be achieved with adjustment of
the height of the reservoir bank. Higher flow rates can be obtained
with pressurization but are subjected only to the limitation of the
stability and integrity of microelectrode impalement of the oocyte
membrane. Minimum lag time and onset times are important for high
sensitivity experiments. One advantage of the cellular physiology
workstation is a recording chamber which minimizes dead volume and
lag time. While pressurization and vacuum may be used, it is not
required. The physiological workstation is capable of optimized lag
times (valve switching to response onset time) of less than about
100 msec, such as less than 50 msec, and onset times (time to go
from about 5% to about 95% of maximum amplitude) of between about
70 milliseconds to about 140 milliseconds are obtained with about
100 .mu.M .gamma.-aminobutyric acid (GABA) responses in oocytes
injected with chick brain poly(A).sup.+ RNA. The solution exchange
time is about one second for 50% exchange and about 8 seconds for
90% exchange as measured by depolarization induced by switching
from normal Ringer to high potassium Ringer. An agar bridge may be
used to establish electrical contact with the reference electrode
through an attached KCl well.
[0062] In one embodiment, a two-electrode voltage clamp is used
with two intracellular microelectrodes pulled on a Flaming-Brown
micropipette puller (Model P80/PC; Sutter Instrument Co.). These
electrodes have input resistances of about 2 mega-ohms to about 4
mega-ohms when filled with a solution comprising about 3M KCl.
Microelectrode positioning and impalement of the oocyte may be
performed under micromanipulator control.
[0063] The recording chamber may further comprise means for
controlling gas levels such as oxygen, nitrogen, and carbon dioxide
levels. In addition the recording chamber may further comprise
means for temperature control.
[0064] Configuration of the Recording Apparatus
[0065] The recording chamber herein described is designed to be
clamped directly onto the microscope stage. The small size and
novel design of this chamber permits two such chambers to be
clamped side-by-side onto the stage of an unmodified Nikon SMZ-10
microscope for visual monitoring of the cells during
experimentation. With slight modification and extension of the
microscope stage, up to 5 such recording chambers could be so
utilized. The microscope head is mounted on a sliding bracket to
facilitate panning of the viewing field across the row of parallel
recording chambers. For simultaneous recordings from multiple
oocytes, each of these recording chambers could have two
independent micromanipulators for positioning of the
voltage-recording and current-injecting electrodes. At least about
six micromanipulators can be positioned around the microscope
stage. Ten or more micromanipulators might be positioned about the
recording chamber by the careful positioning and by the use of
small footprint micromanipulators.
[0066] The recording chamber may optionally comprise means for
receiving and automatically positioning a cell within said
recording chamber. Positioning means may comprise indentations in
said recording chamber for the cells such as oocytes to settle.
Other positioning means may also comprise robotic means, and
artificial intelligence means for the proper positioning of the
cells. Cells may be held in place after positioning by impaling
probes which may or may not be a biosensor. The simplest impaling
probe for immobilizing a cell may be a glass needle. Other forms of
immobilizing cells such as oocyte may comprise, for example,
adhesives, vacuum and indentations.
[0067] The recording chamber may optionally comprise means for
positioning said one or more biosensors to detect a response from
the cell. Means for positioning may be in the form of a template
with biosensors spaced regularly to proximate cells positioned by
the automatic cell positioning means. The biosensors may be, for
example, electrodes, patch clamps or microscopes. The positioning
of biosensors such as, for example electrodes, may involve the
puncture and penetration of the cellular membrane. If the cells are
of uniform size, such as Xenopus oocytes, the depth of penetration
may be preset and fixed. Alternatively, the positioning and the
depth of penetration may be determined by an automatic positioning
system tailored for the specific cell type. The automatic
positioning system may comprise for example, feedback and robotic
mechanism which may be computer controlled for determining the
proper position and depth of the probes.
[0068] Injecting Means
[0069] The physiological workstation may comprise optional means of
injecting one or more injection solutions into said oocyte between
the culturing and measuring step. The injection solution may
comprise an agent. The agent may be a chemical, a protein or a
nucleic acid.
[0070] Examples of agents that may be injection include proteins,
DNA, RNA, PNA, receptor agonists, receptor antagonists,
neurotransmitter, neurotransmitter analogues, enzyme inhibitors,
ion channel modulators, G-protein coupled receptor modulators,
transport inhibitors, hormones, peptides, toxins, antibodies,
pharmaceutical agents, chemicals and combinations of these agents.
Specific agents which are of interest include purinergics,
cholinergics, serotonergics, dopaminergics, anesthetics,
benzodiazepines, barbiturates, steroids, alcohols, metal cations,
cannabinoids, cholecystokinins, cytokines, excitatory amino acids,
GABAergics, gangliosides, histaminergics, melatonins,
neuropeptides, neurotoxins, endothelins, NO compounds, opioids,
sigma receptor ligands, somatostatins, tachykinins, angiotensins,
bombesins, bradykinins, prostaglandins and combinations of these
agents.
[0071] Perfusion Control System
[0072] Agent solutions are held in a plurality of plastic
reservoirs, 18, each of which has a capacity of about 15 ml to
about 50 ml. Reservoirs are constructed so as to maintain a
constant flow rate regardless of the level of solution in each
reservoir. A constant flow rate is important to ensure
reproducibility of responses since onset of response is influenced
by agent application rate. As depicted in FIG. 1, each reservoir 18
contains glass siphon 44 extending down through its cap into the
solution and vent line 46 also extending into the solution that
equilibrates chamber pressure for maintenance of constant flow
rate. Solution-flow is by gravity feed and the flow rate can be
controlled by adjusting the height of the reservoirs. Other method
of solution flow such as vacuum, pressure or pumping may also be
used. Dropwise removal of solution through the efflux line creates
negative pressure in the chamber which is equilibrated by means of
the vent line. In one embodiment, up to 16 different agent
solutions can be prepared and placed in a rack designed to hold 16
reservoirs. Additional reservoirs may also be provided depending on
the number of ports in manifold 20.
[0073] Multi-barrel manifold 20 receives input lines from the
reservoirs 18 and provide a point of convergence for the different
solutions. Flexible tubing, such as Tygon.TM., of about 0.9 mm
inner diameter, carries solution from each solenoid valve of valve
controller 16 to a separate barrel on manifold 20, where the lines
converge to an output port that can be connected directly to
recording chamber 12. Other models having additional barrels may
also be used. These manifolds are custom-made from glass capillary
tubing fitted with plastic tubing adaptors and have been made in 8-
and 16-barrel models. These designs minimize internal dead volume
so as to enable rapid agent application and minimal dilution of
solutions.
