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BRAIN SLICES TO STUDY NEURAL CIRCUIT FUNCTION
McIlwain first discovered that if a brain is rapidly removed from the skull and rapidly
cooled, a "slice" or slab of brain tissue could be cut that would survive for many hours
in the proper organ culture environment in vitro. Whereas originally developed for
studies of metabolism, neurochemistry, and neurotransmitter release from the cerebral
cortex (see A Critical Analysis of Neurochemical Methods for Monitoring
Transmitter Dynamics in the Brain), this technique has now been widely applied to
many brain regions for the study of electrophysiology of neurons and local brain
circuits.
Description
With minor variations for different brain areas, the method for removal and
incubation of a brain slice is straightforward. The techniques for slices of various
types from various brain areas are described in detail elsewhere (ref. 4). The brain is
rapidly removed from the skull and placed in ice-cold saline or artificial cerebrospinal
fluid (ACSF) saturated with carbogen (95% O2, 5% CO2) gas. Using either a
vibrating microtome or a tissue chopper, a thin (why thin????) (usually 100–400
M) slice of fresh brain is cut through the area of interest. This slice is rapidly placed
in cold carbongenated ACSF and transferred to a slice recording chamber containing
ACSF. Recording is usually performed after 1–2 hr of incubation to allow recovery
from the insult of the surgery. Although recording chambers vary somewhat in their
design, they all have the ability to continuously perfuse the slice with fresh ACSF and
oxygen and to add drugs to the ACSF perfusate at known concentrations (as well as
by the pipette methods described in the section on in vivo studies). The two most
common slice techniques are (i) submerged slices, in which the tissue is fully
submerged in the bath with continuous superfusion, or (ii) "interface" slices, in which
the bath fluid extends just to the upper surface of the tissue (to interface with a layer
of warm, moist carbogen gas). While in the recording chamber, the temperature is
best maintained in the physiological range so that normal processes may be studied.
With practice, the brain-slice preparation in most cases will remain viable and yield
excellent electrophysiological recordings as well as neurochemical measurements for
12 hr or more.
Finally, we would be remiss to omit the isolated brain in vitro (36). Seeming like
science fiction, in this method the entire brain (or brainstem plus cerebellum) is
removed and kept alive in an incubation environment where various
electrophysiological experiments can be performed. This exciting preparation has
many advantages of the slice in terms of recording stability and ease of intracellular
recordings, yet is nearly fully intact like the in vivo brain. While technically difficult
and not yet used extensively for pharmacology questions, this approach holds great
promise for future neuropharmacology studies. A related but technically more feasible
preparation is the isolated brainstem–spinal cord, which has been used to great
advantage in studies of the physiology of respiration (37).
Example Studies
The brain-slice technique has been widely used over the last two decades, so that
slices of most brain areas have been studied. One of the first, and by far the most
widely studied, of these preparations has been the hippocampal slice. This
preparation has been a key factor in working out mechanisms underlying long-term
potentiation, postsynaptic effects of various transmitters at the membrane level, and
actions of a variety of drugs (Madison and Nicoll, (ref 38) and Siggins and colleagues
(ref 34, 39)).
Another preparation that has been used to great advantage in understanding the
cellular effects of opiates is the locus coeruleus (LC) slice. The extensive studies of
opiates in LC slices by Aghajanian and colleagues and by North, Williams, and coworker are described in more detail in Foote and Aston-Jones).
Advantages and Disadvantages
The brain-slice method allows the repeatable application of known concentrations of
drugs to the cells being studied, an important advantage in drawing conclusions
concerning receptor identity and postsynaptic mechanisms. A second important
advantage compared to most in vivo methods is the relative ease of obtaining longterm, stable intracellular recordings without anesthetics or immobilizing agents, so
that the effects of drugs on membrane properties of identified neurons can be directly
ascertained. An equally important advantage of the brain slice for electrophysiological
studies is that the local circuits and cytoarchitecture of the tissue are relatively intact.
