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|Neuropsychopharmacology: The Fifth Generation of Progress|
Behavioral Techniques in Preclinical Neuropsychopharmacology Research
Behavioral Techniques in Preclinical Neuropsychopharmacology Research
J. E. Barrett and K. A. Miczek
Behavioral measurements of the effects of drugs have been a central component of the field of neuropsychopharmacology since its inception some 40 years ago. The discovery of chlorpromazine and the subsequent demonstration that this drug produced differential effects on avoidance and escape behavior provided a strong impetus for the development of assays for evaluating the behavioral effects of potential antipsychotic drugs (9, 12). The early growth of neuropsychopharmacology coincided also with the development and spread of the field of operant conditioning. Indeed, many of the techniques and procedures used to control and monitor operant behavior were adopted and enthusiastically endorsed by behavioral pharmacologists emphasizing the importance of behavior in determining the effects of drugs (15, 29, 44). Early researchers understood quickly the utility and the power of behavioral techniques in demonstrating specific and systematic effects in the integrated organism, and the field of behavioral pharmacology emerged as a discipline within the larger context of neuropsychopharmacology.
In a general sense, behavioral research in this context can be viewed as having two major objectives. In the first case, behavior is used to answer questions where the primary interest is in pharmacology, and a behavioral measure is studied to evaluate drug effects in much the same way as any other experimental preparation. For example, a behavior such as locomotor activity or lever pressing maintained by food permits a comparison of dose–response relationships and pharmacological antagonism, and it can also be developed as a method for "screening" new clinical compounds provided that it meets certain other criteria that are described in more detail below. In the second case, the study of behavior has been the primary interest, and drugs have been used to dissect and elucidate certain behavioral phenomena. For example, several studies have shown that existing environmental conditions or prior behavioral experiences can modify profoundly the qualitative and quantitative effects of a drug (4, 35, 37). The sensitivity of behavior to these influences has implications for understanding the significance of environmental conditions on the neuropharmacological substrates at which drug effects occur and unify behavioral and neuropharmacological analyses of drug action. Whether pharmacological or behavioral endpoints are of primary interest, both research areas have contributed substantially to (a) our understanding of drugs affecting the central nervous system and (b) our appreciation of how those effects can be determined by preexisting and current environmental conditions.
Research using behavioral techniques has continued to evolve over the several decades since the emergence of neuropsychopharmacology as a scientific discipline. As the field of neuropsychopharmacology continues its seemingly inevitable progression towards more molecular analyses, it will be of continued importance to maintain the experimental and conceptual rigor that has characterized the study of behavior as it has developed within the broader context of this field. It will also be critical for behavioral pharmacologists to adapt and address the information stemming from this progress. Although the actions of drugs affecting the central nervous system are studied at many different levels, it is inevitable that a thorough analysis will eventually address issues of a behavioral nature. Experiments involving the study of the actions of molecular signaling systems at the cellular level take on added significance when those processes can be related meaningfully to the integrated activities of the behaving organism. As our understanding of molecular mechanisms and targets of drug action increases, it will be of increasing importance to understand the functional relationships of those processes to the expression of various behaviors.
There is a particular irony in the current situation in which it appears that as the understanding of molecular events underlying synaptic transmission and neuroregulation has assumed increasing complexity and sophistication, the procedures used to evaluate the relationship of those events to behavior are, in many cases, often rather simple. The fact that some of the current efforts to integrate molecular biology with behavioral pharmacology appear to have regressed to the use of simpler procedures should not be interpreted as a disregard for the sophisticated advances that have occurred in behavioral pharmacology over the past several decades. In some sense, this situation is similar to that which existed early in the field of neuropsychopharmacology where relatively simple procedures were used initially to assess the actions of drugs such as chlorpromazine, chlordiazepoxide, and imipramine. These drugs were brought into the experimental behavior laboratory after unequivocal evidence of clinical efficacy. The establishment of clinical activity could then be used to develop more complex procedures that would differentiate among the compounds possessing different clinical utility and different neuropharmacological actions. Indeed, behavioral procedures such as conditioned avoidance and "conflict" or punishment have been extraordinarily successful in predicting clinical efficacy and activity for the antipsychotic and anxiolytic drugs, respectively. Newer procedures have also been introduced for the evaluation of antidepressant activity, providing a reasonably broad array of behavioral procedures with relatively good predictive value for numerous clinical disorders (46, 49). Despite these significant advances, there is a continuing need for new procedures that address emerging disciplines, and there is also a necessity for continued analysis and refinement of existing techniques. Until newer research directions involving, for example, "knockout" studies, transgenic animals, or other techniques become firmly established (see Basic Concepts and Techniques of Molecular Genetics and Genetic Stategies in Preclinical Subtsance Abuse Research), it is perhaps best to rely on relatively simple and straightforward procedures. Some of these directions are discussed at the end of this chapter.
