Multielectrode Arrays

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Multielectrode Arrays

• Joe Kostansek, Greg Loney, Ariel Simonton

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• Bundles of electrodes situated together to
  facilitate recording of field potentials and in
  some instances stimulation of brain areas
• These bundles are capable of recording from
  hundreds to thousands of neurons at a time and
  are typically considered chronic
• Typically PCA is used to segregated individual
  neurons or individual neuronal phenotypes

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• 1958 Strumwasser
• Utilized single 80-mm stainless steel wires to
  record from awake, behaving squirrels
• Recordings lasted for a week or longer
• Strumwasser concluded that the constant waveform
  and amplitude implied that he was recording from
  the same neurons repeatedly
• Techniques have largely remained unchanged for
  the last several decades

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• Due to the small diameter of microwires, position
  is not fixed and fluctuates with movement, BP,
  etc
• With the advent of silicon based electronics and
  reduced price in the 1970s, fixed arrays now
  become possible
• Due to strength of silicon and thus increased
  surface tension, more electrodes are able to be
  implemented and implanted
• Michigan-Array is one of the first silicon based
  arrays. Due to increased density of probes,
  researchers are now able to record from soma and
  dendrites simultaneously

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• Utah-array Developed in the late 1980s-early
  1990s
• Each Individual silicon tong contains a
  platinum insulated microelectrode thus allowing
  for higher resolution at each individual
  recording point
• Notice the scaled position of each tong. Due to
  increased resolution, researchers are know able
  to record from various layers of cortex, which
  may contain different cell types, with a greater
  degree of accuracy
• Rigidity of array causes some problems in vivo

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• Polymide-based electrode cuff
• Highly flexible
• Rings of electrodes allow for continuous
  recordings, especially of peripheral nerves

• Sieve electrode
• Nerve is cut and allowed to regenerated through
  the holes in the sieve
• Often coated with BDNF or other NTFs in order to
  facilitate regeneration and approach

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Multielectrode Arraysin vivo
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Advantages

• Ability to record or stimulate hundreds to
  thousands of neurons
• Can correlate activity across many neurons
• Can be used in awake, behaving animals to study
  higher processes
• Memory formation
• Sensory integration
• Chronic or Acute
• Potential human clinical uses
• Deep brain stimulation
• Seizure control
• Restoring motor control

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Disadvantages

• Cannot look at single ion channels
• Better suited for recording action potentials
• Typically implanted chronically
• Can cause immune response and inflammation
• May lead to neurodegenerative effects
• Not a disadvantage, but Provides a LOT of data,
  its necessary to have sophisticated sorting
  software to properly analyze results

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• Analysis of Multielectrode Array Data

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Sample recording from the amygdala

• Individual spikes are recorded into a program and
  can be bundled (as seen here) depending on
  waveform properties.
• Some arrays have the ability to individually move
  each wire.
• Beneficial for recording from multiple layers of
  cortex

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Single Neuron Analysis

• First step analysis of individual recorded
  neurons
• Commonly used raster plots and peristimulus time
  histograms (PSTH)
• These figures graph individual firing patterns
  over time so that they may be correlated to
  either a stimulus or behavioral event.
• Example data Shows an individual neurons
  response to mechanical stimulation of a digit on
  the hand of an owl monkey

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Neural Ensemble Response

• Next step Visualizing neural activity as a whole
• Population Peristimulus Time Histograms (PPSTH)
• Spatiotemporal Maps
• Linear Time Series Analysis
• Artificial Neural Networks

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Population Peristimulus Time Histogram

• X-axis
• Individual neurons arranged rostral/caudal or
  some other method of organization
• Y-axis
• Peristimulus time
• Z-axis
• Instantaneous firing rate (spikes/sec)
• Spike counts per time bin

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Spatiotemporal Population Map

• Magnitude of neuronal firing of standard
  deviations away from spontaneous firing rate