This allows straightforward identification of the neuron being studied by visualization
of its position with respect to known landmarks and other characteristics (e.g., the LC
is clearly evident in the slice as a translucent group of cells adjacent to the fourth
ventricle). This contrasts with studies in cultured neurons, where the neurochemical or
nuclear identity of the neuron under study may be difficult or impossible to determine.
Finally, the relatively intact local anatomy of the slice preparation also allows one to
study synaptic responses of brain neurons as in some culture preparations (see above).
Finally, the physiology and pharmacology of neurons can be studied in semiisolation from the confounds of the ongoing behaviors of a freely moving animal.
Perhaps the most significant disadvantage here again is that the slice is a relatively
isolated preparation, and neurons in the slice lack many normal afferent inputs and
efferent targets. Of course, neurons in the slice are also isolated from any circulating
influences such as hormones or steroids. Thus, properties observed in slice studies
must always be considered with the caveat that results may reflect the artificial nature
of the preparation and may differ from those obtained in the intact organism.
Similarly, the slice is, of necessity, situated in an artificial environment rather than the
natural and more complex milieu of the brain. The properties of neurons observed
vary widely with minor changes in the slice environment, so that results may be
heavily biased by the particular experimental conditions employed in an individual
lab (for example, depending upon whether interface or submersion slice chambers are
used). Also, by being isolated from the behaving organism, neurons within the slice
are not amenable to study in the intact, functioning circuits in which they normally
reside. Responses of neurons to transmitters are often best revealed when the neuron
is challenged by other afferents, which may be lacking in the slice. Similarly, one
cannot use this preparation to test the role of a set of neurons in a particular circuit
function or behavior. Thus, slice studies are, of necessity, limited to the cellular level
of analysis. However, when used in combination with studies in the intact organisms
(reviewed next), experiments in the brain slice provide a powerful adjunctive analysis
of an important range of phenomena.
Relevance to Neuropsychopharmacology
The same advantages for neuropsychopharmacology listed above for cellular
techniques apply to approaches using brain slices, because these techniques also allow
a cellular-level examination of function. However, slices have the additional
advantage that neuronal structure is better preserved than in isolated or culture
situations, so that drug development can be carried out more easily at identified
neurons and synapses.
Slices can also take advantage of animal models of disease to examine underlying
cellular changes in identified neurons and synapses. One example here are models of
drug abuse (using chronic drug administration) where slice studies have provided
important insights into underlying neuronal changes (e.g., see Electrophysiological
Properties of Midbrain Dopamine Neurons, Dopamine Autoreceptor Signal
Transduction and Regulation, and Mesocorticolimbic Dopaminergic Neurons:
Functional and Regulatory Roles). Other animal models (e.g., transgenic mice) could
also be profitably explored using slice methods.
IN VIVO SINGLE-CELL ELECTROPHYSIOLOGY TO STUDY
NEURAL CIRCUIT FUNCTION
Description
The intact, functioning brain is readily explored with microelectrodes in anesthetized
animals. In this approach, the animal is anesthetized, most commonly with a
barbiturate, urethane, chloralose, or halothane. 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 contrast to the intracellular recordings discussed above. In extracellular recordings,
the tip of a microelectrode (typically 1–10 m 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).
There are many experimental applications of in vivo single-cell electrophysiology.
Below we briefly describe three: iontophoresis and local drug application, stimulation
recording, and antidromic activation.
Iontophoresis and Local Drug Application (From Greek phorēsis, a carrying).
In neuropharmacology experiments it is often useful to study the direct effects of
neurotransmitter agents or drugs on neurons in the intact brain to mimic or alter
responses to synaptically released transmitters. The ability of an exogenously applied
agent to mimic the actions of the endogenously released transmitter is one of the
cardinal criteria for establishing the identity of a neurotransmitter at a particular
synapse (5). Also, such direct application of drugs obviates interpretive problems of
systemic drug application, where direct effects might be confounded with indirect
effects mediated by the drug acting at multiple sites in the CNS or periphery.