The main objective of this chapter is to provide a foundation for, and an overview of, the use and assessment of behavioral techniques in the study of drug action. The scope will necessarily be somewhat limited because several excellent monographs are available that provide detailed information on both behavioral principles and procedures in behavioral pharmacology (e.g., see refs. 26 and 59). This chapter will also attempt to address the means by which more recent techniques can be meaningfully and productively incorporated into the field of behavioral neuropsychopharmacology.
PRINCIPLES OF BEHAVIOR
A detailed review of guiding principles underlying the study of behavior is beyond the scope of this chapter. However, it is of critical importance to appreciate the complex processes involved when neuropsychopharmacological research is escalated to include behavioral questions. One of the more striking aspects of many who evaluate behavioral research often has been the lack of appreciation for seemingly simple principles that, upon intensive scrutiny and experimental analysis, are of overwhelming importance. Often, for example, great care is taken in the preparation of solutions, mixtures of brain homogenates, and other experimental details, while considerably less attention is given to matters surrounding the acclimation of animals to the experimental testing conditions, handling, and so on despite the fact that these variables also can be of critical significance. Some of these concerns can, of course, be overcome by studying variables that are strong enough to override the influence of such intrusions, but, in many cases, this is not possible because the variables controlling behavior exert subtle but disruptive influences.
Substantial progress has been made in the development of techniques that permit the objective and quantitative study of behavior that is stable over time, manipulable over a range of controlling parameter values, reproducible within and across species, and sensitive to a number of pharmacological and environmental interventions. In the field of neuropsychopharmacology, the adoption and widespread use of these procedures has had the multiple benefit of broadening our understanding of behavior, elucidating the principles and mechanisms of drug action, and demonstrating the dynamic interactions between behavior and the neurochemical substrates influencing both behavior and drug action.
Reflexive Behavior and Conditioned Reflexes
One basic type of behavior that has been utilized in research on the behavioral effects of drugs is respondent or reflexive behavior. This type of behavior is elicited by specific stimuli and usually involves no specific training or conditioning in that the responses are typically part of the behavioral repertoire of the species and are expressed under suitable environmental conditions. Although factors responsible for the occurrence of these behaviors presumably lie in the organism's distant evolutionary past, certain unconditioned responses, called reflexes, can be brought under more direct and immediate experimental control through the use of procedures first discovered and systematically explored by Pavlov. Such procedures consist of expanding the range of stimuli capable of producing or eliciting a response. For instance, considerable use has been made of a procedure for the study of anxiolytic drugs in which a stimulus paired with the delivery of electric shock enhances the response to a loud auditory stimulus that elicits a startle response (13). When the startle reflex is reduced by the presentation of a brief stimulus presented immediately before the eliciting startle stimulus, "prepulse inhibition" results; this phenomenon has been useful in the evaluation of neuroleptic drugs (52). For the study of drugs that impair or enhance memory, conditioned reflexes such as those by the nictitating membrane of the rabbit eye have proven informative (24). These behaviors depend primarily on antecedent events that elicit specific responses. Typically, these responses do not undergo progressive differentiation in that the responses to either a conditioned or unconditioned stimulus are generally quite similar. Such procedures do not establish new responses, but the range of stimuli to which that response occurs is expanded. Protocols for eliciting conditioned and unconditioned reflexes have been automated, and the measurement techniques for reflexes in response to startle, eyeblink, or nociceptive stimuli result in precise and accurate data.