[0074] The perfusion controller system functions to translate
digital output from an analog/digital input/output (MacADIOS II)
card 60 (FIG. 4), which is connected to computer 10, into signals
which can be utilized to switch relays and solenoid valves that
control perfusion. Solution flow between the constant-flow
reservoirs and multi-barrel manifold is preferably controlled via
16 miniature teflon-coated solenoid valves (Lee Valve Co.; Essex,
Conn.). These valves are particularly suitable because of their
corrosion resistance, biocompatibility and power requirements.
Other valves exhibiting these characteristics may also be used.
These valves may be actuated by a direct current voltage supplied
from a direct current power source which would eliminate electrical
hum. Direct current power sources may be, for example, a direct
current power supply or a battery. The value of the direct current
voltage may be, for example, 12 volts. The valves are interfaced to
the instrumentation software through an analog/digital input/output
module 16 serving as valve controller 16. The digital I/O module
preferably comprises a 16-channel backplane (OPT022, #PB1 6HC)
fitted with DC output modules (OPT022, #ODC5) to which the solenoid
valves are connected. The digital I/O module connects to the
digital out port of the analog/digital input/output (MacADIOS II)
card via a flat ribbon cable and has an external 7.5V power supply
(FIG. 4). The perfusion control system containing the solenoid
valve assembly and the digital I/O module, is housed in a box into
which flow lines enter from the agent reservoirs and exit to the
multi-barrel manifold. This gravity fed perfusion control system
incorporates solenoid valves to allow computer controlled
switching.
[0075] In another embodiment, the perfusion control system selects
between five different agent valves using transistor-based circuits
in digital I/O module 16 to switch between buffers and agent inputs
in response to transistor-transistor logic (TTL) signals sent on
two digital lines from a data acquisition card such as a MacADIOS
II card. Perfusion automation at this point provides the ability to
turn on a buffer valve at the start of data acquisition, switch to
a preselected agent valve at an indicated time, then switch back to
buffer for agent washout, all under control of computer 10. This
perfusion control system enables timely agent application protocols
and removes the necessity of having to manually switch valves at
appropriate times, tedious manipulations prone to experimental
error and generation of noise.
[0076] According to another embodiment of the invention a chamber
for internal perfusion of oocytes is depicted in FIG. 3. A glass
perfusion chamber may be drawn from a Pasteur pipette tip that has
been melted down to form a narrow aperture. Increase of fluid level
in the chamber leads to formation of a high resistance seal between
the devitellinized oocyte membrane and glass. Multi-port manifold
20 feeds into a perfusion line 46 which is threaded close to the
oocyte 32 for agent delivery and thus external perfusion. Perfusate
is removed through line 48 which maintains a constant fluid level
by means of a peristaltic pump 50. Electrodes 28 and 30 may be in
electrical contact with oocyte 32 by a fluid retention sleeve 52
through which a perfusion cannula 54 may be advanced for oocyte
membrane rupture and internal perfusion. Chloridized silver wire 56
provides conduction between reference electrode 38 in well 40 and
the chamber fluid. The internal perfusion controller of FIG. 3
allows introduction of drugs or enzymes into the oocyte cytoplasm
and control of intracellular composition. The use of this perfusion
controller allows control over external and internal perfusion.
[0077] Computer Control and Data Analysis Means
[0078] Instrumentation software, currently based on the SuperScope
II v1.43 programming environment (GW Instruments; Somerville,
Mass.), preferably operates on computer 10. SuperScope II provides
a sophisticated graphical environment which facilitates the
development of virtual instruments that are used for data
acquisition and instrument control. On-screen representations of
buttons, dials and sliders can be programmed to activate desired
software routines using an object-oriented programming language and
are used to build application-specific instruments. Other
programming environments, for example, LabView.TM. by National
Instruments (Austin, Tex.) or similar products or a general purpose
programming language such as C++ can also be used to achieve to
achieve similar interface. An instrument for oocyte
electrophysiology may integrate agent delivery, instrument control,
data acquisition and waveform analysis through an on-screen,
mouse-driven interface. FIG. 6 shows a screen shot from one
embodiment of such an instrument. While the virtual instrument
programs such as SuperScope II or LabView provides significant
convenience for the user, instrumentation software may also be
written by those of skill in the art.
[0079] A virtual instrument of oocyte electrophysiology is depicted
in FIG. 6. The virtual instrument was created using the SuperScope
II development environment (GW Instruments, Inc.) to allow complete
experimental control through a graphical user-interface. On-screen
markers (M1-M4) can be moved via the mouse to set the duration of
the PREPULSE PHASE (the interval between markers M1 and M2), the
DRUG APPLICATION PHASE (the interval between markers M2 and M3),
and the WASHOUT PHASE (the interval between markers M3 and M4). For
each phase, the perfusion controls 1 are set to select the valves
controlling the wash solutions. The VC COMMAND and STEP SIZE
controls 2 are used to control the voltage-clamp amplifier. The
EPISODES PER TRIAL selector 3 is used to define the number of
episodes to be acquired, after which the trial is initiated using
the BEGIN button 4. Waveforms are displayed in real-time in the
VOLTAGE and CURRENT windows 5 as they are acquired. Journal 6
automatically logs transcript of experimental session and provides
waveform analysis. The protocol selection area 7 is used to select
predefined protocols, while the file/log management section 8
provides file handling and data output.
[0080] The automation routines that may be implemented allow the
entire recording session to be controlled through the on-screen
interface by changing control knobs using the mouse. Automated
protocols have been developed that can initiate and carry out
dose-response, reversal potential, modulator effect and repetitive
application experiments with a single keypress. Waveform analysis
routines automatically measure parameters such as response
amplitude, onset time and desensitization time constant and log
this information directly to disk. Appendix A contains a code
listing of the software implementation which may be operated on
computer 10.
[0081] Computer 10 may be any computer, computer workstation,
dedicated processor, microprocessor or dedicated micro-controller.
In an embodiment computer 10 is a MacIntosh IICi computer (Apple
Computer; Cupertino, Calif.), a 68030 based computer having a
minimal configuration of 8 MB RAM and an 80 MB hard drive. Voltage
and current traces are acquired through analog to digital
conversion means and similarly valves and perfusion controllers may
be controlled through digital to analog conversion means. The
analog/digital conversion means may be, for example, an
analog/digital input output expansion card which may be installed
into computer 10.