Curtis first used the iontophoretic technique, showing that charged drug molecules in
a solution would be carried out of the tip of a micropipette by electrical current flow
of the same polarity as the charge on the drug ion. Thus, by passing current through a
glass micropipette, one can apply drug into the local area of the neuron being
simultaneously recorded by another, adjacent pipette. This technique, denoted initially
as microelectrophoresis but later as iontophoresis, was further developed in the CNS
by Krnjevic and Salmoiraghi and colleagues (ref. 5). In brief, a multibarrel glass
micropipette is manufactured so that 5–7 tips are adjacent to one another. A single
micropipette is used for recording neural impulses extracellularly; this may be one
barrel of the multibarrel pipette assembly, or (better) it may be an adjacent pipette
affixed (glued) to the iontophoretic multibarrel electrode so as to protrude 10–20 m.
The iontophoretic barrels are filled with drug or salt solutions. It is important that one
barrel be filled with NaCl, so that current of opposite polarity to that being passed
through a drug barrel can be applied at the same time as the drug ion, to neutralize
stray currents and minimize artifacts. Once stable impulse activity from a neuron is
recorded, current (usually 5–200 nA) of appropriate polarity is then applied to the
barrel containing the drug of interest and the resulting effect on neural activity is
monitored, usually using a rate meter-type recording of firing rate. When the drug is
not being applied (during control conditions), a "backing" or "holding" current of
appropriate intensity (usually 5–20 nA) and polarity to attract the drug ion is applied
to prevent unwanted leakage of drug from the tip. It is most advantageous to use an
iontophoretic device designed to apply the drugs at precise intervals and automate the
control procedures.
An important variant of iontophoresis is local application of drugs from micropipettes
by pressure (denoted micropressure application). In this method, a multibarrel
micropipette similar to the iontophoretic assembly is employed, but controlled
automated pneumatic pressure instead of electrical current is used to eject drug from
the tip. This method is often necessary to apply large or uncharged molecules (e.g.,
large peptides) that do not readily move with iontophoresis. It is possible to configure
the pipette so that both iontophoresis and micropressure techniques can be used from
the same barrel. If similar results are obtained with both methods of local drug
delivery, it is less likely that the results are due to artifacts associated with either
technique alone. See ref. 40 and Physiological and Anatomical Determinants of Locus
Coeruleus Discharge: Behavior and Clinical Implications for recent examples of this
combined method.
Local micropressure application has several advantages over iontophoresis. One of
the major drawbacks of iontophoresis is that one does not know the concentration of
drug applied. This is because the drug is carried by current, and for most drug
solutions the physiochemical properties determining the relative transport of drug
molecules in an electrical field in the micropipette glass are not known. It is possible
that very high concentrations are ejected even with low currents (for easily ionized
drugs), while even high currents may eject very little of another, poorly ionizable
drug. There is also considerable release variability across micropipettes. In contrast,
with local pressure application the solution ejected is the same concentration as that in
the pipette. While the absolute concentration at the recorded cell is uncertain due to
diffusion and dilution in the extracellular milieu, at least the highest possible
concentration is known. This is important, because the receptor specificity of drugs is
dependent upon their use within a certain concentration range. By increasing the
volume of solution ejected, pressure application may also allow a larger area of tissue
to be infused than with iontophoresis. For this reason, pressure is typically the method
of choice when trying to locally antagonize synaptically mediated events that may
reflect inputs onto distal or remote dendrites of the neuron being recorded. However,
there are caveats with this method, such as artifacts due to pressure (movement), pH,
or osmolarity changes; iontophoresis usually allows a greater range of drugs to be
applied to the same neurons, and it is often associated with more stable and successful
recording.
It is important for both microiontophoresis and micropressure application of drugs
that the proper controls for current, pH, and volume effects be conducted, and that
drug application follows a regularly timed protocol to minimize "warm-up" effects
and possible experimenter bias (ref. 5).
Stimulation - Recording
This is the simple but requisite procedure for discerning the functional effect of an
afferent input to a neuron. There are two methods available for this purpose: (i) the
classical approach of electrically stimulating the afferents while recording the target
neuron and (ii) a more recent method (especially important for studies in such
complex tissues as brain) using local chemical (instead of electrical) stimulation to
activate the input source.
With the former method, pulses of electrical stimulation are applied to a stimulating
electrode to activate neurons that project to the area where a target cell is recorded.
