Brazilian Journal of Medical and Biological Research (1997) 30: 289-304 Ethology and anxiety 289 ISSN 0100-879X

Animal models of anxiety: an ethological perspective

R.J. Rodgers, Ethopharmacology Laboratory, Department of Psychology, B.-J. Cao, University of Leeds, Leeds LS2 9JT, England A. Dalvi and A. Holmes

Abstract

Correspondence In the field of anxiety research, animal models are used as screening Key words R.J. Rodgers tools in the search for compounds with therapeutic potential and as • Animal models of anxiety Ethopharmacology Laboratory simulations for research on mechanisms underlying emotional behav- • Ethology Department of Psychology iour. However, a solely pharmacological approach to the validation of • Defence University of Leeds such tests has resulted in distinct problems with their applicability to • Risk assessment Leeds LS2 9JT • systems other than those involving the benzodiazepine/GABA re- Plus-maze England A • ceptor complex. In this context, recent developments in our under- Behavioural profiling Fax: 44 (113) 233-5749 • Pharmacology E-mail: [email protected] standing of mammalian defensive behaviour have not only prompted the development of new models but also attempts to refine existing Based on paper presented at ones. The present review focuses on the application of ethological the XI Annual Meeting of the techniques to one of the most widely used animal models of anxiety, Federação de Sociedades de the elevated plus-maze paradigm. This fresh approach to an estab- Biologia Experimental, Caxambu, MG, Brasil, August 21-24, 1996. lished test has revealed a hitherto unrecognized multidimensionality to plus-maze behaviour and, as it yields comprehensive behavioural Research supported by the profiles, has many advantages over conventional methodology. This Medical Research Council, assertion is supported by reference to recent work on the effects of the Royal Society, the Wellcome diverse manipulations including psychosocial stress, benzodiazepines, Trust, and the University of Leeds. GABA receptor ligands, neurosteroids, 5-HT1A receptor ligands, and panicolytic/panicogenic agents. On the basis of this review, it is suggested that other models of anxiety may well benefit from greater

Received December 3, 1996 attention to behavioural detail. Accepted December 19, 1996

Introduction conveniently be classified as either condi- tioned or unconditioned responses to stimuli Animal models form the backbone of which appear capable of causing anxiety in preclinical research on the neurobiology of humans (Table 1). Although conditioning psychiatric disorders, and are employed both models permit fairly precise experimenter as screening tools in the search for novel control over behavioural baselines, they of- therapeutic agents and as simulations for ten necessitate food or water deprivation, the studies on underlying mechanisms (1-8). use of electric shock and considerable time More than 30 animal models of anxiety are investment in the training of subjects. In currently in use and, while some are based contrast, while prone to more variable on physiological (e.g., hyperthermia) or en- baselines, models involving unconditioned docrine (e.g., plasma corticosterone) re- (i.e., spontaneous) behaviour generally have sponses to stress, the vast majority are be- a higher degree of ecological validity, are havioural in nature. Behavioural models may less susceptible to confounds arising from

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interference with learning/memory, hunger/ (3,5,7,8). Such problems have led to wide- thirst or nociceptive mechanisms and, at least spread (and, in our view, unwarranted) criti- in principle, allow for a truly comprehensive cism of the models themselves rather than a ‘behavioural profiling’ of experimental in- more logical acceptance of the fact that ‘phar- terventions. macological validation alone does not make In view of traditional approaches to the a test a model of anxiety’ (4, p. 323) and that pharmacotherapy of human anxiety disor- existing procedures should more accurately ders, it is perhaps understandable that the be considered ‘models of benzodiazepine major approach to validation of animal mod- psychopharmacology’ (2, p. 22). els of anxiety has been pharmacological. The need for a new strategy in preclinical Thus, while many of these procedures have a anxiety research is not only indicated by the reasonable degree of face validity, their ma- apparent limitations of existing animal mod- jor claim to status rests upon selective els, but also by the need for novel, safe and responsivity to agents with established effi- effective treatments for the full range of cacy in the clinical management of anxiety anxiety-related disorders. In this context, disorders (predictive validity). As benzodi- there has been growing medical and public azepines have been predominant in this field concern about the side-effect profile of com- for almost 4 decades, pharmacological vali- monly prescribed benzodiazepine anxiolytics dation has, in practice, involved the use of which, acutely, may include cognitive im- chlordiazepoxide or diazepam as a ‘gold pairment and, chronically, the development standard’. Although this approach has worked of normal-dose dependence (9). In addition well in identifying the anxiolytic potential of to these problems, which may or may not be