[0082] One preferred analog/digital input/output conversion means
is a MacADIOS II data acquisition card (GW Instruments; Somerville,
Mass.). This NuBus based card has an additional 12 bit A/D
converter daughterboard, 2 analog output channels 8-bit digital I/O
port, can be configured with additional daughterboards for enhanced
functionality to facilitate independent acquisition of 2-channel
data and a 16 bit digital I/O daughterboard that is used to trigger
digital TTL lines for control of solenoid valves. The MacADIOS II
card is interfaced to laboratory equipment through a MacADIOS APO
analog I/O panel which provides electrical connections, such as BNC
connections, directly to the card. As additional channels are
needed, one or more secondary analog/digital converters or
additional analog/digital input/output conversion cards may be
added. In one embodiment, a second 12-bit A/D converter was
installed to facilitate independent acquisition of 2-channel data
at high speeds, while a 16-bit digital I/O daughterboard allowed
individual addressing of a total of 24 digital output lines for the
control of solenoid valves and other devices. Other data
acquisition cards may also be used. While other computers and
instrumentation software may be used, the graphical interface of
the MacIntosh and the SuperScope II file format simplify
manipulation and plotting of waveforms. A 68030 based computer can
digitize a 2-channel electrophysiological data at about 100 Hz and
recorded directly to 90 MB removable data storage devices
(Bernoulli cartridges; Iomega, Inc.) to facilitate convenient
storage and retrieval. The implementation of the cellular
physiology workstation is not computer specific, as computer
technology and storage technology improve, the cellular physiology
workstation may be implemented on the improved computer and storage
platforms. Functions and protocols on the physiological workstation
can be developed as the need arises. Computer programs and data
analysis routines not available in the SuperScope II environment
can be written in the C programming language for import into the
existing virtual instrument.
[0083] Sample data representative of traces produced by a system
according to the invention is shown in FIG. 7-FIG. 14. In FIG. 7,
responses are shown for several ligand-gated ion channels that were
expressed after injection of the oocytes with rat brain mRNA.
Traces show responses to about 100 .mu.M .gamma.-aminobutyric acid
(GABA) (A), about 100 .mu.M kainate (B), and about 100 .mu.M AMPA
(C). Standard buffer solutions were used with a holding potential
of about -100 mV. Automation enables extended recording sessions
with minimal operator intervention.
[0084] FIG. 8 depicts the results of a test to determine the
accuracy of a cellular physiology workstation according to the
present invention. In FIG. 8, Averaged kainate dose-response data
from 4 oocytes injected with GluR6 cRNA which yields an EC.sub.50
of about 0.5 .mu.M under Vh of about -100 mV, favorably compares to
an EC.sub.50 of about 1 .mu.M as previously reported.
[0085] FIG. 9 depicts the effect of pregnenolone sulfate on kainate
dose-response curve in oocytes injected with rat brain poly A.sup.+
mRNA. Kainate dose-response curves were generated with and without
the neuroactive steroid, pregnenolone sulfate (PS), and under Vh of
about -100 mV to determine the mechanism of action. A decrease in
Emax suggests a noncompetitive mechanism. This experiment
demonstrates the utility of a system according to the invention for
performing dose-response experiments with and without
modulators.
[0086] FIG. 10 depicts the measurement of pregnenolone sulfate IC50
in oocytes injected with rat brain poly A.sup.+ mRNA under Vh of
about -100 mV. Pregnenolone sulfate was applied in increasing
concentrations with 100 .mu.M kainate to characterize its
inhibitory effect.
[0087] FIG. 11 depicts the effects of pregnenolone sulfate and
5.beta.3.alpha.S on recombinant GluR6 kainate receptors. All
measurements were performed under Vh of about -100 mV. Recombinant
receptors may be rapidly characterized by utilization of automated
methodologies according to the invention. The inhibitory effects of
neuroactive steroids PS and 5.beta.3.alpha.S are shown in this
experiment. These steroids decrease the maximal response to kainate
with no change in kainate EC50.
[0088] FIG. 12 depicts steroid, pregnenolone sulfate and
5.beta.3.alpha.S, IC50 determined for recombinant GluR6 receptors
under Vh of about -100 mV. Increasing concentrations of two
steroids were applied with 10 .mu.M kainate to determine the
steroid IC50.
[0089] FIG. 13 depicts the kainate concentration dependence of
pregnenolone sulfate inhibition. To further characterize the
mechanism of inhibition for neuroactive steroids, pregnenolone
sulfate was applied with increasing concentrations of kainate to
determine the percent inhibition observed. It was found that
concentration dependence of 100 .mu.M pregnenolone sulfate
inhibition of kainate induced currents. The currents were expressed
as percent change from kainate D-R. Percent change was calculated
as ((I.sub.kain+PS/I.sub.kain)-1).multidot.100.
[0090] FIG. 14 depicts inhibition of kainate responses by
stimulation of metabotropic glutamate receptors. The ability of a
system according to the invention to do repetitive applications of
agent solutions was used to make the finding that about 500 .mu.M
of the metabotropic agonist, tACPD, can inhibit responses to
kainate of about 100 .mu.M in oocytes injected with rat brain poly
A.sup.+ mRNA.
[0091] Detailed specification one implementation of the cellular
physiology workstation is listed in Table 1.
1TABLE 1 Parameters of perfusion system and recording chamber of a
cellular physiology workstation Parameter Measured value rise time
(5% to 95%) about 70 msec to about 140 msec solution exchange time
(90%) about 8 second lag time about 100 msec flow rate about 1.5
ml/minute to about 3.0 ml/minute chamber volume about 75 .mu.l to
about 100 .mu.l dead volume about 1 .mu.l
[0092] Simultaneous Perfusion of Parallel Recording Chambers
[0093] The cellular physiology workstation may also be directed to
simultaneous recordings from multiple oocytes such as, for example,
simultaneous and coordinated perfusion of two or more recording
chambers. Several approaches can be taken to accomplish this
objective.
[0094] First, valve outputs can be divided into two or more
channels using tubing leading into separate manifolds. Solution
from each constant-flow chamber flow into a single valve, where it
diverges into two or more channels. With this approach, only one
set of solutions has to be made up prior to experimentation which
reduces the problem of slight differences in concentration obtained
from making up multiple, but distinct batches.