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Linear Time Series Analysis

• Best for continuous stimuli or behavioral
  variables
• Uses neuronal inputs and behavioral outputs in a
  modified linear regression equation
• X(t) matrix
• Columns Single neurons
• Rows Time segments
• If data is 3D, then x,y,z is analyzed in a
  separate matrix and incorporated with X(t) matrix

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Artificial Neural Networks (ANNs)

• Very important tool to look at neural ensemble
  activity in relation to sensory input or
  behavioral output
• Require no a priori assumptions
• Useful for both categorical and continuous stimuli

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Optimized Learning Vector Quantization

• Three layers input, hidden, output
• Input Data
• Hidden Two artificial neural units
• Output Prediction
• A method of analysis in which the program
  learns which neural patterns are associated
  with a given output
• Can follow with principal component analysis

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Summary

• Multielectrode arrays are very useful tools for
  recording data from many neurons
• Creates a spatiotemporal summary of neural
  activity
• Information recorded from multielectrode arrays
  may be analyzed for individual cells as well as
  populations
• Advances in understanding of sensory perception,
  learning and memory, and other higher processes
  can be contributed to the introduction of
  multielectrode arrays

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Nucleus accumbens neurons are innately tuned for
rewarding and aversive taste stimuli

• Roitman, MF, Wheeler, RA Carelli, RM

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Methods

• Rats were implanted with intraoral catheters, and
  microelectrode arrays in the anterior digastric
  muscle and nucles accumbens
• Rats received 30-cue trials (light tone) of
  both sucrose and QHCl, presented after a variable
  delay
• EMG activity recorded from anterior digastric
  muscle
• Individual neuronal activity recorded from nucles
  accumbens

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A B EMG activity data in response to intraoral
infusions of sucrose and QHCl, respectively C
D Sample EMG traces indicating learning of
taste-cue pairings for sucrose and QHCl,
respectively E F Average latency to first EMG
burst for cue-paired sucrose and QHCl,
respectively. Bars below the line indicate that
first burst occurred during cue, bars above the
line indicate that first burst occurred during
infusion G Note large amplitude bursts
following QHCl infusion H EMG activity
displayed a trend to decrease as a function of
infusion duration C
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Four classes of cells Raster plot and spike
frequency bins (histograms) of four
representative cells A Sucrose inhibitory B
Sucrose excitatory C QHCl inhibitory D QHCl
excitatory
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Average firing rates of the 4 identified classes
of cells Sucrose inhibitory (39 0f
102) Sucrose excitatory (13 of 102) QHCl
inhibitory (10 of 98) QHCl excitatory (30 of 98)
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Representative sucrose inhibitory cell Note the
opposite pattern of firing for both types of
stimuli
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Firing rates for all four identified cells
plotted against EMG activity for each 100 ms bin
of pre-infusion (6 s black) and post-infusion (6
s gray). Sucrose inhibitory negatively
correlated Sucrose excitatory positively
correlated QHCl inhibitory negatively
correlated QHCl excitatory positively correlated
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Four new classes of cells Raster plot and spike
frequency bins (histograms) of four
representative cells A Sucrose-cue
inhibitory B Sucrose-cue excitatory C
QHCl-cue inhibitory D QHCl-cue excitatory
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Average firing rates of the 4 identified new
classes of cells Sucrose inhibitory (16 0f
102) Sucrose excitatory (26 of 102) QHCl
inhibitory (12 of 98) QHCl excitatory (27 of 98)
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Cue-invoked firing is significantly correlated
with learning A Firing rate increases as a
function of trial repetitions B Firing rate is
negatively correlated with latency to first burst
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Conclusions