In some cases the elicited behavior is elicited by the administration of a drug, and then the particular behavior is used to define or assess pharmacological activity. For example, the administration of high doses of amphetamine can elicit stereotyped behavior; 5-HT1A agonists, such as 8-hydroxy-di-n-propylamino tetralin (8-OH-DPAT) can elicit the "serotonin syndrome" consisting of head weaving, reciprocal forepaw treading, and hindlimb abduction. When there is a particular behavior elicited by a drug, this often suggests that the behavior is mediated by a specific neurotransmitter receptor. Drug-induced behavior has been quite useful for studying the pharmacology of various neurotransmitter systems. Technically, drug-induced behavior is assessed by trained observers who employ rating scales. Video-tracking systems have been developed to automate the detection of stereotyped behavior.
In contrast to respondent behavior, operant behavior is controlled by consequent events in that it is established, maintained, and further modified by its consequences. Operant behavior occurs for reasons that are not always specifiable. Such responses may have some low probability of occurrence, or they may never have occurred previously. Novel or new responses are typically established by the technique of "shaping," in which a behavior resembling or approximating some final desired form or characteristic is selected, increased in frequency, and then further differentiated by the provision of a suitable consequence such as food presentation to a food-deprived organism. This technique has been used widely to develop operant responses such as (a) lever pressing by rodents, humans, and nonhuman primates or (b) key pecking by pigeons. Behavior that has evolved under such contingencies may bear little or no resemblance to its original form and can perhaps only be understood by careful examination of the organism's history. Although some behaviors often appear unique or novel, it is likely that the final product emerged as a continuous process directly and sequentially related to earlier conditions. The manner in which operant responses have been developed and maintained, as well as further modified, has been the subject of extensive study and has had a tremendous impact on the development of behavioral neuropsychopharmacology. Many of the potent variables that influence the occurrence of behavior, such as reinforcement, punishment, and precise schedules under which these events occur, also are of critical import in determining how a drug will affect behavior.
Ethological Analysis of Behavior Patterns
Experimental procedures that engender more complex species-specific behavior patterns in animals have been the focus of the ethological approach. These types of behaviors have evolved in situations of survival. Selection pressure has resulted in the development of sensory and motor functions, sexual behavior, care of the young, social cohesion and dispersion, and interactions with other species in the ecological niche. These elaborate behavior patterns are the result of phylogenetic and ontogenetic processes; they are typical for the species and require no explicit conditioning for their expression, although they can be modified by experience. It is possible to reproduce under controlled conditions the essential features of situations promoting the display of those elements of the behavioral repertoire that are characteristic of exploration, foraging and appetite, reproduction, maternal care, attachment to and separation from the group, as well as aggression and defense (8, 32, 36).
Particularly relevant to neuropsychopharmacology are the ethological approaches to the study of drug action on emotional behavior that derive their rationale from Darwin's argument of emotions having evolved just as did an organism's morphology. Ethological analyses of emotional behavior attempt to delineate its distal and proximate causation in the phylogeny and ontogeny of the organism and to determine its function. When experimental techniques succeed in engendering emotional behavior with an adaptive function such as distress calls by an individual who is separated from the group, then selective drug action on this behavior points to modulation of functionally significant biological mechanisms. Adequate measurements of the behavioral expressions of affect require detailed familiarity with the species-typical displays in order to avoid impressionistic and anthropomorphic accounts, colored by the bias of the observer. Quantitative ethological methods have proven most informative when comprehensive analyses, usually on the basis of audio and video records, incorporate the traditional behavioral measurements of latency, frequency, and duration parameters, as well as a quantification of the temporal and sequential pattern (39). Increasingly more sophisticated levels of analysis assess not just the presence or absence of these behaviors, but also whether or not the species-typical acts, postures, displays, and gestures are fully developed in intensity, latency, and patterning. Precise analyses of the salient elements in an animal's repertoire detect behaviorally selective drug action; that is, they assess the "behavioral cost" for a desired drug effect. As the availability of agents to treat various neurological and psychiatric disorders increases, and the selectivity of the drugs available to treat those disorders improves, it will be possible to use this information to design even more sensitive and selective procedures for the evaluation of pharmacological activity.