[0095] Second, the constant-flow chambers are manufactured with
multiple output lines. Each output line feeds into a separate valve
and, subsequently, into a separate manifold. This approach requires
a greater expenditure in valves and associated control circuitry,
but minimizes hydrodynamic problems associated with division of
solution flow.
[0096] Lastly, separate sets of constant-flow chambers can be used
for each recording chamber. Though easier to implement, this
requires a greater daily expenditure of time required to prepare
solutions as well as the expense of additional valves.
[0097] Electrophysiological recordings from Xenopus oocytes are
typically performed using a voltage-clamp amplifier in
two-electrode voltage-clamp mode. This method utilizes both a
voltage-recording electrode and a current-injecting electrode for
the control of membrane voltage. Electrophysiological recordings
from additional oocytes may require two additional electrodes per
oocyte, as well as appropriate headstages and micromanipulators for
positioning of electrodes.
[0098] The present design of commercially available voltage-clamp
amplifiers, however, only provides inputs for two microelectrode
headstages. A multichannel amplifier for electrophysiology can be
used to allow simultaneous recordings from multiple cells. Several
approaches can be taken.
[0099] First, simultaneous recordings can be based on commercially
available voltage-clamp amplifiers. One amplifier and two
intracellular electrodes can be used with each recording chamber in
a typical recording configuration. Electrophysiological traces are
acquired from the multiple amplifiers using the currently existing
data acquisition system, as described below, which has the capacity
to capture up to 10 channels of analog data.
[0100] Low-cost amplifiers that have a smaller size and lack the
extensive features found on higher cost amplifiers may also be
used. Several of these amplifiers can be combined into a
multi-channel device that readily handle simultaneous recordings.
Electrophysiological traces from these separate amplifiers would
feed into the currently existing data acquisition system.
[0101] Another embodiment incorporates an amplifier that is
designed to handle multichannel data and facilitate simultaneous
recordings. This device can have inputs for up to about 10, or
more, microelectrode headstages and the appropriate circuitry for
electrode zeroing, bridge balancing and adjustments for
capacitative currents and series resistance. Designed from the
outset as an amplifier for simultaneous recordings from multiple
cells, this instrument is able to acquire multi-channel data at
high speed and smoothly integrates into a fully automated system
for simultaneous recordings.
[0102] Another embodiment is based on designing a novel instrument
that accepts input from about 10 microelectrode headstages and
feeds the data into standard, commercially available voltage clamp
amplifiers. This device would have its own circuitry to maintain
the proper holding potential for a given cell while the device is
cycling through the other cells. This type of device eliminates the
need and expense of purchasing multiple amplifiers.
[0103] One embodiment of the invention utilizes the MacADIOS II
board by GW Instruments, Inc., for data acquisition and instrument
control. This NuBus-based board is configured with two
analog-to-digital converters (ADC) that can acquire 2-channel data
at about 25 KHz, but is also capable of multiplexed data
acquisition of about 10 or more channels at about 3 KHz, which is
of sufficient resolution for Xenopus oocyte electrophysiology.
Thus, the design of the automated workstation for electrophysiology
enables simple scale-up which may not require additional computer
or data acquisition boards. Electrophysiological traces from the
additional recording chambers would be either displayed in separate
windows or superimposed for independent viewing and analysis.
[0104] An application of a cellular physiology workstation with
highly parallel monitoring and perfusion capabilities is the rapid
generation of dose-response data from multiple oocytes. A series of
agent solutions of increasing concentration can be prepared in the
constant-flow chambers of the perfusion system and the lines and
manifold primed to load an agent to be tested. By sweeping the
microscope head across the stage of parallel recording chambers,
RNA-injected oocytes are successively positioned in each recording
chamber and impaled with both voltage-recording and
current-injecting microelectrodes. Electrode zeroing, bridge
balancing, and adjustments for capacitative currents and series
resistance are each independently performed as required on each
voltage-clamp amplifier. For a typical dose-response experiment,
multiple voltage-clamp amplifiers are simultaneously stepped to a
holding potential appropriate for the ionic current under study.
This can be accomplished by distributing the output of the
digital-to-analog converter (DAC) from the MacADIOS II board to the
separate voltage-clamp amplifiers. The perfusion system may be set
to deliver the desired agent solutions to the multiple recording
chambers simultaneously. Depending upon experimental design,
multiple oocytes may be simultaneously exposed to the same agent to
replicated a single experiment on multiple oocytes, or each oocyte
can be exposed to a different agent to facilitate rapid screening
of large drug libraries. Current and voltage recordings are
acquired from each amplifier and fed into appropriate windows in
the SuperScope II virtual instrument. At the conclusion of the
protocol, automated routines perform waveform analysis on each
current recording.
[0105] While the automated electrophysiological workstation is
particularly suited to oocyte electrophysiology, the approach
described in general can be readily adapted to patch-clamp
electrophysiology, calcium imaging studies, confocal microscopy and
other applications where perfusion control and data acquisition
need to be tightly integrated. Any type of biosensor capable of
producing an electrical output, such as a sensor capable of
measuring concentrations of substances within the cell, can be used
in place of or in addition to the voltage-measuring electrode.
Biosensors are well known to those of skill in the art and are
reviewed for example by Lowe (Lowe, C. R. Biosensors, Trends in
Biotechnology, 2:59-65, 1984) and by Byfield and Abuknesha
(Byfield, M. P., Abuknesha, R. A. Biosensors & Bioelectronics
9:373-400 1994). Other automation aspects that may be optionally
incorporated into the automated cellular physiology workstation are
digitally controlled voltage-clamp amplifiers, and robotics and
machine vision to automate the tasks of oocyte placement and
microelectrode positioning to result in a fully automated
electrophysiological assay system.
[0106] In another embodiment, the present invention relates to a
high-throughput, parallel electrophysiology system which will
enable simultaneous recordings from multiple cells. The embodiment
to be discussed here is a 4 recording chamber embodiment, however,
one of skill in the art will recognize that scaling up to a larger
number of recording chambers is a matter of routine
experimentation.
[0107] In the 4 recording chamber embodiment, the dissecting
microscope discussed in connection with embodiments described above
is replaced with video cameras. A dedicated camera is positioned
above each of the 4 recording chambers. A number of video systems
have been evaluated and a commercially-available, inexpensive color
CMOS camera has proven adequate. The CMOS camera/lens system
employed can give 10-20.times. magnification at a working distance
of about 4 inches. Video images from each of these cameras feed
into a commercially-available video controller box that is used to
select between video feeds. For oocyte impalement, the operator
stands in front of a video monitor and a pair of joysticks which
are used to control electronic micromanipulators. Microelectrode
positioning and oocyte impalement are accomplished on the basis of
the video image. The operator can switch between the different
video feeds from the 4 recording chambers with the video controller
box, and can switch between 4 different micromanipulator pairs with
a micromanipulator controller box.