other benzodiazepine/GABAA receptor-re- manageable (10), it is widely acknowledged lated agents (‘me-same’), an obvious draw- that the benzodiazepines are only truly ef- back is with the identification of compounds fective in the treatment of one of the many which may achieve anti-anxiety effects anxiety-related conditions recognized in through unrelated mechanisms (‘me-differ- DSM-IV (11), i.e., generalized anxiety dis- ent’). An excellent example of the pitfalls of order. As such, there is also a demonstrable adopting a purely pharmacological approach need for the development of animal models to validation has been the general insensitiv- for different anxiety states and not just those ity of existing models to the clinically effec- which respond to benzodiazepines. Although

tive 5-HT1A partial agonist, it seems likely that existing models may, to a greater or lesser extent, already be tapping Table 1 - Some commonly used animal models of anxiety. different facets of anxiety (1), it is not yet clear how individual tests relate to specific Adapted from Refs. 1-8. dPAG, Dorsal periaqueductal gray matter. clinical conditions. Nevertheless, in consid-

Unconditioned responses Conditioned responses ering the broader question of improvements in animal modelling, it seems prudent to bear Anxiety/defence test battery Active/passive avoidance in mind that a ‘....balance must be struck Elevated plus-maze and zero-maze Conditioned emotional response (CER) Fear/defence test battery Conditioned taste aversion between the proliferation of newer models Free exploration Conflict tests (pigeons and primates) and the refinement of existing ones’ (12, p. Holeboard Defensive burying 49). Human threat (primates) dPAG stimulation Light/dark exploration Fear potentiated startle Open field Four plate test Defensive behaviour: a way forward Social competition Geller-Seifter conflict Social interaction Learned helplessness Historically, it is somewhat paradoxical Ultrasonic vocalization (pups) Ultrasonic vocalization (adult) that attempts to develop animal models of

Braz J Med Biol Res 30(3) 1997 Ethology and anxiety 291 anxiety have paid relatively little attention to tance in anxiety research and, in this context, behaviour or, more specifically, to the issue recent studies have pointed to a remarkable of behavioural validation. While acknowl- similarity among vertebrates in the neural edging its hidden or subjective aspects, hu- systems (e.g., amygdala, periaqueductal gray man anxiety is invariably reflected in overt matter) involved in detecting danger and behavioural disturbances including, for ex- producing defence responses (23,24). As this ample, avoidance, escape, non-verbal vocal- system is so strongly conserved in evolution, ization and/or hypervigilance (13-15). When it has been convincingly argued that we can also observed in animals, such responses learn much about human defence or fear suggest (but of course do not prove) a com- reactions by studying other creatures (25). mon affective state. At minimum, therefore, At the behavioural level, it has been the human and animal responses may be said known for quite some time (26) that animals to be analogous, thereby providing neces- are capable of displaying diverse defensive sary face validity for the animal model. Con- reactions in response to external threats, e.g., struct validity, on the other hand, implies a predator or an aggressive conspecific. In that the human and animal responses are mammals (e.g., rats), such behaviours clas- homologous (common substrate) and, fur- sically comprise freezing, flight, defensive ther, that the response in question has clini- threat/attack and even death-feigning, and cal significance for the disorder being mod- are dependent both upon threat imminence elled (16). Herein lies a significant problem (27) and escape opportunity (28). This rep- in that, in the absence of a detailed under- ertoire is most clearly observed in feral ani- standing of the substrates of human anxiety, mals, although several features (e.g., freez- it becomes impossible to rigorously estab- ing) may be elicited by appropriate stimula- lish homology between animal and human tion of laboratory rats (29). However, recent response patterns. Nevertheless, the truly research has shown that the rodent defensive remarkable parallels between fear/anxiety repertoire is even more elaborate than sug- reactions in humans and animals, together gested by this classical description. Thus, with the ease with which we seem able to laboratory rats will bury dangerous objects empathize with frightened (as opposed to (30), and emit ultrasonic vocalizations when depressed or schizophrenic) animals, sug- injured (31), separated from their mother gest that at least some animal response pat- (32) or exposed to a natural predator (33). terns may ultimately fulfil the homology cri- Furthermore, in potentially dangerous situa- terion. tions (for example, when a predator has been In The Expression of the Emotions in seen but is no longer present), laboratory rats Man and Animals (1872), Charles Darwin (34) and mice (35) have been reported to (17) laid an important foundation for view- engage in a cluster of behaviours collec- ing the defensive patterns of other species as tively referred to as risk assessment. These essential evolutionary precursors to human responses, originally identified in specially fear and anxiety reactions (18). More re- constructed visible burrow systems, are char- cently, a number of clinical accounts have acterized by cautious approaches to a sur- conceptualized human anxiety disorders as face area where a predator (cat) has briefly disorders of defence (e.g., 19-22), in which been presented and include i) scanning the the key feature concerns inappropriate acti- danger area from tunnel openings, ii) vation of defensive behaviour arising from stretched attend, or flatback, postures di- erroneous assessment of danger. As such, an rected towards the danger area and iii) understanding of the neurobiology of defen- stretched, or flatback, locomotion upon ini- sive behaviour assumes particular impor- tial re-entry into the danger area. Very simi-

