Download Unit Extracellular recordings in anesthetized animals In these

Basic Techniques in Electrophysiology
In vivo recordings
•Electroencephalographic recordings in humans and
animals
•Extracellular Recordings in anesthetized animals
•Extracellular Recordings in vivo in free-moving animals
•Intracellular Recordings in anesthetized animals (Currentclamp)
•Voltage clamp in anesthetized animals
Electroencephalographic recordings
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 1,000–100,000 times, or 60–100 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.
The EEG can help diagnose seizure
disorders like epilepsy, head
injuries, brain tumors, encephalitis,
some kinds of infections, metabolic
disturbances, and sleep disorders.
EEG has several limitations:
1) 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.
2) 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.
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.
Steriade M, Timofeev I.
Neuron. 2003 Feb 20;37(4):563-76
Traces of electroencephalographic recordings of mice during seizures. Nicotineinduced seizures in wild type (WT) mice result in spike-wave discharges, whereas
seizures in homozygous (Hom) mutant don’t. 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.
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.
In these experiments, impulse activity of neurons is typically
recorded extracellularly. In extracellular recordings, the tip of a
microelectrode (typically 1–10 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 0.1–1 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.
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.
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.
Iontophoresis
However, there are also several disadvantages of in vivo
preparations:
1) 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.
2) 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.
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.
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.
Free Moving
Intracellular Recordings in vivo
Use “sharp” glass electrodes ( tip
opening ~ 1 µM, resistance 25-150
MΩ). Measure changes in voltage (
see current-clamp techniques).
intra 1
intra2
Lee et al, Neuron 2006
WholeClamp
cell recordings
in vitro Clamp
Current
/ Voltage
Current Clamp
Measure voltage
Change current
Voltage Clamp
Measure current
Change voltage
K+ channel
currents
K+
K+
K+
K+
K+
K+
K+
Extracellular/Intracellular/Patch
Extracellular
(field or single unit)
Intracellular
Patch Clamp
Field Recordings
The local field potential represents the sum of synaptic activity across
cells
Single channel recording
Whole cell current recording
Perforated Patch Recording
Used to prevent dialysis of the intracellular contents of the
recorded neuron
Channel Recordings - heterologous expression
systems
Tissue culture
Recording from tissue
slices
Slice culture
Gene gun
Slice culture/co-culture
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
In vivo
In vitro
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20
0
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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-biascurrent, 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, ε, 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