Schedules of Reinforcement
One of the more important techniques to emerge from the field of behavioral research is the study of behavior using schedules of reinforcement. Many features of operant conditioning, including its frequency of occurrence, idiosyncratic form, and susceptibility to further modification and intervention, depend on the schedule of reinforcement. A schedule of reinforcement is, by definition, a prescription for the initiation and termination of reinforcing or discriminative stimuli in time and in relation to an organism's responses (42). Most schedules are variations on conditions that arrange for a consequent event to follow a response either after a specified number of responses (ratio schedules) or after a specified period of time (interval schedules). Under both ratio and interval schedules, reinforcement can be arranged to follow either a fixed or a variable number of responses or a fixed or a variable period of time. Under most laboratory conditions, and indeed under most environmental conditions, behaviorally relevant consequences occur intermittently. Properties of behavior that have useful analytical dimensions, such as the rate and patterning of responses, often can be seen only under conditions where consequent events are intermittently scheduled. Although it may seem paradoxical, behavior is actually strengthened and intensified by carefully scheduled intermittent rather than continuous reinforcement. Schedules of reinforcement have allowed and encouraged the creation of reliable quantitative, reproducible features of behavior and have permitted the study of behavioral processes that are of general importance.
In addition to their ability to establish and maintain durable behavior that occurs with reliability and orderliness, schedules of reinforcement offer other practical advantages. When food presentation is arranged intermittently under a schedule to engender and maintain behavior, it is possible to sustain behavior over long periods of time with less concern about satiation, thereby permitting adequate evaluation of the time course of pharmacological activity. If the maintaining event is the self-administration of a drug, it is possible to capture the essence of the dynamic aspects of drug abuse because stimuli can be paired with the drug injection and the behavior can be brought under the control of those stimuli as well as the drug (28). There has been a progressive evolution of newer approaches and procedures to address new developments or issues in the field of drug abuse such as "craving" and withdrawal. The behavioral techniques used in this field have served as the foundation not only for the evaluation of drugs of abuse but also of the principles (such as reinforcement) that initiate, perpetuate, and maintain the process of addiction and dependence (see Animal Models of Drug Addiction).
BEHAVIORAL METHODOLOGY AND DRUG EVALUATION
The number of methods employed to evaluate various behavioral processes and to examine the behavioral effects of drugs has increased dramatically in recent years. One main reason for this increase has been the growing availability of a number of drugs that, on the basis of either neurochemical or traditional receptor binding studies, would be expected to possess certain types of activity, thereby warranting study using sensitive and selective in vivo techniques. There is also heightened interest in newer procedures because many of the new chemical entities are based on molecular techniques that involve the use of cloned receptors; these developments, while offering tremendous potential, also remain to be validated using behavioral techniques. Finally, in recent years a number of drugs have appeared to be clinically effective yet their activity in traditional animal "models" has been inconsistent or absent, thus prompting the need to reevaluate existing procedures and/or develop new ones. The field of behavioral pharmacology is far from static; these developments, coupled with the results obtained in clinical studies, promise to result in newer techniques that provide better sensitivity and selectivity for studying the behavioral effects of drugs.
Among the more compelling principles in the study of operant behavior are those of reinforcement and punishment. Though commonly used terms, these principles refer precisely to processes that, respectively, increase and decrease specific responses following the presentation or termination of some event. Reinforcement and punishment are descriptive, empirical processes that refer to relationships between behavior and its consequences. The use of punishment procedures, as will be discussed below, has provided one of the dominant methods for evaluating drugs effective in the treatment of anxiety. Recent work on the neurobiology of anxiety and the development of anxiolytic drugs with novel mechanisms of action also illustrates some of the successes and challenges of preclinical behavioral research. Procedures such as those developed in the early period of modern experimental psychopharmacology by Geller and Seifter (20) that began to characterize the behavioral actions of anxiolytic drugs have been refined further (10, 45, 47) and have added to the utility of these procedures. The use of punished behavior (sometimes referred to as a conflict procedure), in which the food-maintained responding of an animal is suppressed by a stimulus such as electric shock, has provided a sensitive and systematic procedure for the study of dose-dependent effects of benzodiazepine-type anxiolytics that could be prevented or reversed by benzodiazepine receptor antagonists (5). A hallmark of these procedures is that they enabled the monitoring of graded drug effects over time and on repeated occasions on behavioral parameters predictive of clinically relevant anxiolytic actions as compared to those indicative of sedative or suppressive effects. These features satisfy fundamental pharmacological and behavioral criteria and are obtained in various animal species, including humans. Moreover, these procedures have continued to play an integral role in the analysis of drug activity with the more recent development of partial benzodiazepine agonists that have lower positive intrinsic efficacy but retain significant therapeutic advantages (23, 34). Undoubtedly, these procedures will continue to play an important role in the development of anxiolytic drugs that are devoid of unwanted side effects.