Extracellular recordings are typically used to measure the functional effect of the
input. Most commonly, responses are measured in displays called peri-stimulus time
histograms (PSTHs), where neural activity recorded for many successive stimulus
trials is accumulated, synchronized with the stimulus presentation. By accumulating
activity in such a histogram, even relatively weak responses can be revealed due to
the summation over many trials. This type of analysis allows quantification of
response magnitude, onset latency, and duration. These parameters can then be
compared before and after drug administration to determine, for example, the effect of
a particular receptor antagonist on the response to activation of an input and thereby
help determine the likely transmitter candidate in that afferent. The reader is referred
to the review by Ranck (41) for a detailed treatment of factors determining types of
neural elements activated with different parameters of electrical stimulation.
A major drawback of electrical stimulation is that both cell bodies in the area of the
stimulating electrode, and fibers of passage derived from cells located in other areas,
will be activated by the stimuli applied. Therefore, the origin of the responses
obtained is uncertain. This problem is surmounted by using local chemical
stimulation, which activates the input neurons by infusion of a neural activator such as
glutamate or one of its analogues into the area of the cell bodies or dendrites. Because
stimulation by this method relies on receptor activation, and receptors are thought to
reside only on somata and dendrites of neurons, this approach does not activate
passing axons that originate from neurons elsewhere. However, while the origins of
responses are better identified with this method, the temporally imprecise activation
by chemical microinfusion does not allow accurate determination of response
latencies. A second important limitation of chemical stimulation is that neurons can
be inactivated by stimulating chemicals such as glutamate, if too high a concentration
is applied. That is, too much of a chemical activator (e.g., glutamate) can depolarize
neurons into a state of depolarization block, where the neuron is maintained in a
depolarized state and thus cannot generate action potentials due to persistent
inactivation of Na+ channels. Thus, what is thought to be stimulation can actually
inhibit neurons of interest. A procedure that minimizes such concerns is to apply a
range of concentrations of the chemical activator and examine the corresponding
dose–response curve in the target cell. This should reveal the minimum dose for
obtaining an effect, which with glutamate is presumably due to excitation of neurons
near the site of infusion. Another way around this potential problem is to record the
response of neurons in the infusion site during application of the chemical stimulant.
This not only allows direct confirmation of the effect of the infused agent on those
cells, but also gives the time that activity in the local neurons is affected; this can
greatly improve the temporal accuracy of such stimulation–response studies. Without
such procedures, results obtained with local chemical stimulation are difficult to
interpret.
Local Synaptic Inhibition or Decoupling
A similar method is used in combination with stimulation-recording experiments to
test the involvement of a particular brain region as a circuit element mediating an
evoked response. Instead of infusing an excitatory neurochemical, however, the
solution microinfused is one that inactivates or synaptically decouples local neurons.
A composite recording/infusion pipette can be used so that neuronal recordings help
localize the area desired for infusion as well as verify the effect of the infusion on
neurons at the infusion site. One approach is to locally infuse a local anesthetic (e.g.,
lidocaine) to block local activity while conducting the stimulation-recording
experiment (42). However, anesthetics block impulse conduction in passing fibers as
well as in local somata, so that this approach does not test the role of local neurons as
a possible relay in the circuit response being examined. Alternatively, the infusion
solution contains either (i) a strongly inhibitory neurotransmitter agent (e.g., GABA,
or a GABA agonist such as muscimol) or (ii) a synaptic decoupling agent (e.g.,
divalent ions such as low Ca2+ ; high Mg2+). By inhibiting neurons in the infusion
area, the first approach prevents them from responding to synaptic inputs, while in the
latter approach interference with Ca2+-dependent neurotransmitter release
"synaptically decouples" the local area infused. Because passing fibers are not thought
to be sensitive to these agents, in either case the result is that local neurons but not
passing fibers are functionally removed from circuit activity, and their role in the
circuit response being examined can be directly tested. See (ref 43) for an example of
the use of these methods to investigate the role of the ventral medulla in sensory
responses of locus coeruleus neurons.