• Individual, naïve neurons in the nucleus
  accumbens demonstrate unique patterns of firing
  to prototypically rewarding an aversive stimuli
  and are hihgly correlated with reflexive
  behaviors associated with these stimuli
• These same neurons demonstrate a reversed
  pattern of activation in response to stimuli of
  the opposite valence
• Neuronal activation demonstrates a pattern of
  adaptation reminiscent of learning
• Nucleus accumbens neurons may be innately tuned
  to encode predictions and aggregate motor output
  associated with rewarding and aversive taste
  stimuli

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Cortical Excitation and Inhibition following
Focal TraumaticBrain Injury

• Ming-Chieh Ding, Qi Wang, Eng H. Lo, and Garrett
  B. Stanley

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Background

• Brain injuries causing swelling of the cortex
  leads to
• Increased extracellular K
• Altered firing rates
• Neuronal injury/death
• Stroke
• Changes from brain injury can lead to overall
  changes in network inhibition and excitation
• Purpose To assess the effects of compression
  injury on excitatory and inhibitory networks in
  vivo.

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Methods

• Male Long-Evans rats
• Microarray is implanted into the barrel cortex
  (primary somatosensory cortex)
• 90 minute recovery time
• Array
• 8 x 8 silicon electrodes
• 1mm length
• 400um spacing
• 100-400 kohm impedance
• Stimulus
• Mechanical stimulation of vibrissae
• Compression
• 1mm steel cylinder, 1mm of compression

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1. Experimental setup
2. PSTH of all electrodes with stimulus of C2
   vibrissae
3. Recordings from two single electrode channels
4. Cortical activation

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• A Stimulus delivery
• B Channels responsive to vibrissae deflection at
  different inter-deflection intervals
• D Attenuation of second stimulus response
  depending on IDI

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• Neurons have an attenuated response to the second
  stimulus at shorter IDIs

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Neural response after compression

• After compression, there is a slow recovery of
  baseline neural activity.
• Postcompression intensity often exceeded
  precompression intensity

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Neural response after compression

• Increased activity post-compression, after a
  specific amount of time.

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Neuron Response Profiles

• Principal Channel
• Channel with the largest response magnitude to a
  given stimulus
• Precompression Significant
• Significant channel before compression
• Postcompression Significant
• Significant channel post-compression, but not
  pre-compression

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• Spike magnitudes significantly different between
  principal, pre-compression and post-compression
  significant channels
• Post-compression significant channels showed the
  largest relative change in spike magnitude over
  time

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• Paired pulse whisker stimulation after
  compression, 50ms IDI
• Before compression, response at this IDI is
  suppressed
• After compression, response to the same stimulus
  IDI eventually leads to excitation

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• A Principal channel latency does not change
  before and after stimulus
• C Vector strength a measure of temporal
  precision does not change in principal or
  precompression neurons
• D Vector strength increases in postcompression
  significant channels

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Conclusions

• After compression in the rat brain, neurons
  displayed a change in response to vibrissae
  stimulation
• Neurons displayed a period of inhibition after
  compression, followed by excitation greater than
  seen pre-compression.
• Some neurons were not responsive at all before
  compression, but became active after compression
• Na and K levels unbalance after injury
• May cause lower threshold for depolarization
• Hyperexcitability

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In vitro Multi(Micro)-Electrode Arrays (MEAs)

• What is it?
• Instead of implanting into the organism and
  dealing with the difficulties of live animals,
  the in vitro approach allows for cultured
  cells/tissues to be used.
• First done in myoneural junctions and gastropods
  (1980s, linear method) ? technology has improved
  technique dramatically (planar, 3D, perforated,
  thin, etc.). Extracellular recordings ? field
  potentials, spikes
• Two types
• Acute slices ? neurons dissociated, spontaneously
  form networks
• Organotypic slices ? network integrity remains

The cells/tissues grow directly onto the
recording electrodes

• Advantages/Disadvantages
• Long term recordings (weeks to months if done
  carefully)
• Works like most electrophyisology ? differences
  array, analysis of data
• Multiple electrodes some experimental, some
  controls, simultaneously stimulate/record from
  different sites
• Non-invasive to the cell (no rupturing of cells)
• High spatial resolution (very low for single
  cells)
• Expensive, tough to maintain/clean