Newer Developments in Behavioral Methods to Study Anxiolytic Drugs
Some of the impetus for other recent developments in the field of anxiolytic behavioral pharmacology appears to have been the discovery of drugs acting through novel mechanisms where conventional punishment procedures appeared less effective. This was the case, for example, with 5-HT1A-type drugs such as buspirone which, though clinically active, were not easily detected using typical rodent models of anxiety such as the Geller–Seifter procedure, and there was a need for different or more sensitive methods to detect the actions of these drugs (6). In this case, punishment procedures using the pigeon provided a useful means of evaluating the novel 5-HT1A anxiolytics and antidepressants. This area of research and the use of animal models for other clinical disorders is discussed in Animal Models of Psychiatric Disorders).
A result of these expanded efforts, however, has been the heightened interest in different methods for evaluating anxiolytic drug activity, including methods using ethologically based approaches (30). An intriguing ethological approach to the study of anxiolytics has focused on vocal responses in mammals in situations where they appear to communicate an affective state. The range of vocalizations includes the following: (a) monotonous ultrasounds emitted by rat pups that are separated from their littermates, (b) modulated calls by juvenile rhesus monkeys, and (c) the more extensive vocal repertoire emitted by adults exposed to environmental and social demands (18, 27, 41, 60). Vocal responses have been effectively modulated in a systematic, dose-dependent, antagonist reversible fashion by prototypic anxiolytics as well as by several classes of substances with potentially anxiolytic effects such as those with actions on serotonergic, N-methyl-D-aspartate (NMDA), and peptidergic receptor subtypes. The behavioral specificity of these drug effects can be assessed by comparing effects on vocalizations with those on laryngeal, respiratory, and thermoregulatory changes.
Drugs as Discriminative Stimuli
One of the more popular methods to emerge in behavioral pharmacology has been the use of drugs as discriminative stimuli. In essence, this procedure consists of establishing a drug as a stimulus in the presence of which a particular response is reinforced. The use of a drug to gain discriminative control over behavior is very different from that mentioned earlier in which a drug elicits a reflexive-like behavior. When a drug is developed as a discriminative stimulus, it is often said to "set the occasion" for a response, indicating that it does not by its administration merely produce the response but makes the response more likely to occur because of past consequences in the presence of that stimulus. Typically, when a drug is established as a discriminative stimulus, a single dose of a drug is selected and, following its administration, one of two responses are reinforced (usually, with rodents or nonhuman primates this consists of pressing one of two simultaneously available levers, with reinforcement scheduled intermittently after a fixed number of correct responses). Alternatively, when saline is administered, responses on the other device are reinforced. Over a number of experimental sessions, a discrimination develops between the administration of the drug and saline, with the interoceptive stimuli produced by the drug seen as "guiding" or controlling behavior in much the same manner as any external stimulus such as a visual or auditory stimulus. Once established, it is possible to perform several additional studies to investigate aspects of the drug stimulus in the same way as one might investigate other physical stimuli. Thus, it is possible to determine "intensity" gradients or dose–effect functions as well as generalization functions that are directed towards determining how similar the training drug dose is to a different dose or to another drug that is substituted for the training stimulus. It has also been possible to use drug discrimination techniques as a means for exploring changes in neurotransmitter function following exposure to neurotoxins or other types of interventions that may alter receptors in the central nervous system (7, 11).
One of the more striking aspects of this technique is the finding of a strong relationship between the generalization profile and the receptor binding characteristics of various drugs. For instance, animals trained to discriminate a benzodiazepine anxiolytic (such as chlordiazepoxide) from saline respond similarly to other drugs that also interact with the receptor site(s) for benzodiazepine ligands (5, 48). However, anxiolytic drugs that produce their effects through other mechanisms, such as the 5-HT1A compounds, do not engender responding similar to that occasioned by benzodiazepines, suggesting that it is actually receptor-mediated activity that is established using this technique and not the action of the drug on a hypothetical psychological construct (3, 33, 43).