[0108] Video-enablement will set the stage for full automation of
the electrophysiology process where video images will be fed into a
computer via a frame-grabber card. Image analysis software will
automatically determine where the microelectrodes are in relation
to the oocyte and guide microelectrode positioning and
impalement.
[0109] Image analysis software is used to identify and outline the
circular shape of the oocyte on the video image. At a 10-20.times.
magnification that the CMOS cameral will enable, the oocyte will be
taking up about 50% of the field of view, allowing for easy
identification. Similarly, the glass microelectrodes will be
identified in the video image based on the distinctive outline of
the electrodes. The microelectrodes are controlled by
commercially-available electronic micromanipulators that are
computer controllable. The image analysis software is therefore
able to move the electrode tip into an appropriate position above
the oocyte, for example, positioning the tips at a point halfway
between the oocyte center and edge. In this manner, the
microelectrodes will be moved into position directly above the
oocyte in an automated manner. This approach can be utilized for
either 2 separate microelectrodes, or for a single unit
"double-barrel" microelectrode.
[0110] At this point, the microelectrodes will be positioned above
an oocyte. As the electrode is lowered toward the oocyte,
monitoring of the electrode junction potential will allow
determination of tip location in the following manner.
2 Phase Resistance Air Infinite In solution Very low Contacting
oocyte Increasing Impalement Low
[0111] Therefore, automated routines are developed to automate the
process of microelectrode descent and oocyte impalement. This phase
does not require any video-based information, but electrode
impalement can be followed on the video monitor. If desirable,
video-based tracking of membrane dimpling that occurs during
impalement could be used to provide additional information for
automated impalement. The membrane invaginates as the
microelectrode pushes inward and then pops back upon penetration.
Image analysis software for tracking these events can be developed
routinely.
[0112] More advanced analysis can also be undertaken. For example,
automated testing of cell viability and response can be addressed.
Upon impalement, an automated determination can be made if resting
potential and membrane resistance fall within acceptable predefined
ranges. The impaled oocyte can be perfused and allowed to
equilibrate for a predetermined period of time prior to such
testing. Control responses can be obtained from the cell to
determine if receptor expression levels are within an acceptable
range. All these determination are made in an automated manner.
[0113] In addition, advanced waveform analysis routine can be
developed. Specific analysis modules are developed for analysis of
responses from ligand-gated ion channels, voltage-gated ion
channels, G-protein coupled receptors, etc. Each module will have
routines that will enable the measurement of relevant parameters of
a typical response including amplitude, onset time, desensitization
rate, etc.
[0114] The system is also useful for automated plotting. For
example, automatic generation of concentration-response and
current-voltage plots on the basis of averaged data from multiple
experiments can be enabled in the system. Such features will
greatly reduce the time and effort necessary to generate plots from
electrophysiology data.
[0115] Other embodiments and uses of the invention will be apparent
to those skilled in the art from consideration of the specification
and practice of the invention disclosed herein. All references
cited herein, for whatever reason, are specifically incorporated by
reference. The specification and examples should be considered
exemplary only with the true scope and spirit of the invention
indicated by the following claims.
EXAMPLES
Example 1
[0116] Oocyte Isolation.
[0117] Female, oocyte positive Xenopus laevisfrogs, purchased from
Nasco, Inc. were kept on an about 12 hour light/about 12 hour dark
cycle. Frogs were maintained on a diet of chopped calf liver fed
about every three days. Prior to surgery, frogs were anesthetized
in a solution containing about 0.15% Tricaine for about 30 minutes.
Ovarian sections were removed through a lateral abdominal incision,
after which the incision was sutured with about 4 to about 5
stitches and the animal allowed to recover in isolation for about 3
hours to about 4 hours. Ovarian lobules containing follicular
oocytes were immediately placed in calcium-free ND96 solution (96
mM NaCl, 1 mM MgCl.sub.2, 2 mM KCl, 50 mM Hepes, 2.5 mM pyruvate)
and cut into groups of about 10 to about 20 oocytes. Following a
treatment with collagenase (Sigma, type II, 2 mg/ml) at about 2
mg/ml for about 2 hours at room temperature, individual oocytes
were obtained free of their follicular layer. Selected Dumont stage
V and VI oocytes were then transferred to 60.times.15 mm glass
petri dishes containing ND96 (96 mM NaCl, 1 mM MgCl.sub.2, 2 mM
KCl, 50 mM Hepes, 2.5 mM pyruvate) and maintained in an incubator
at about 18.degree. C. to about 20.degree. C. On the following day,
batches of about 20 oocytes to about 30 oocytes were injected with
about 30 nl to about 80 nl prepared RNA solution using an
electronic microinjector (Drummond Instruments, Inc.).
Example 2
[0118] RNA Preparation.
[0119] RNA was prepared for injection into oocytes by extraction of
mRNA from brain tissue and by synthesis using in vitro
transcription of linearized DNA templates encoding recombinant
receptor subunits.
[0120] The extraction technique of RNA preparation uses brain mRNA
from chick embryos of about 19 day old as starting material.
Extraction was performed using the Dynabeads Oligo-dT (.sub.25)
isolation kit (Dynal, Inc.) which utilizes magnetic beads having an
attached poly-thymidine oligomer to allow magnetic separation of
poly(A).sup.+ RNA from cell homogenates.
[0121] In vitro transcription was performed using plasmids
containing the GluR3 (flop) and GluR6 cDNA as starting material.
Plasmids were linearized with restriction endonuclease XhoI (GluR3)
or XbaI (GluR6) prior to in vitro transcription with T3 RNA
polymerase using a commercially available kit (Message Machine;
Ambion, Inc.; Austin, Tex.).
Example 3
[0122] Electrophysioloqy.
[0123] About 3 days to about 5 days after RNA injection,
electrophysiological recordings were carried out using an
Axoclamp-2A voltage clamp amplifier (Axon Instruments, Inc.).
Experiments were performed in two-electrode voltage clamp mode
using two intracellular microelectrodes of about 1 to about 3
mega-ohm resistance filled with a solution of about 3M KCl. A
close-up view of an oocyte under impalement in voltage-clamp mode
is shown in FIG. 7.