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Want to start using this technique?
Microscope - One challenge among in vitro MEAs
has been imaging them with microscopes that use
high power lenses, requiring low working
distances on the order of micrometers. In order
to avoid this problem, thin-MEAs have been
created using cover slip glass. These arrays
are approximately 180 µm allowing them to be used
with high-power lenses.
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Want to start using this technique?
Temperature Controller
Pelltier device Heating element
Amplifier
60 channel Upright/inverted Blanking circuit
64 Channel Stimulator
Use to stimulate your cultures With a variety of
factors electricity, Solutions, etc.
MEA and Base
PCI Data Acquisition Card
Capable of recording from up to 128 channels
simultaneously Exceeding transfer rates of 6
MHz. Channels sampled at 50 kHz
Software, Air tables, computers, oscilloscopes,
audio, amplifiers, perfusion systems,
analog/digital converters etc.
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Want to start using this technique?
The MEA
Standard Set-ups 8 x 8 or 6 x 10 electrodes.
Titanium oxide electrodes that have diameters
between 10 and 30 µm. These arrays are normally
used for single-cell cultures or acute brain
slices. 60 electrodes are split into 6 x 5
arrays separated by 500 µm. Electrodes within a
group are separated by 30 um with diameters of 10
µm. These can be used to examine local responses
of neurons while also studying functional
connectivity of organotypic slices Want good
spatial resolution? HD-MEA is your answer. It
allows signals sent over a long distance to be
taken with higher precision. These arrays usually
have a square grid pattern of 256 electrodes that
cover an area of 2.8 by 2.8 mm. Other
types Perforated The perforated MEA design
applies negative pressure to openings in the
substrate so that tissue slices can be positioned
on the electrodes to enhance contact and recorded
signals. Thin, multi-welled, hexagonal, 3D
(penetrates farther into cultures)
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Want to start using this technique?
Too expensive to buy, you can make your own!
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Want to start using this technique?
neural networks Animat
So what now?
epilepsy synaptic plasticity (LTP, PPF, etc.)
development regeneration biological rhythms
network oscillators cardiac physiology robotics
Other techniques Histology/ICC Calcium
imaging Patch-clamp optogenetics
A computer generated animal, in a virtual world.
Cortical neurons from rats are dissociated and
placed on a MEA capable of both recording and
stimulating neural activity. Distributed
patterns of neural activity are used to control
the animats behavior in a simulated environment.
The computer acts as its sensory system
providing electrical feedback to the network
about the Animats movement within its
environment. Changes in behavior ? neural
plasticity.
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http//upload.wikimedia.org/wikipedia/commons/thum
b/5/55/Circadian_rhythm_labeled.jpg/350px-Circadia
n_rhythm_labeled.jpg
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Some Useful Background Knowledge
Primary endogenous oscillator that controls
circadian rhythms of numerous behavioural,
endocrine and physiological processes. The SCN
network synchronizes its component cellular
oscillators, reinforces their oscillations,
responds to light input (RHT) by altering their
phase distribution, increases their robustness to
genetic perturbations, and enhances their
precision.
The basis for cell-autonomous circadian
oscillations are positive and negative feedback
loops as shown here. These loops drive rhythms in
protein expression of several clock components.
http//people.usd.edu/cliff/Courses/Behavioral20
Neuroscience/Biorhythm/BRfigs/BRAfferent20SCN20f
igures.html Welsh et al, Suprachiasmatic Nucleus
Cell Autonomy and Network Properties. Ann. Rev.
Physiol. 2010
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Some Useful Background Knowledge