While the utility of receptor-selective drug discrimination procedures have become increasingly apparent to pharmacologists and medicinal chemists, the behavioral significance of stimuli correlated with drug administration has only recently been of experimental interest. Recently, the discriminative stimulus properties of "anxiogenic" drugs such as pentylenetetrazol (PTZ) or benzodiazepine inverse agonists, as well as other compounds such as barbiturates and psychomotor stimulants, have been of interest as a means of assessing subjective states (31). For example, rats trained to discriminate PTZ from saline responded on the lever appropriate for PTZ administration when threatened by an aggressive opponent or when exposed to the odor of a cat (19, 55, 56). In addition, the fear-like state of the threatened animal, as assessed by PTZ-appropriate responding, was antagonized by benzodiazepine anxiolytics. Such observations suggest that the drug-induced "anxiogenic" stimulus overlaps considerably with a behavioral fear-like state that is engendered by specific environmental events. Thus, it may be possible to mimic certain behavioral "states" by the administration of a drug, and the drug-related state then can be used to assess certain environmental conditions that may engender the same set of stimuli as those encountered in the environment. This technique has also received attention in attempting to determine whether withdrawal from a drug also produces an anxiogenic response similar to that engendered by PTZ (16; see also Animal Models of Drug Addiction). These studies warrant further exploration and evaluation but provide a possibly useful means for assessing potential "emotional" states induced by specific environmental conditions.
Related Areas of Behavioral Research—Challenges and Problems
The area of drug abuse research has relied heavily on behavioral methodology and has broadened considerably in scope over the past several years. Drugs of abuse have been analyzed using the same conditioning principles outlined above and have relied heavily on behavioral procedures to analyze the processes of tolerance, dependence, withdrawal, and addiction (see Animal Models of Drug Addiction). An area of continuing development, and perhaps the most complex and as yet unresolved, is that pertaining to the field of learning and memory ("cognition"). As yet, there are no prototypical drugs available and, as a result, no validated tests or procedures that engender experimental confidence. Despite a plethora of models and procedures, as well as a pressing need to evaluate potential drugs for the treatment of age-related dementia such as Alzheimer's disease, it is difficult to evaluate these drugs because such models appear to lack predictive value and also because there is, as yet, little understanding of the mechanisms involved in the disease process. Many of these behaviors—that is, those in which behavior is in "transition," such as during sensitization, tolerance, and habituation, as well as in complex forms of learning and maturation—require special research strategies and techniques because exposure of the individual animal to these conditions may produce long-lasting or even irreversible effects.
Another formidable challenge to preclinical research is the development of experimental and analytic procedures that adequately capture behavioral events that are episodic, infrequent, or cyclic in nature. Many affective disorders are characterized by explosive and intense behavior such as psychotic outbursts or depressive phases that may intrude into the behavioral repertoire only once or twice a year. In preclinical research, productive experimental protocols have only begun to emerge that engender intense aggressive behavior (40) or that encompass the assessment of behavioral and biological rhythms (53). While the significance of these phasic and infrequent events for neuropsychopharmacology is apparent, they too require novel experimental and analytical methodologies.
One of the main problems in each of these areas of research—anxiety, drug abuse, learning and memory, and aggression—is that the proliferation of procedures frequently has surpassed their careful experimental evaluation and validation. For example, in the study of anxiolytic drug effects, there are at least 30 different procedures ranging from the traditional Geller–Seifter conflict procedure to procedures utilizing ultrasonic vocalization, conditioned startle responses, or open field activity. During the last decade a range of procedures were introduced that attempted to avoid elaborate conditioning protocols and used exploratory and social behavior suppressed by aversive consequences such as bright light or an unfamiliar environment (17, 30, 54). While these procedures offer the convenience of being rapid and simple, and also have the appeal of increased face validity, these advantages have to be contrasted with the problems surrounding extensive involvement of the experimenter (e.g., more subjective measurements, time to perform evaluations and analyze results), single use of each subject due to rapid habituation, and the apparent variability depending on a host of uncontrolled variables. Furthermore, another potential difficulty is that the uncritical selection of a procedure for the evaluation of a drug may yield information that is of limited utility. The problem is complex because of the acknowledged difficulty of any type of in vitro or in vivo drug evaluation that may be quite good for the discovery of a drug that functions through the particular mechanism for which the test was designed, but which may not detect comparable potential clinical activity of a drug that functions through a different biochemical mechanism to which the screens are not sensitive. This dilemma can only be resolved by a close and detailed understanding of the behavioral, biochemical, and clinical issues as progress in each of these areas continues to unfold.