Stimulation
Blocking
Antidromic Activation
Antidromic activation has at least two major uses: (i) to confirm a monosynaptic
projection to an area and (ii) to determine the time required for conduction of an
impulse along a projection pathway. In this procedure, an electrical stimulation
electrode is placed in the projection area of a neuron while a microelectrode in the
soma region records neurons that are "backfired" from the target area. This technique
is based upon the fact that axons will conduct impulses in both directions. Although
under most conditions impulses are generated at the source of the axon (soma) and
only travel orthodromically, antidromic ("backwards") conduction is a powerful and
convenient property. By stimulating the target area and recording an antidromically
driven response elsewhere, one can conclude that the neuron recorded sends an axon
to the region being stimulated. This is often done to confirm anatomical evidence of a
projection. Note, however, that because this employs electrical stimulation a positive
response does not indicate that the recorded neuron terminates at the site of
stimulation, only that the neuron sends a fiber (possibly en route elsewhere) to that
region; additional (orthodromic) tests must be performed to indicate a synaptic input
(e.g., as described under Stimulation Recording, above). In addition, such activation
yields the time required for the impulse to travel the length of the axon (conduction
velocity is the same in both directions along the axon). This latency of impulse
conduction is useful in interpreting stimulation-recording experiments, because
synaptic (orthodromic) responses at a similar latency may be due to a direct
monosynaptic (versus indirect, polysynaptic) projection from the input neuron
stimulated.
There are three tests that should be performed to confirm that a driven impulse is
antidromic: (i) It should have a very constant latency of activation (synaptically
driven responses usually exhibit a few milliseconds of "jitter"); exceptions to this
constant latency rule can occur for very-small-diameter unmyelinated fibers (ref. 44);
(ii) the driven response should faithfully follow high frequency activation above 100
Hz (e.g., two stimuli at a 5-msec interpulse interval should drive two spikes); and (iii)
orthodromic (spontaneously occurring) spikes should collide with and eliminate the
driven spike (collision test).
Example Studies
Because these methods have been in wide use for several decades, there are literally
hundreds of specific examples that could be described. The reader is referred to the
text by Shepherd (7) for additional references and descriptions of applications of these
methods in sensory and motor systems of the brain. These methods have been used to
determine the origin of the hyperactivity of locus coeruleus neurons during opiate
withdrawal (45) and to demonstrate that systemic nicotine potently activates the LC
indirectly (46) (see also Physiological and Anatomical Determinants of Locus
Coeruleus Discharge: Behavior and Clinical Implications).
Advantages and Disadvantages
The advantages of in vivo electrophysiology compared to the in vitro methods
described previously 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.
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 organismic 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. Antidromic
activation can more directly confirm projections found in anatomical experiments,
and stimulation-recording studies can establish the functional effect and
neurotransmitter of a pathway, again confirming results seen with anatomical tracttracing and immunohistochemical experiments (for anatomical approaches to these
issues see Cytology and Circuitry).
However, there are also several disadvantages of in vivo preparations. In addition to
the relative difficulty in performing many of the intracellular and whole-cell studies
described above (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.
Relevance to Neuropsychopharmacology
Several applications of in vivo electrophysiological methods lend themselves
particularly well to clinically relevant questions of special interest to
neuropsychopharmacology. In addition to the points above, the in vivo preparation
allows the study of neural activity and drug responses in animal models of human
disorders. Studies of locus coeruleus activity in opiate withdrawal (see Physiological
and Anatomical Determinants of Locus Coeruleus Discharge: Behavior and Clinical
Implications, Intracellular Messenger Pathways as Mediators of Neural Plasticity, and
Opioids) are obvious examples of this application, but others abound. There are
animal models of other abused drugs (e.g., alcohol and cocaine) and several disorders,
including schizophrenia, depression, and anxiety, all of which promise to make (or
already have made) significant contributions to neuropsychopharmacology, and which
require electrophysiological testing in the intact organism (for descriptions of animal
models see Behavioral Techniques in Preclinical Neuropsychopharmacology
Research, Central Norepinephrine Neurons and Behavior, Serotonin and Behavior: A
General Hypothesis, Adaptive Processes Regulating Tolerance to Behavioral Effects
of Drugs, Animal Models of Psychiatric Disorders, and Genetic Stategies in
Preclinical Subtsance Abuse Research). In addition, because these techniques are
needed for study of effects of systemically administered drugs, they can be an
important step in new drug development.