Glycine
Glycine
Welsh et al, Suprachiasmatic Nucleus Cell
Autonomy and Network Properties. Ann. Rev.
Physiol. 2010
Glycine is present in the SCN ? can act as a
classical inhibitory NT and an excitatory
neuromodulator Circadian release of glycine In
slices, high concentrations of glycine can reset
the clock What is glycines function in the SCN?
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Figure 1. Voltage-clamp recording of
glycine-induced current in neurons of acute SCN
slices
A Application of glycine (5 s) at a holding
potential of 0mV generated an outward current in
83 of neurons remaining neurons were
insensitive. Concentration dependent effects
(threshold 10µM). ?Characteristics of responses.
B Concentration response curve. Fitted to Hill
equation EC50 at 780µM
C - Extracellular recordings cell-attached
mode showed a concentration dependent
suppression of spontaneous firing activity in SCN
neurons sensitive to glycine.
D Agonists of glycine receptors (beta-alanine
and taurine) induce currents with similar
characteristics to glycine-induced currents
(applied to the same cell) Evidence that SCN
neurons in acute coronal brain slices of mice
exhibit a glycine-induced current.
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Figure 2. Glycine activates strychnine-sensitive
GlyRs in SCN
A Typical response to 1mM glycine (outward
current) at a holding potential at 0 mV (upper
trace). Typical response is reduced by the
coapplication of strychnine (5µM) (middle
trace) Recovery after washout (lower trace)
B Extracellular recordings in an acute SCN
slice. Strychnine reduced the duration of the
inhibition of spontaneous electrical activity
C Glycine antagonists strychnine (5µM), PMBA
(100µM) and ginkgolide B (1µM) reduce
glycine-induced currents by 51, 56 and 34
respectively.
D - Suppression of the amplitude of the current
decreases with increased concentrations of
strychnine.
E - Gabazine knocks out the GABA component of
the glycine responses.
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Figure 3. Ion selectivity and specificity of the
glycine-induced current
A B - Distinguish currents induced by glycine
and GABAA receptors application of 100µM GABA
and 1mM glycine. I-V relationship reversal
potentials of the cells used in A GABA-induced
-50.1mV, glycine-induced -49.2mV. Average
glycine trace -47.1mV Nernst potential for
chloride at their conditions -51mV.
E - Strychnine GABA had no effect on GABA
responses ? effects of strychnine are caused by a
specific block of GlyR. Gabazine suppressed GABA
currents. F Glycine does not act on nAChRs
no difference in current amplitude when
tubocurarine (blocker) is applied.
C Co-application of saturating amounts of GABA
and glycine resulted in currents that were
smaller than the sum of glycine GABA. Slow
deactivation glycine current.
D - 3/71 neurons tested for GABA and glycine
were insensitive to GABA but yielded a
glycine-induced current.
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Methods in vitro Multi(Micro) Electrode Arrays
(MEAs)
Organotypic Slices
250-350µm thick coronal slices containing the SCN
from 2-5 day old animals Placed in a culture
dish with culture medium (1mL DMEM/F12
supplemented with 10 fetal calf serum, 2.5mM
glutamax, 10mM Hepes, and 100µg/mL
penicillin-streptomycin) which was exchanged
3x/week Incubated _at_37C in 5CO2-95 air for more
than 2 weeks Before recording, the slice was
placed onto a nitrocellulose-coated MEA
(Multichannel Systems, Reutlingen) Recording
medium same as culture but Hepes was elevated to
20mM and the NaHCO3 was reduced to 0.56 g/L.
Exchanged continously at 20µL/min using SP 260PZ
syringe pump (WPI). Maintained on MEAs for up to
3 weeks under flow-through culture conditions
Can be kept for weeks in culture Can monitor
the output signal of the circadian clock
(electrical activity) for periods up to 3 weeks
http//www.staff.uni-mainz.de/golbs/Methods.html
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Methods in vitro Multi(Micro) Electrode Arrays
(MEAs)
Multi-electrode Array Recordings
Recorded long-term firing rate from organotypic