As mentioned earlier in this chapter, there have been a number of recent developments in the field of molecular neurobiology that, in conjunction with behavioral techniques, provide powerful experimental tools for examining the relationship between receptors and behavior. Two techniques that will undoubtedly receive more widespread use in neuropsychopharmacology involve antisense oligonucleotides and the generation of animals with specific gene mutations.
Antisense Oligonucleotide Approaches in Neuropsychopharmacology
Significant advances have occurred in the molecular biology of receptor subtypes for several neurotransmitter systems, including dopamine and serotonin. Although in some cases the functional significance of the different receptor subtypes is reasonably well understood [e.g., the 5-HT1A receptor appears to be involved in 5-HT synthesis and release that is related to anxiety and depression (62)], for the most part the functional relevance of numerous receptors remains unclear. The use of antisense oligonucleotides to target and inhibit the information flow from gene to protein allows for the development of selective pharmacological tools for better understanding receptor-mediated activity in the central nervous system and also permits the development of therapeutics targeted towards specific genes (57). Recently, an antisense oligodeoxynucleotide of the rat neuropeptide Y1 (NPY-Y1) receptor was used to evaluate the role of this receptor for which there is as yet no specific receptor antagonist (58) (see Basic Concepts and Techniques of Molecular Genetics and Neuropeptide Y and Related Peptides). The addition of this antisense oligodeoxynucleotide to rat cortical neuron cultures reduced the density of Y1 receptors while having no effect on Y2; in addition, there was a concomitant reduction in the decrease in cAMP that typically occurs after Y1 receptor activation. Injection of the oligodeoxynucleotide directly into the lateral cerebral ventricle of rats also resulted in a reduction of cortical Y1 receptors, but produced little effect on Y2 receptors. These animals also were tested in an elevated plus maze used frequently to measure exploratory behavior and to study the effects of anxiolytic and anxiogenic drugs. The maze, which consists of two open and two closed arms, assesses the tendency of rodents to explore the two compartments; normally, less time is spent in the open arms, a result believed to reflect the fear of open spaces by rodents. Anxiolytic drugs increase the proportion of time spent in the open compartments, whereas anxiogenic drugs reduce this time. In the antisense-treated animals there was a decrease in the number of entries and amount of time spent in the open portion of the elevated maze, reflecting an anxiogenic-like effect. Thus, these results support (a) the hypothesis that NPY is involved in anxiety and (b) prompt additional studies using other types of behavioral procedures to extend these findings and to also evaluate NPY involvement in other areas (such as feeding) where it also has been implicated.
A similar approach using antisense was adopted to study the function of the dopamine D2 receptor (61). Intracerebroventricular administration of an oligodeoxynucleotide antisense to the D2 dopamine receptor messenger RNA in mice resulted in a reduction in the levels of D2 receptors and D2 receptor mRNA but had little effect on D1 receptors or D1 mRNA. In antisense-treated mice that also received 6-hydroxydopamine lesions of the corpus striatum, the customary contralateral rotations produced by the D2 dopamine receptor agonists quinpirole and N-propyl-N-2-thienylethylamine-5-hydroxytetralin were significantly inhibited. Importantly, effects of the dopamine D1 receptor agonist 1-phenyl-2,3,4,5-tetrahydro1H-3 benzazepine-7,8-diol HCl or the muscarinic cholinergic receptor agonist oxotremorine were not affected.
These studies with NPY and dopamine receptors suggest that it is possible to use antisense techniques to selectively block specific receptors in the central nervous system, and they indicate that this approach may be a valuable alternative to the use of traditional receptor antagonists to probe the behavioral significance of those receptors. The further use and extension of these powerful technologies to include increasingly more complex behaviors promises to yield (a) a better understanding of the roles of specific receptors in neuropharmacology and (b) more selective tools and therapeutics.