In vitro neuron-chip interface
Behavioral Electrophysiology: Researching the Spark of Cognition
22/11/06
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.
EXTRACELLULAR
INTRACELLULAR
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. An
analogous relationship exists between physics and chemistry: While the principles of
physics are critical to our understanding of chemistry, they are not sufficient to fully
understand or predict the properties of chemical reactions. It is fundamentally
necessary to conduct experiments in chemistry per se or, as is the case at hand, in
cognitive neuroscience.
Hence, most studies in the electrophysiology of cognitive processes involve recording
single neurons in behaving animals. These methods will be briefly described below,
followed by a description of methods used to locally manipulate neurons in behaving
animals to test hypotheses generated by correlations found with recording
experiments.
Single-Cell Recording in Behaving Animals
These methods rely on the same principles as described above with some
modifications for behaving animals. Most such 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. These chambers often allow lateral repositioning of the
recording microelectrode for multiple penetrations, while the micropositioner allows
the electrode to be lowered into the brain to the desired target along a particular track.
Additional details on such methods can be found in refs. 47, 48, 49.
The most common types of microelectrodes used for recording from neurons in
behaving animals are (a) etched tungsten or platinum–iridium wires, insulated with
either glass or lacquer except for ~20 m from the tip, or (b) thin microwires that are
typically 25–62 m in diameter and lacquer-insulated except for the bluntly cut tip.
Neurons of different brain areas are recorded more easily with one or another type of
electrode; for example, locus coeruleus neurons in awake rats and monkeys (43) are
more easily recorded using the more flexible microwires. In general, microwires are
advantageous for experiments entailing long-term recordings from neurons in deep
structures in behaving animals, whereas etched, stiff microelectrodes are
advantageous for studies where penetration of the dura mater is needed or where
numerous penetrations in a small area are desired.
Two general approaches are used with microwire electrodes. Perhaps most commonly
the microwires are simply implanted and glued in place with no further movement of
the wires possible after surgery. In this method, a large number of wires (often more
than 40) are implanted and each wire is monitored daily over the course of months for
unit activity. Activity occurs on enough wires over time that many long-term and very
stable recordings can often be obtained (e.g., see refs. 50 and 51). The second method
is to attach a small number of microwires (two to six) to a movable microdrive which
allows the wires to be repositioned vertically, and sometimes laterally as well, for new
penetrations after surgery (e.g., see ref. 43). This approach has the advantage of
obtaining many more recordings from a single subject than the fixed wire approach,
an important consideration when subjects are in limited supply (e.g., monkeys) or
when extensive training is required for each animal recorded.
High-gain amplification of signals from the head of a moving animal often yields
considerable movement related electrical artifacts; these are the bane of a behavioral
electrophysiologist. These problems are typically overcome by including a miniature
first-stage amplifier in the fixed implant on the animal's head, so that recordings from
the microelectrode can be amplified and converted to low-impedance signals before
traveling over long distances of flexing cables.
As with all extracellular recordings, it is important to know whether activity seen on
an individual electrode is generated from one neuron only, or from several nearby
neurons simultaneously recorded. Results of the latter, termed multiple-cell recording
or multi-unit recording (MUA), are more difficult to interpret because neurons in the
multiple cell population may be physiologically heterogeneous. In that case, opposite
changes in different cells recorded may appear as no change in the multiple-cell data.
In addition, it is more difficult to ensure the stability of the recorded signal over time
with multiple-cell activity.
In recordings in awake monkeys, the animal's head is usually fixed in place by a post
that is cemented to the skull and anchoring screws, so that such movement artifacts
are minimized. This technique also allows precise measurement of the direction of
gaze by monitoring eye position, an especially valuable aspect in studies where
monitoring and controlling attention is important. However, this approach does not
allow free movement of the animal and may produce uncontrolled effects of
immobilization stress.