slices of SCN/PVN using a MEA-1060 recording
system (Multichannel Systems, Reutlingen) Two
types of HD-MEA used with two different
layouts Two fields of 30 electrodes with a
diameter of 10µm and 30µm spacing fields
separated by 500µm Only one field covered by
the SCN (rendering other tissue (PVN) covered by
other field) One field of 60 electrodes with
10µm diameter and 40µm spacing SCN covered
entire field in this case Extracellular signals
amplified 1200x and sampled at 32kHz on 60
channels simultaneously Noise detected and
removed by threshold algorithms APs exceeding
said voltage threshold were digitized and stored
as time-stamped spike cut-outs using the MC Rack
software (Multichannel Systems).
Ehab Tousson and Hilmar Meissl, 2004, J
Neuroscience
58
Figure 4. Glycine induced changes in the firing
rate of SCN neurons in organotypic cultures
A An increase in firing rate of SCN neurons due
to application of 1mM glycine C A decrease in
firing rate of SCN neurons due to application of
1mM glycine ? Suggests a counteraction of the
effect of glycine on glycine receptors. Both
responses are found throughout the circadian cycle
B In cells that were excited by glycine,
application of 5µM strychnine reduced their
spiking activity
? Excitation in the SCN and inhibition in the
PVN. Possible that differences in response to
glycine and other hypothalamus cells could depend
on circadian time.
Slice culture has rhythmic neuronal firing
23.860.37h
D The proportion of cells that were inhibited
by glycine was more prominent at CT 4 than at CT
16 (24 vs 6) At CT 4 a small subset of SCN
neurons (5) had a biphasic response to glycine
E F Simultaneous recording from the SCN and
one of its targets (PVN) using high-density MEAs
with two recording fields revealed opposite
responses to glycine.
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Figure 5. Glycine phase-shifts the circadian
rhythm of the firing rate of SCN neurons
A Vehicle application (aCSF) with no resulting
phase shift (0.20.1 h) in circadian oscillation
of the firing frequency.
B A phase advance (1.70.2 h) resulting from
the application of 1mM glycine 3 h before (CT4)
activity peak (CT7).
Circadian activity of the firing rate of the
cells were measured for 3 days. Then 1mM
glycine was applied to the bath and the activity
was measured for 4 days. Phase shifts were
calculated for the activity recorded on
individual electrodes as well as for the average
activity of all electrodes (in gray) ? both
methods showed similar results.
C - A phase delay (-1.40.2 h) resulting from
the application of 1mM glycine at CT 16 shortly
before the nadir of SCN neuronal activity
Glycine has the ability to phase-shift rhythmic
neuronal activity in the SCN by activation of
strychnine-sensitive glycine receptors.
D Phase response histogram for vehicle and 1mM
glycine at CT 4 and CT 16. Phase advance at CT 4
and phase delay at CT 16.
E Phase response histogram for glycine
coapplied with glycine receptor antagonists
strychnine and PMBA at CT 4 and CT 16 (applied
for 5 s before application of glycine)
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Conclusions/Thoughts
Glycine is able to phase-shift rhythmic neuronal
activity in the master clock by the activation of
strychnine-sensitive glycine receptors Glycine
can function as both an inhibitory and excitatory
NT in the SCN depending on circadian
time (possible mechanism circadian fluctuation
of the chloride equilibrium potential) A weak
glycinergic innervation of the SCN, as well as
intrinsic release of glycine from the SCN
could lead to a precise fine-tuning of GABA- and
NMDA-mediated synchronization and influence phase
resetting of the clock.
MEA allowed the researchers to observe long-term
oscillations in the SCN with/without the
treatment of the SCN with glycine/drugs The
organotypic slices of the SCN/PVN allowed the
researchers to have a in vitro system that was
very close to the in vivo system They were able
to record throughout the entire SCN system
(core/shell) and even extensions to the PVN to
see how one system affects the other