[0124] Oocytes were usually clamped at a holding potential of about
-60 mV and stepped to about -100 mV during agent application. GluR6
injected oocytes were treated for about 10 minutes with a solution
of about 10 .mu.g/ml concanavalin A to prevent fast desensitization
of kainate responses.
Example 4
[0125] Application of GABA to Oocytes.
[0126] In this experiment, a solution comprising about 100 .mu.M
GABA was applied to an oocyte expressing GABA.sub.A receptors to
test the function of the automated workstation. Oocytes were
immobilized in the recording chamber, impaled with
voltage-recording and current-injecting microelectrodes, and
allowed about one to about two minutes to recover to a resting
membrane potential of about -40 mV to about -50 mV. The amplifier
was switched into voltage clamp mode, typically at a holding
potential of about -60 mV, prior to the start of experimentation
protocol.
[0127] A typical experimental protocol may comprise the steps of
prepulse, agent application, and washout phases and the function of
each phase may be set up in advance by defining the positions of
on-screen markers M1-M4, which can be easily moved via a user input
device such as a computer mouse. The PREPULSE VALVE selector is
used to select a prepulse solution which may be used to
pre-equilibrate with a modulator before coapplication of modulator
and agonist. The modulator is applied during the interval M1 to M2.
The DRUG VALVE panel of buttons is next used to select the solution
that is to be applied during the interval M2 to M3. Finally, the
WASH VALVE selector is used to select the solution to be applied
during the washout phase of the protocol, defined as the interval
M3 to M4. The WASH VALVE selector also controls the WASH TIMER,
which perfuses the oocyte for a preset amount of time between
successive episodes.
[0128] After the perfusion controls were set, the VC COMMAND slider
is used to select the voltage offset that will be sent to the
amplifier to determine the holding potential at the start of the
first episodes. The oocytes were held at about -60 mV and stepped
to about -100 mV at the start of data acquisition to increase
electrochemical driving forces; this entails a voltage offset being
sent to the amplifier of about -40 mV. Experiments usually consist
of multiple episodes per trial, where an episode is defined as a
single cycle of data acquisition. The EPISODES PER TRIAL selector
is used to select the number of episodes to be acquired, after
which the experiment is started by pressing the BEGIN button. At
the initiation of data acquisition, the holding potential is
stepped to the preset voltage, and perfusion commences with the
selected solutions. At the end of data acquisition, the current and
voltage traces were saved to disk, and the oocyte is perfused with
wash solution for a predetermined amount of time. Each episode is
logged to the journal window, where analysis routines measure and
log waveform parameters. From this basic protocol, more complex
protocols were developed that automatically execute repetitive
application, dose-response, reversal potential and voltage-stepping
experiments.
Example 5
[0129] Repetitive Application Experiments.
[0130] The EPISODES PER TRIAL selector is used to specify a
repetitive application protocol, in which multiple cycles of agent
application are desired. FIG. 15 shows the results from an
experiment in which 100 .mu.M GABA was applied repeatedly to an
oocyte injected with chick brain poly(A).sup.+ RNA. A protocol of
about 30 seconds was used, consisting of a 10 second prepulse with
buffer solution, a 10 second application of 100 .mu.M GABA, and a
wash phase of about 10 seconds, followed by a wash cycle of about
60 seconds with buffer solution prior to the next application of
agent. An increase in current amplitude was evident that reached a
plateau over the course of about one hour experiment in which 100
.mu.M GABA was applied 30 times. These experiments demonstrate the
utility of the workstation that has been developed, as the
experiment is performed automatically without any operator
intervention. This type of protocol is useful for following a
response over an extended period of time which is useful for
looking at time-dependent processes such as rundown of receptor
mediated responses. This protocol can also be used to study the
effects of compounds that have a slow time course of action, for
example, compounds that affect the phosphorylation state of
receptors, such as kinase inhibitors or membrane permeant cAMP
analogues. Lastly, the repetitive application feature makes it
possible to easily compare averaged data taken before and after an
experimental manipulation. By avoiding comparison between single
responses, averaging of data reduces errors due to response
variability, noise and time-dependent changes in response
amplitude.
Example 6
[0131] Dose-Response Determination.
[0132] An automated workstation for electrophysiology was designed
to fully automate dose-response experimentation. Typically, the
generation of dose-response data is usually achieved by application
of increasing concentrations of a given agent to a responsive cell.
FIG. 8 shows the results of a dose-response experiment in which
increasing concentrations of kainate were applied to an oocyte
expressing homomeric GluR3 kainate receptors formed from cloned
receptor subunits. For the kainate response from this cell, an
EC.sub.50 of about 27 .mu.M was calculated. This type of experiment
is initiated by pressing the DOSE/RESPONSE button, which selects a
protocol designed to sequentially step through a series of agent
solutions, beginning at the valve specified by the ALTERNATE VALVE
selector. The EPISODES PER TRIAL selector is then used to specify
12 episodes, corresponding to the number of concentrations to be
tested. The dose-response protocol closely follows the operation of
the repetitive application protocol, with the exception that this
protocol increments the DRUG VALVE selection after each
episode.
[0133] FIG. 16 shows a high-resolution dose-response curve
generated by the Cellular physiology workstation. In this
experiment, increasing concentrations of kainate are sequentially
applied to an oocyte expressing rat GluR3 receptors. An automated
protocol steps through up to about 15 different agent
concentrations to rapidly generate dose--response curves. An
EC.sub.50 of about 90 .mu.M was determined for GluR3 receptors
expressed in Xenopus oocytes. Oocyte was held at about--100 mV
during kainate application and washed for about 60 seconds between
episodes. Total duration of this experiment, which was performed
automatically without operator intervention, was about 15
minutes.
[0134] FIG. 15 depicts the reproducibility of dose-response curves
generated by the cellular physiology workstation. Four separate
GABA dose-response determinations were made about 20 minute apart
to document system performance. EC.sub.50 determinations yielded
similar results (26 .mu.M, 19 .mu.M, 20 .mu.M, and 21 .mu.M) over
the course of this 70 minute experiment. Oocytes were held at about
-100 mV during application of about 5 .mu.M, about 10 .mu.M, about
50 .mu.M, about 100 .mu.M and about 500 .mu.M GABA, and washed for
about 30 seconds between episodes. Dose-response curves were
determined on an oocyte expressing GABA.sub.A receptors after
injection with chick brain poly A.sup.+ RNA.