A key element in behavioral electrophysiology is the computer system that is used to
acquire and analyze the data. Because so many events are typically recorded
simultaneously (e.g., two or more cells, EEG, EMG, markers for different sensory
stimuli and behavioral events, video time markers, X and Y eye positions), it is
necessary to have a system that can rapidly record large amounts of data on-line with
millisecond temporal resolution. In the last few years, affordable microcomputers
with sufficient speed and disk storage have become available to accomplish this data
storage task. In addition, sophisticated software is required so that neuronal activity or
other data can be tabulated (typically in PSTHs) with respect to an arbitrarily chosen
type of event out of the many recorded. For example, it may be desired to construct
PSTHs of neuronal activity synchronized with a particular type of sensory stimulus
out of the many presented, or synchronized with a particular type of sensory stimulus
that elicits a specific behavioral response (e.g., to examine neuronal activity
associated with non-target conditioned cues for trials in which the animal mistakenly
elicited a behavioral response). The most challenging (but important) aspect of
developing such software is to make it easy to use and easy to abstract results from
very large data files, but also make it sufficiently flexible so that new subroutines can
be written and integrated to analyze electrophysiological data. The latter is an everpresent need, so that typically the behavioral physiologist must be quite computerliterate!
Example Studies
Recordings of neuronal activity in animals performing structured behavioral tasks
have proven invaluable in understanding the neural bases of various types of cognitive
activity. Of the many studies using this approach, one example for activity of locus
coeruleus neurons in a monkey performing an attention task is found in ref. 47 (see
also Pharmacology and Physiology of Central Noradrenergic Systems, Physiological
and Anatomical Determinants of Locus Coeruleus Discharge: Behavior and Clinical
Implications, Noradrenergic Neural Substrates for Anxiety and Fear: Clinical
Associations Based on Preclinical Research, and Serotonin and Behavior: A General
Hypothesis). Similarly, recent studies by Schultz and colleagues recording putative
dopamine neurons in waking monkeys (52) hold great promise for further
understanding this system and its importance in neuropsychopharmacology. For other
examples of the use of behavioral electrophysiology to decipher the neurobiology of
cognition, the reader is referred to work by Goldman-Rakic and colleagues (47) and
by Wise, Desimone, and colleagues (48, 49).
Acute Manipulation of Specific Neurons and Neural Groups in Behaving Animals
Description
Studies of the causal role of a brain area in a specific behavior or cognitive process
commonly employ lesions of one type or another. The simplest approach is to remove
or destroy the area of brain by excision or electrolytic lesion and tissue coagulation.
However, these approaches have serious drawbacks that limit the interpretability of
results obtained, most prominently (i) passing fibers originating from neurons located
elsewhere are lesioned and (ii) recovery of function may occur (via adaptive changes
in remaining brain structures) during the weeks needed for the animal to recuperate
from such a gross insult. This effect may lead to false-negative results concerning the
role of the lesioned brain structure. The reader is referred to the elegant studies of
Newsome and colleagues (53) for an example of recovery of function following
chronic lesion manipulations.
A better approach to such causal behavioral studies is to (a) acutely manipulate (e.g.,
activate or inactivate) the neurons in question using local infusions of selective
chemical agents or cryogenic manipulation and (b) examine the effect on behavior
during the acute effect. This approach avoids the two concerns listed above. These
studies employ techniques similar to those described under Stimulation Recording
(above) to locally infuse drugs into a target group of neurons. Optimally, the
investigator employs a combination recording/infusion electrode that allows neuronal
recording from cells in the immediate area during infusions of chemical agents. This
permits several advantages: (i) The desired infusion site can be precisely localized by
recording characteristic neuronal activity prior to infusion, (ii) the time of onset and
offset of altered activity in the target neurons can be monitored and compared to the
timing of behavioral changes, and (iii) individual infusion trials can be directly
confirmed as to their effectiveness in altering neuronal activity at the infusion site in
the desired fashion.
