Basic Techniques in Electrophysiology

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Basic Techniques in Electrophysiology In vivo
recordings
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• Electroencephalographic recordings in humans and
  animals
• Extracellular Recordings in anesthetized animals
• Extracellular Recordings in vivo in free-moving
  animals
• Intracellular Recordings in anesthetized animals
  (Current- clamp)
• Voltage clamp in anesthetized animals

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The recording is obtained by placing electrodes
on the scalp, usually after preparing the scalp
area by light abrasion and application of a
conductive gel to reduce impedance. Each
electrode is connected to an differential
amplifier, which amplifies the voltage (typically
1,000100,000 times, or 60100 dB of voltage
gain), and then displays it on a screen or inputs
it to a computer. The amplitude of the EEG is
about 100 µV when measured on the scalp, and
about 1-2 mV when measured on the surface of the
brain.
Electroencephalographic recordings
The EEG can help diagnose seizure disorders like
epilepsy, head injuries, brain tumors,
encephalitis, some kinds of infections, metabolic
disturbances, and sleep disorders.
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• EEG has several limitations
• Scalp electrodes are not sensitive enough to pick
  out individual action potentials, or whether the
  resulting electrical activity is releasing
  inhibitory, excitatory or modulatory
  neurotransmitters. Instead, the EEG picks up
  synchronization of neurons, which produces a
  greater voltage than the firing of an individual
  neuron.
• Secondly, EEG has limited anatomical specificity
  when compared with other functional brain imaging
  techniques such as functional magnetic resonance
  imaging (fMRI). Some anatomical specificity can
  be gained with the use of EEG topography, which
  uses a large number of electrodes to triangulate
  the source of the electrical activity.

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Each horizontal tracing corresponds to an
electrode pair placed on a particular area of the
patient's scalp, forming a regular grid-like
pattern. By noting the set of channels where
abnormal waves occur (such as those marked in
red), the neurologist is able to infer the parts
of the brain where the abnormality is located.
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Steriade M, Timofeev I.Neuron. 2003 Feb
2037(4)563-76
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Traces of electroencephalographic recordings of
mice during seizures. Nicotine-induced seizures
in wild type (WT) mice result in spike-wave
discharges, whereas seizures in homozygous (Hom)
mutant dont. A mecanotransducer attached to the
bottom of the cage detects frequency and
intensity of movement. WT mice do not have
seizures when injected with 2 mg/kg nicotine.
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Unit Extracellular recordings in anesthetized
animals
The intact, functioning brain is readily explored
with microelectrodes in anesthetized animals. In
this approach, the animal is anesthetized, most
commonly with a barbiturate. The animal is then
placed in a stereotaxic instrument which
positions the skull in an exact position and
orientation with respect to submillimeter scales
in three dimensions on the instrument. By
positioning the microelectrode tip at a desired
coordinate along these scales, determined by
reference to a stereotaxic atlas of the brain of
that species, any site within the brain can be
found and cellular activity recorded. X-ray or
magnetic resonance imaging methods may also be
used for this purpose in human studies.
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In these experiments, impulse activity of neurons
is typically recorded extracellularly. In
extracellular recordings, the tip of a
microelectrode (typically 110 mm in diameter) is
positioned immediately adjacent to, but outside
of, a neuron. When in close proximity to the
neuron, current fields generated by action
potentials in that cell are detected by the
microelectrode as small voltage deflections
(typically 0.11 mV).
The advantages of in vivo electrophysiology
compared to the in vitro methods are obviously
due to the more intact preparation in vivo. With
these in vivo methods, one can study brain
regions or neurons in their intact state with its
normal complement of inputs and targets, and in
their natural milieu of circulating hormones and
factors. The cells being studied usually have not
been severed or damaged, as is almost always the
case with slice studies, and have developed
normally in the intact organism, in contrast to
the culture preparation. These considerations
lend additional credibility and fewer caveats to
results concerning neuronal activity in vivo.
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Unit Extracellular recordings in anesthetized
animals
Use glass or metal electrodes. The animal is
fixed to an steretoaxic apparatus. Gives
information regarding the frequency and mode of
firing and cellular responses to drug application
or electrical stimulation. Records from one
single neuron.
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• There are several experimental questions that
  require an intact organism, and they cannot be
  pursued in vitro.
• For example, to mimic the clinical situation it
  is important to determine the effect of a
  systemically administered drug (e.g., abused
  drugs like ethanol or opiates) upon activity in a
  particular brain region. In this way even if the
  drug has several sites of action in the brain,
  one sees the "net effect" of human-like drug
  exposure on the neurons of interest.
• The intact in vivo preparation is also
  necessary for determining the effect of certain
  physiological manipulations on particular neurons
  (e.g., effects of changes in cardiovascular
  activity or steroid levels).
• Similarly, the effects of functionally
  defined inputs typically must be examined in the
  intact organism (e.g., sensory or painful
  stimuli). Finally, a significant advantage of the
  in vivo preparation for electrophysiology is that
  it is more readily correlated with anatomical
  studies than in in vitro models.

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Iontophoresis
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• However, there are also several disadvantages of
  in vivo preparations
• In addition to the relative difficulty in
  performing many of the intracellular studies
  (and therefore in obtaining data on membrane
  mechanisms of drug action), the researcher does
  not have as much knowledge as in the in vitro
  preparations of actual drug concentrations at the
  cell under study. Therefore, drug and transmitter
  responses are less confidently identified with a
  specific receptor or channel.
• In addition, there may be other confounds, such
  as the presence of anesthetics (or in awake
  animals, immobilization stress) that could alter
  the normal electrophysiological responses to
  drugs and transmitters.

