Evolution of microelectrode array plates fabricated at UNT
MMEP 8.256: Same plate
as MMEP-8 but with all 256 electrodes located in a center area to record
from one network. Amplifier contacts are also the same, allowing use of
common electronic data acquisition equipment.
Plate dimensions: 56 x 90 mm
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All
plates consist of optically flat glass and transparent indium-tin oxide
conductors. Plates are spin insulated
with methyltrimethoxysilane
resin and de-insulated at the tips of the conductors with single laser
shots. Except for the masks, all
fabrication is performed by students working with the Center.
MMEP 4: 64 microelectrodes arranged in an 8 x 8
format with equal electrode separation of 150 um.
MMEP 5: 64 microelectrodes serving two separate
recording matrices of 32 electrodes each.
MMEP 6: 64 microelectrodes arranged to form three
recording areas with 16 electrodes each connected
by two linear
conduits each with 8 electrodes.
NOTE: MMEPs 4-6 have the same amplifier connections
MMEP 8: 8-network plate with 32 electrodes per
recording matrix. Compatible with Plexon Inc. 256 preamplifier circuit board.
Summary
of Methods and Examples of Results
Figure
1.
Examples of neuronal circuits on microelectrode arrays. Transparent conductors allow extensive
optical access to the network morphology.
(A) Neuronal network derived from murine spinal cord tissue (98 days
in vitro), grown on the recording matrix of a 64-electrode array
plate. Bodian stain. (B-D) Living neurons on MEAs Gold-plated exposed ITO conductors are
shown by arrows in (B). The conductors are 8 mm wide. (all bars = 40 mm).
Figure
2. (Right) Electronic display of
time stamp patterns (A), waveforms (B), and electrode layout (C).
(Plexon Inc., Dallas).
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EXAMPLE 1. Zinc Toxicity (Parviz, M. and Gross,
G.W. (2007) Quantification of zinc toxicity using
neuronal networks on microelectrode arrays.
NeuroToxicology 28: 520-531.)
Figure 3. (A) Common network response to high
concentra-tions of zinc. Each data point represents the average spike (left
ordinate) and burst (right ordinate) rate in 1 min bins for all
discriminated units. Addition of 200
mM
zinc at 38 min results in an immediate excitatory period lasting 70 min
followed by activity decay for about 30 min and complete, irreversible
activity loss. (B1-B6) Consecutive pictures of the same neuron taken at
approximately 30 min intervals. At
220 min (B6, right panel), the neuron is swollen and necrotic in
appearance.
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Figure
4 (below). Pooled
data from spinal cord and frontal cortex cultures. A double log plot reveals linear
functions for 50% and 90% activity decreases. All experiments (n=23) were
conducted in serum-free and albumin-free medium. Activity losses were irreversible.
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DMEA+O-CO-S(CH2)4S-CO-DMEA+
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Screening of 7 newly
synthesized weak AChE blockers for alleviation of Alzheimer’s syndrome synthesized at the
University of Perugia, Italy by Prof. Vincenzo Talesa.
BIOCHEMICAL DATA CONFIRMED
BINDING TO AChE.
However, two out of seven compounds
were irreversible inhibitors of activity (unexpected secondary binding.)
Keefer,
E.W., Norton, S.J., Boyle, N.A.J., Talesa, V., and Gross, G.W.
(2001) Acute toxicity screening of
novel AChE inhibitors using neuronal networks on microelectrode
arrays. NeuroToxicology 22: 3-12.
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Figure 5. Desired spike and burst response profiles activity
increases at defined concentrations with reversible toxicity at higher
concentrations. Such drug efficacy
range information can be obtained in a few hours and used to guide and
minimized subsequent animal experiments.
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Figure 6. Unexpected irrever-sible toxic effect
at 300 mM. Activity could not be recovered by
three medium changes and during 24 hr monitoring.
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EXAMPLE 2. Screening Novel AChE Blockers
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Comparison of ethanol
effects on mammals and FC cultures
Mammal Conc. Effect
humans 5-15 slight impairment of attention and judgment
humans 10-22 impairment of speech and balance
humans 15-30 significant sedation
rats 20 sedation
FC cultures 10 first
measurable change in activity pattern
(slight) excitation
FC cultures 15 first
overt change in spike and burst production
(inhibition)
humans 30-55 mental confusion
mice 40 loss of righting reflex
FC cultures 48.8 EC50
for spike production
humans 100 coma and death
mice 120 coma, hypothermia
FC cultures 100-140 cessation of
all spontaneous activity
_________________________________________________________
(human
and animal data from Charness et al., 1989; Little, 1991).