Gene Inactivation Techniques: Mutant Mice and "Knockout" Studies
With the development of technology for targeting and manipulating specific genes, it is possible to produce animals with specific mutations in any gene that has been cloned. The methodology for this approach involves the introduction of a desired gene mutation into embryonic stem cells and the eventual development of a line of mutant animals (22). A number of studies have now shown that inactivation or "knockout" of certain protein kinases produce animals deficient in performing certain tasks. For instance, studies of mice with mutations of a-Ca2+-calmodulin-dependent kinase II (a-CaMKII), a synaptic protein that is enriched in the hippocampus, have been shown to be deficient in learning a spatial navigational task and are also deficient in demonstrating long-term potentiation (LTP), believed to be a synaptic marker of learning and memory (50, 51). Behavioral experiments with the mutant and wild-type mice were conducted using the Morris water maze task, a procedure in which mice are placed in a pool of water and must swim to a platform that is located below the water level. In some cases the platform is indicated by a marker, whereas in other cases it is not; both tasks are believed to assess the learning involving spatial stimuli. The a-CaMKII-deficient mice took longer than the wild-type control subjects to learn the location of both the marked and hidden platform, suggesting an impairment in spatial learning ability. Other test procedures were also used to characterize possible differences between the mutant and wild-type animals. For example, animals with hippocampal lesions not only show deficits similar to those of the mutant mice in these studies in the water maze task, but also demonstrate characteristic increases in locomotor or exploratory behavior, effects shown also by the a-CaMKII mutant animals. Other studies have now also demonstrated impaired learning of spatial tasks involving the water maze, as well as impaired LTP induction in mice with mutations of fyn, a tyrosine kinase gene, and in mice lacking the g subtype of protein kinase (PKC), an enzyme involved in signal transduction in a number of physiological systems (1, 2, 21). Due, in part, to the fact that this research is in its nascent phases, there are still several questions that remain, and certain concerns have already been raised about the nature and specificity of the deficits and about the view that something as complex as learning can be reduced to a single gene (14). However, these developments remain one of the most promising directions for the integration of behavior, pharmacology, and cell biology that may provide insight into an area of research that has been rather stagnant for a lengthy time period.
Finally, knockout studies have also been conducted with specific neurotransmitter receptors and provide additional compelling evidence of the potential utility of this technique in neuropsychopharmacology (25). The 5-HT1B receptor, which is the rodent homologue of the 5-HT1Db (see Molecular Biology of Serotonin Receptors: A Basis for Understanding and Addressing Brain Function, Serotonin Receptor Subtypes and Ligands, and Serotonin Receptors: Signal Transduction Pathways), has been demonstrated in several studies to play an important role in feeding, aggression, and locomotor activity (62). Mutant mice, generated by homologous recombination, lacking the 5-HT1B receptor show diminished locomotor activity compared to wild-type animals and do not respond to the typical locomotor-enhancing effects of the 5-HT1A/1B agonist RU 24969. Additionally, the mutant mice are also hyperaggressive, suggesting that the pharmacological analyses of the role of this receptor as subserving a role in locomotor activity and aggression are due to activity of this receptor subtype.
These developments in molecular biology and genetics provide a new set of tools with which to explore the complex interrelationships between neuropharmacological systems and behavior and promise a fertile means for generating insight into processes that previously could only be specultative. At present, the behavioral techniques have been rather basic when compared to some of those outlined in this chapter, but, as was suggested earlier, it is necessary to use uncomplicated procedures initially and then move to more complex techniques. Undoubtedly, this will occur as the field of molecular neuropsychopharmacology continues to grow and as more individuals embrace the combined approaches of behavior and molecular neurobiology.
SUMMARY AND CONCLUSIONS
The techniques and principles of behavioral analysis and drug action summarized in this chapter point to the richness and potential benefits derived from combining the careful study of behavior with the analysis of drug effects. A drug is not simply a molecule with static, unitary effects but can exert an array of behavioral effects depending on several well-characterized features of the previous and immediate environment. The study of drugs within the context of neuropsychopharmacology contributes both to an understanding of behavior and to the systems that subserve drug action and behavior. There are an overwhelming number of procedures that have been developed to study the behavioral effects of drugs over the period of time since the discipline of neuropsychopharmacology was initially established. Many have been used repeatedly and have served the science well, helping to establish a prominent place in the field for characterizing the behavioral effects of drugs and for understanding neuropharmacological mechanisms underlying behavioral processes. With the advent and incorporation of new molecular techniques into the field of neuropsychopharmacology, it will be a challenging time to adapt and extend existing procedures to provide the type of synergy possible when different fields merge.
We wish to thank Kathleen O'Sullivan for her assistance in the preparation of the manuscript.