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Unit Extracellular Recordings in Behaving Animals
In addition to interactions between individual
neurons, there are other, more complex
organizations in the nervous system. Neurons are
typically associated in functionally related
groups and circuits. Function at the behavioral
level is a product of these neuronal networks
rather than simply the product of properties of
individual neurons. There are networks and
circuits specialized for sensory and motor
functions, and others specialized for associative
activities. It seems highly likely that the
elements of such neuronal networks have evolved
within the context of network function(s) to have
specific and perhaps unique properties tailored
for that network.
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As has been stated above for in vivo techniques,
many questions concerning neuropsychopharmacology
require experiments in the intact animal. This is
perhaps most true for questions regarding
cognition. While molecular and cellular
experiments are important for understanding
details of processes involved in mental
dysfunction or drug responses, they are unable to
integrate such results to ultimately and
completely explain cognitive functions such as
attention, perception, emotion, or memory.
Hence, most studies in the electrophysiology of
cognitive processes involve recording single
neurons in behaving animals.
These methods rely on the same principles as
described above with some modifications for
behaving animals. Most studies employ
extracellular recordings from a metal
microelectrode held in a miniature
micropositioner on the animal's head. The
micropositioner is typically attached to a
chronically implanted steel chamber or cylinder
that is stereotaxically positioned and
permanently cemented to the skull in a prior
surgery.
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Free Moving
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Intracellular Recordings in vivo
Use sharp glass electrodes ( tip opening 1
µM, resistance 25-150 MO). Measure changes in
voltage ( see current-clamp techniques).
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intra 1
intra2
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Lee et al, Neuron 2006
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Current Clamp / Voltage Clamp
Whole cell recordings in vitro

• Current Clamp Voltage Clamp
• Measure voltage Measure current
• Change current Change voltage

K channel currents
K
K
K
K
K
K
K
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Extracellular/Intracellular/Patch
Extracellular (field or single unit)
Intracellular
Patch Clamp
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Field Recordings
The local field potential represents the sum of
synaptic activity across cells
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Single channel recording
Whole cell current recording
http//qcbr.jabsom.hawaii.edu/LCMS/LCMS/LCMS/Topic
/About/File/PCMov.html
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Perforated Patch Recording
Used to prevent dialysis of the intracellular
contents of the recorded neuron
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Channel Recordings - heterologous expression
systems
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Tissue culture
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Recording from tissue slices
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Slice culture
Gene gun
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Slice culture/co-culture
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In vitro pitfalls(There is no such thing as a
perfect preparation)

• In vitro is not in vivo
• Is something missing?
• Loss of extrinsic inputs

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In vivo
In vitro
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single microelectrode penetrates the cell. The
voltage recorded is the sum of the voltage drop
(Ve) across the electrode and the membrane
potential (Vm). The voltage is buffered by a
high-impedance, low-bias-current, buffer
amplifier and then applied to a sample-and-hold
amplifier. The sample-and-hold amplifier
preserves for the whole of the cycle interval (T1
plus T2) the value of the recorded voltage (Vms)
that is present at the moment labeled sample in
the figure. Vms is compared to the command
voltage (Vcmd ) in the differential amplifier.
The difference voltage, e, is amplified by the
differential amplifier and applied to the
current-passing input of the electronic switch.
The electronic switch alternates the input path
to a voltage-controlled current source. The
function of the voltage-controlled current source
is to generate a specified current in the
electrode. During the current passing interval
(T1) the voltage-controlled current source passes
a current into the electrode that is proportional
to the output of the differential amplifier, with
a magnitude determined by the transconductance GT
. In this example, at the beginning of the cycle
the current is a depolarizing current pulse. The
square pulse of current in the electrode causes
the electrode voltage to rise at a rate
determined in a non-simple fashion by the series
resistance of the electrode, the input impedance
(mostly capacitance) of the cell, the capacitance
through the wall of the electrode, stray
capacitances to ground and the capacitance at the
input to the buffer amplifier. For the sake of
simplicity, the change in the electrode voltage
is shown as if it were a simple exponential. The
current pulse interval is normally a few
electrode time constants but, importantly, it is
much shorter than the membrane time constant.
There is no need for the voltage on the electrode
during the current pulse to reach steady state
and in most cases in order to maximize the
switching rate the current pulse is terminated
before steady-state conditions are reached.
Because the duration of the current pulse is
much shorter than the time constant of the cell
the Vm change shown in Figure 3 appears linear.
During the zero-current interval (T2) the
command input of the voltage-controlled current
source is zero (i.e., connected to ground) thus
its output current is zero. During T2, the value
of Ve decays towards zero and given sufficient
time the buffer amplifier records Vm alone. In
practice, the T2 interval must be sufficiently
long for the residual value of Ve to fall to a
fraction of a millivolt. Given that at the start
of the T2 recording interval the value of Ve
might be several volts, a large number of
electrode time constants must be allowed for Ve
to decay sufficiently. The polarity of the
differential amplifier is such that the closed
loop performance tends to drive e towards a small
value. Under steady-state conditions, Vms moves
in small increments about the mean value
(Vms,ave). The difference between Vms,ave and
Vcmd is the steady-state error, e. This error
exists because in order to maintain stability the
open-loop gain of the voltage clamp amplifier
must be finite. The open-loop gain is the product
of the voltage gain of the differential amplifier
and the transconductance, GT, of the
voltage-controlled current source. For a
detailed discussion of the equations that
describe the operation of the dSEVC see Finkel
and Redman 1984 and 1985.
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