[0135] In a separate experiment, to determine reproducibility and
reliability of agonist responses, the cellular physiology
workstation was used to determine current responses to 30
consecutive applications of 100 .mu.M GABA (FIG. 17). Current
responses to 30 consecutive applications of 100 .mu.M GABA are
shown in an oocyte expressing GABA.sub.A receptors after injection
with chick brain poly A.sup.+ RNA. Slight increases in current
amplitude are observed during the course of this 45 minute
experiment, which was performed automatically without operator
intervention. Oocytes were held at about -100 mV during agent
application and washed for about 1 minute with Ringer solution
between each of the 30 episodes.
Example 7
[0136] Reversal Potential Determination.
[0137] The virtual instrumentation that was developed also provides
control over the voltage-clamp amplifier, thereby making it
possible to automate experiments in which holding potential may be
varied, such as those determining reversal potentials and examining
current-voltage relationships. A reversal potential for a
receptor-mediated response is the voltage at which no net current
is observed upon activation of the ionic conductances associated
with the receptor. Reversal potentials are typically determined by
applying a given agent at various holding potentials, plotting
current vs. holding potential, and calculating the voltage at which
the current reverses direction. FIG. 18 shows the results of an
experiment in which a reversal potential of about -15 mV to about
-10 mV was determined for the kainate response in oocytes
expressing GluR6 kainate receptors. The holding potential was
progressively increased from about -80 mV to about +25 mV in 11
steps of about 10 mV. The oocyte was returned to a holding
potential of -60 mV and washed for about 30 second after each agent
application. The direction of the kainate-induced current was found
to reverse between about -15 mV and about -10 mV. This type of
automated protocol can also be used to determine current-voltage
relationships, in the absence and presence of a receptor modulator
and to investigate mechanisms of action of modulatory drugs. For
these types of voltage experiments, the STEP SIZE slider is used
with a multi-episode trial to increment the VC COMMAND offset,
which controls the holding potential of the amplifier.
Example 8
[0138] Voltage-Stepping Response.
[0139] The examples described above used a perfusion system for
applying a receptor ligand during the period between the M2 and M3
markers to generate a receptor-mediated response. The cellular
physiology workstation may also be programmed for examining
voltage-gated ion channels. For example, the Xenopus oocyte
membrane has a number of well characterized voltage-dependent
conductances, including an endogenous chloride current (I.sub.C1-),
an endogenous calcium-dependent chloride current
(I.sub.Cl-.sub.(Ca++)) , and an endogenous sodium current
(I.sub.Na+), that can sometimes interfere with other currents of
interest and may sometimes be subtracted out. FIG. 19 shows traces
from an experiment using the cellular physiology workstation to
examine endogenous, calcium-dependent chloride current
(I.sub.Cl-.sub.(Ca++)) found in native Xenopus oocytes. Voltage was
stepped from about -100 mV to about +20 mV, each time returning to
a holding potential of about -60 mV, to determine the
current-voltage relationship for this conductance. In these types
of experiments, voltage is stepped from thy amplifier's holding
potential to a voltage determined by the VC COMMAND and STEP SIZE
sliders during the M2-M3 interval. Predefined protocols are in this
way established for voltage-dependent conductances of interest, and
can be easily selected through on-screen buttons. This type of
protocol can be used to screen recombinant voltage-dependent ion
channels against libraries of agents such as drug libraries.
[0140] Outlined herein is a set of hardware and software for
high-throughput, parallel electrophysiology enabling simultaneous
recordings from multiple cells. The software will be developed
using the LabVIEW (TM) Professional Development System, Version 6i
for Windows 95/98/2000/NT. The completed application package to be
delivered will consist of all source code VIs along with compiled
executable modules. The Professional Development System includes an
Application Builder, which is used to create stand-alone executable
routines that may be distributed among several PCs without the need
for additional site or software licenses. The hardware proposed
herein is based on a combination of National Instrument's E-series
plug-in PCI-bus data acquisition boards (for analog inputs and
outputs) and FieldPoint products (for support of the digital
outputs for switching solenoid valves). This combination of
installed and remote hardware optimizes the cost/performance
tradeoffs for each kind of functionality. The installed PCI boards
can stream data continuously through buffered acquisition at the
desired maximum 3 kS/sec, while the FieldPoint modules allow for
the higher channel counts and somewhat slower response times
required for solenoid valve controls. For this application,
communications between the host PC and FieldPoint modules will be
accomplished via an RS-485 serial link.
[0141] The host PC will be housed in a standard 19-inch laboratory
equipment rack. Optimally, the FieldPoint modules are located in
close mechanical proximity to the respective solenoid valves they
control in or to minimize cabling complexity.
[0142] The system proposed will facilitate a variety of functions.
For example, when first entering the program a data acquisition
server engine will be launched and will run as a background task.
The purpose of this server engine is to acquire data continuously
into a buffer at the maximum required sampling rate, and serve
contiguous slices of that buffered data as requested to individual
cell software modules. A menu screen is displayed on the host PC.
The menu screen prompts a user to select a desired cell (e.g., Cell
1-4) to configure or operate and may use this screen as a central
navigation point from which to access any running cell software
module. In this manner, individual cell software modules may run
independently and asychronously of one another, and data may be
collected at different rates as desired.
[0143] The menu screen discussed above may contain a button
entitled "Go To Recipe Editor" which will enable customization of
an experimental protocol. Each step in an experimental protocol is
defined by which of a variety of solenoid valves are open or
closed, the resting and testing voltage clamp potential, and the
respective duration of the three phases (prepulse, drug
application, and washout) which comprise each episode. An
appropriately created Recipe file that delineates each of these
values will control operation of the hardware during a given
experimental run. The creation and editing of these recipe files is
accomplished through the use of a Recipe Editor VI, whose font
panel is similar to that shown in FIG. 21. For each discrete
parameter, the user simple clicks on the desired checkbox to select
the particular function. Various set points and tolerances are
entered through numerical controls. Intuitive editing commands
(e.g., insert, delete, go to next step, go to previous step) are
provided. When a new step is inserted, all values in that step are
initially copied from the previous step. The used can edit such
settings as desired. In this manner, steps which reflect a small
change from a previous operation are easily created with minimum
effort. Note that provision has been made to accommodate up to 100
solenoid valves, any combination of which may be turned on during
the application phase. New recipes may be created from scratch, old
recipes may be loaded and edited, and any recipe created may be
stored to file for later recall and use from a running cell
module.