Examples
One example of this approach is found in recent studies by Foote, Valentino, and
colleagues in studies of the impact of activity in locus coeruleus neurons on EEG in
rats. As described in more detail in Pharmacology and Physiology of Central
Noradrenergic Systems, local intra-coerulear infusions of the cholinergic drug
pilocarpine to activate locus coeruleus neurons also activated the EEG, whereas local
infusions of clonidine (that powerfully and selectively attenuated locus coeruleus
activity) led to EEG synchronization. Recordings from locus coeruleus neurons
during the infusions confirmed the effectiveness of the manipulations and revealed
that the EEG changes were closely time-locked to the changes induced in LC activity.
A second example of the power of this approach to test hypotheses generated by
electrophysiology in behaving animals is found in the work of Goldman-Rakic and
colleagues. Recording experiments suggested that the dorsolateral prefrontal cortex
may be involved in spatial working memory (47). To test the role of dopamine in this
proposed function, Sawaguchi and Goldman-Rakic (54) infused selective D1
antagonists into this area and found that animals apparently could not remember a
specific location for objects; the same objects in surrounding locations were not
affected. Hence, this method can also be used to test the role of transmitter influences
in a particular brain area on behavioral functions.
Advantages and Disadvantages
The advantages of behavioral electrophysiological methods for
neuropsychopharmacology are numerous. These are the only methods whereby one
can directly correlate neural activity with behavior. This can be especially powerful
when the behavior being measured is itself a measure or reflection of a cognitive
process. In addition, there is no confounding effect of anesthesia on neuronal activity
recorded. Finally, because these studies take place in the intact animal, one can relate
results to anatomical and neurochemical properties of the relevant circuits.
Extending electrophysiology with acute chemical manipulations of specific brain
neurons can directly test the causality of hypotheses generated from the correlative
results of behavioral electrophysiology experiments. This approach obviates problems
of nonspecificity and recovery of function encountered with more conventional lesion
manipulations. By using local infusions of neurotransmitters or related drugs, this
method also provides results that can be directly related to anatomical and
neurochemical results on the same system. Together, these techniques provide a
powerful means of investigating the neural basis of cognitive function.
Example is the localization of the site of plasticity by Thompson's group. They used
recording of the model neuronal response together with behavior in the serial net,
lesions and inactivation of the different sites, and finally stimulation of the different
sites. The use of the different techniques stems from the
understanding that neither of the techniques is pointing to
the function in decisive way.
Disadvantages of this approach are also considerable. First, because these experiments
take place in the behaving animal, it is difficult (if not impossible) to control all of the
possible relevant variables that may affect the activity being recorded (e.g., behavioral
state, stress, training differences, individual differences in task ability, etc.). Second,
the behavioral measures obtained may not be temporally precise, or may only
indirectly reflect the process of interest (e.g., attentional studies). Third, these studies
are slow, technically difficult, and require tedious and long (often months-long)
training of the animals before recording experiments can even begin. Finally, elegant
though they are, these behavioral-recording studies yield only correlative data. It is
necessary to extend such experiments with manipulations of the systems of interest
using activation or inactivation of select groups of neurons (employing methods
described below) to test causal hypotheses of their roles in specific behaviors.
Relevance to Neuropsychopharmacology
The importance of these approaches to neuropsychopharmacology lies in the fact that
many mental disorders are problems of complex cognitive function; the neural bases
of these normal cognitive functions are only partly understood (if at all). Substantial
progress in understanding and developing new treatments for disorders of memory,
attention, drug craving, and the like require a more complete understanding of the
underlying biological processes. As stated above, such an understanding will require
experiments combining neurobiology and cognitive testing, particularly
electrophysiology and local acute manipulations in animals performing sophisticated
cognitive tasks. In addition, studies by Georgopoulous et al. (55) and by Houk et al.
(56), among others (50), are revealing the power of analyzing activity in networks of
neurons to understand the neural bases of cognition and behavior. If combined with
drug administration and testing, such experiments analyzing neuronal populations in
behaving animals could prove to be very valuable for future
neuropsychopharmacological analyses. Such models of normal function can be used
not only to understand the neural processes involved and to develop new drug
treatments of related disorders, but also to test putative drug treatments in animals in a
clinically relevant manner before their application to the human patient.
Visit the following site for demonstration of single cell recording:
http://tonto.stanford.edu/eotn
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