FC:
frontal cortex cultures
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Fig. 7. (A)
Responses of networks to low and high concentrations of ethanol. 160 mM (60 mM past coma) for 2 hr exposures are still
reversible. (B) Dose response
curves from five networks. The mean EC50 is 48.8±5.4 mM. (C) Table comparing responses to
alcohol for humans, mice, and mouse networks in vitro.
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Fig. 8. Left Panel: Two-network module
experiment showing normal responses to botulinum toxin A at 50 ng/ml and
100 ng/ml. Irreversible loss of
activity occurs at approximately 3 hrs after application. Right Panel: Protection of exposed
network by mouse antiserum.
Activity is still strong at 20 h.
Note: the two traces in the right panel are showing mean spike
production (green) and mean burst production (blue) of a single network.
The left panel shows mean spike production per minute
for two independent networks.
Paper in preparation.
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EXAMPLE 3. Histiotypic Mammalian
Network Responses to Ethanol and Responses to Botulinum Toxin A.
EXAMPLE 4. Pharmacologically
Induced Different Network States (Spike and Burst Patterns)
Fig. 9. Network
exposure to 11 different pharmacological states results in reliable pattern
changes expressed here as clusters of burst rate plotted against burst
duration. The native raster plot and that for a much more organized burst
oscillation state (induced by blocking all synapses except NMDA synapses) are
shown in the panels to the right. State
11 at different Ca++ concentrations is expanded in B.
From Keefer, Grawowski and Gross (2001) J. Neurophysiol.
EXAMPLE 5.
Quantitative Pharmacology:
Determination of Dissociation Constants
Determination of Kd values for the GABAA receptor
blockers bicuculline, gabazene, and
Figure 10.
(A) Concentration response curves of the normalized data in absence
and in the presence of 10, 20, 40, 80 µM of bicuculline. Vertical bar
represented mean and SEM of spike activity change. (B):
Schild plot of log (dose ratio-1) vs log bicuculline concentration
(Molar) for the antagonism of muscimol. The fitted solid line is determined
by a linear (least square) fit without slope constraint. PA2=6.3, Kd=0.52
µM
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trimethylol
propanephosphate.
Figure 11.
(A) Muscimol titration in the presence of 0, 10, 20, 40 µM of gabazine. Vertical
bars represent mean and SEM of spike activity change. (B): Schild plot of log (dose ratio-1) vrs
log gabazine concentration (M) for the antagonism of muscimol. Linear fit
(least square) without slope constraint.
PA2=7.9, Kd=0.015 µM
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Fig. 12. Environmental chamber enclosing liquid
handling robot. Chamber maintains
sterility, 10% CO2 for pH control , constant humidity, constant
temperature. Oxygen levels can also
be controlled.
Left: Preliminary tests with two-chamber
module and 64 preamplifiers.
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Fig. 13. Preliminary data from robotic pharmacological experiments. 72 hour robot manipulation of a
single network in an environmental chamber.
The panel shows 10 cycles of medium changes with 40 uM bicuculline
and titration with muscimol. Program
manipulations caused some response profile changes (e.g. cycle 3). System noise (N), is caused by stepping
motors and some vibrations of amplifiers during robot movement.
Improvements in programs, noise abatement, and mixing of test substance
with medium should provide almost identical response profiles. All data points represent “minute means”
(all discriminated, digitized units averaged in one minute bins). Bicuculline was added to wash medium pool
before experiment.
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EXAMPLE 6. Robotic culture maintenance.
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Fig. 14.
Design of 8-network array plate (glass with transparent indium-tin oxide
conductors) and first electrophysiological recording on a microscope stage
(Dec 24, 2005). Note only two
32-channel preamplifiers were available.
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EXAMPLE 7.
8-Network Array Design and First Recording
First simultaneous multinetwork pharmacology experiment
(Dr. Edward Keefer, UT Southwestern Medical Center using CNNS
facilities)
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Control
25 mM
carbachol
25 mM
carbachol + 25 mM
scopolamine 25 mM carbachol + 10 mM dopamine 25 mM carbachol + 10 mM risperidone
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Left: 8-network assembly with 8 VLSI preamplifier modules for 32 channels
each. Above. 5-network simultaneous recording
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