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http://www.acnp.org/g4/GN401000005/CH005.html 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 END