[0144] Whenever the system is following a recipe and performing an
automated sequence of operations, the main display screen will be
displayed. This main display screen is similar to embodiments
described previously. Various mode control buttons, which allow the
user to start, pause or abort the current process, are provided.
The Operating Screen shows all present values of the parameters in
the system (e.g., valve open/closed status, amplifier voltage set
point, collected voltage and current waveforms) and the results of
analysis such as peak amplitude and rise time. Raw voltage and
current data are saved as a delimited text file for easy import
into spreadsheets and analysis packages. Each cell is accessed from
a Start Screen. A further description of the display functions and
modes is listed in the Specification which follows at the end of
this description.
[0145] As previously described, a combination of National
Instruments' PCI plug-in data acquisition boards and FieldPoint
hardware will serve as the primary data acquisition and control
interfaces between the host computer and the actual process
elements. For each host PC, a PCI-6034E (16-bit, 16 channel input
card) and a PCI-6703 (16-bit, 32 channel output card) are
installed. A nominal count of four cells per host PC is initially
targeted, with the potential for future expansion as practical. The
requirement of 2 analog inputs per cell dictates that for each
installed PCI-6034E card, a maximum of 8 cells may be accommodated.
Wiring to and from these PC cards shall be accomplished though the
use of shielded cable assemblies and related modular screw-terminal
blocks, which facilitate easy wiring and servicing. The initial
FieldPoint physical configuration will consist of one "bank" of six
FP-DO-403 16-channel current-sinking output modules, controlled by
(and communicating to the server computer with) an associated
Network Module. One bank of modules will provide 96 discrete
outputs, sufficient for four cells up to 24 solenoid valves per
cell. Additional solenoid valves, as well as future expansion to
higher cells per host PC, can be accommodated in a modular manner
by adding further banks of Network and Output modules. These banks
are DIN-rail mountable in any convenient location. We envision that
an appropriate instrumentation cabinet will be employed to house
both the FieldPoint control elements as well as the associated
solenoid valves. Each bank will be powered by a dedicated modular
DC power supply.
[0146] This project requires the use of an appropriately modem and
powerful PC to serve as a host platform. At a minimum, this PC must
have the following specifications:
3 Processor: 600 MHz Pentium .RTM. III Memory: 256 MB 100 MHz SDRAM
or more Hard Drive: 20 GB or larger Monitor: 17" or larger, 0.28 dp
Video Card: AGP graphics card, 8 MB or more Network Card: 3 Com
C905 C TXM 10/100 or equivalent Modem: V.90/56 K PCI DataFax Modem
or equivalent CD-ROM: 48X max variable CD-ROM Drive Re-writable
Large Media: 100 MB Zip Drive or CD-RW Drive Installed Shrink-wrap
Software: Windows 98 2E/2000/NT 4.0 Recommended: Office 2000 McAfee
VirusScan 4.02 PcAnywhere 32 v9.0
[0147] Specifications
[0148] A minimal implementation of the automated parallel
electrophysiology system would consist of the following:
[0149] An initial design that would enable the control of 4
separate chambers. The modular software design should be easily
scalable as the number of chambers is increased from 4 to 100.
[0150] The ability to acquire, display, and save current and
voltage waveforms from multiple chambers at between 300-3000 Hz for
each waveform for a period between 1 and 600 seconds. We would want
to be able to view, in real-time, simultaneous traces from all
amplifiers on screen as they are acquired.
[0151] Ability to control 4 separate banks of solenoid valves, each
bank initially consisting of 20 valves, expandable to 100 valves.
Each bank would be addressable as to which valve is turned on
during periods that would be set by on-screen movable markers. Each
individual drug application is divided into three phases: 1)
pre-application, 2) application, and 3) wash-out, each of which may
be of varying duration (which may be zero). Independent control of
the solution (valve) and duration is required for each of these
three phases. In addition, it should be possible to program one or
more changes in holding potential to an operator determined
potential, via a command to the appropriate amplifier at any time
during any of these phases, by placement of an on-screen marker on
some kind of timeline or graph. After wash-out, the cell is
returned to its resting potential and allowed time to recover
during the washing phase, during which it is perfused with buffer
for a period specified by separate wash timers (0-240 sec) for each
chamber.
[0152] Basic waveform analysis initially consisting of amplitude
measured from peak response to an averaged baseline for each
response.
[0153] Generation of a textual experiment log that would
automatically log the experimental protocol similar to that seen in
the existing implementation. A log should be generated indicating
the sequence and times of drug application. There should be a
method for the operator to insert comments into the log. The log
should be saved to disk after each drug application. A mechanism
should be provided for the operator to enter labels to identify
specific drug solutions (valves). A method should be provided for
saving all settings to disk, including operator comments.
[0154] On-screen controls for the control of 4 separate
voltage-clamp amplifiers at different voltages if necessary.
[0155] Built-in protocols for dose-response and voltage-stepping
experiments (described in previous materials). Dose-response
protocols should include the ability to specify the sequence of
solution applications (valves to be activated): increasing
numerical sequence, decreasing sequence, or an arbitrary,
operator-defined sequence. As well, an automated method should be
provided for repeating out a "standard" treatment between each test
treatment. For example, a dose response study might involve the
following sequence: 1, 2, 1, 3, 1, 4, 1, 5, 1 . . . (where the
numbers denote specific solutions/valves).
[0156] Ability to do the same experiment in parallel. We would also
want to be able to easily do the same protocol simultaneously on
all 4 chambers. In such a manner, for example, we could do 4
simultaneous dose-response experiments on 4 cells. In such a case,
the same set of drug reservoirs would feed into the 4 sets of
solenoid valves.
[0157] A method for taking one or more chambers off-line, and
suspending drug applications to those chambers while continuing to
conduct experiments with the remaining preparations. As well, we
would want to be able to abort for each chamber in real-time if the
cell goes bad, as we wouldn't want to be wasting drugs and using up
disk space on data from a dead oocyte.
[0158] A list of all the data acquisition hardware and software
that will be necessary for this project.
[0159] Other embodiments and uses of the invention will be apparent
to those skilled in the art from consideration of the specification
and practice of the invention disclosed herein. All references
cited herein, for whatever reason, are specifically incorporated by
reference. The specification and examples should be considered
exemplary only with the true scope and spirit of the invention
indicated by the following claims.
* * * * *