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lar behaviour patterns have been observed in or ratio of total arm entries, and time spent the rat defensive burying paradigm (36,37) on the open arms expressed as a percentage and in mice exposed to conspecific odours or ratio of total arm time), while total arm (38,39), supporting the contention that their entries have frequently (but erroneously, see function is to inform behavioural strategy in below) been employed as a measure of gen- potentially dangerous situations (18,40). As eral activity. many animal models of anxiety are based The advantages of the conventional el- upon exploration of novel (and, hence, po- evated plus-maze are obvious and numer- tentially dangerous) environments, it would ous: ecological validity, economy, speed, be predicted that these situations should also simplicity and bidirectional sensitivity elicit risk assessment. Recent work in our coupled with the fact that the procedure does laboratory and elsewhere has confirmed this not require lengthy training procedures in- prediction. volving the use of food/water deprivation and electric shock (44). However, while there The elevated plus-maze is no doubt that the conventional plus-maze is highly sensitive to the influence of benzo-

The elevated plus-maze is undoubtedly diazepine/GABAA receptor-related manipu- one of the most widely used animal models lations, effects obtained with other anxiety- in contemporary preclinical research on anxi- modulating agents (e.g., buspirone) have been ety (5,41). This test derives from the early very much more variable (5,8,49). Although observation that, in mazes consisting of open this profile has led certain authors to doubt and closed alleys, rats consistently show the utility/reliability of the model (50,51), higher levels of exploration in enclosed al- alternate interpretations of this pharmacolo- leys and, when faced with a choice of alley gical inconsistency are just as plausible. As type, typically avoid those without walls already noted, the way in which tests such as (42). Some 30 years later, this finding led to the plus-maze were originally developed (i.e., the first studies on the potential utility of an benzodiazepine criterion) provided excel- elevated ‘X’-maze as a model of anxiety lent tools for detecting benzodiazepine/ (43). Using a simple maze configuration, GABA-related compounds and it would seem elevated 70 cm above floor level and com- churlish, to say the least, to criticize them for prising two open and two enclosed arms relative insensitivity to agents operating (like arms opposing one another), these au- through entirely different mechanisms. This thors not only confirmed that rats avoid the (not irrelevant) point aside, negative and/or open arms but also demonstrated that open contradictory findings with buspirone may arm avoidance is reduced by diazepam and arise from the use of inappropriate dose enhanced by picrotoxin (an anxiogenic ranges (e.g., the issue of pre- vs post-synaptic agent). Comprehensive validations of the sites of action) or, indeed, from the fact that test (now termed the elevated ‘plus’-maze) animal studies more often than not involve for use with both rats (44) and mice (45) acute administration whereas clinical expe- quickly followed this initial report, and more rience would indicate therapeutic efficacy recent research has suggested its potential only after several weeks of treatment (see utility for other species including guinea also 52). In our view, an equally important pigs (46), wild voles (47) and Syrian ham- consideration arises, somewhat paradoxi- sters (48). From the outset, the primary indi- cally, from the aforementioned advantages ces of anxiety in the plus-maze have been of the plus-maze paradigm. Thus, the fact spatiotemporal in nature (i.e., the number of that a maze is readily constructed, sessions open arm entries expressed as a percentage are short (5 min) and conventional scoring is

Braz J Med Biol Res 30(3) 1997 Ethology and anxiety 293 both fast and easy has been instrumental in the fashionable fallacy of dispensing with the adoption of the test by numerous re- description. The implicit criticism of limited search laboratories (at least 100 according to analysis is also apparent in the writings of a recent survey; 41). Unfortunately, in the the Dutch ethologist, Niko Tinbergen (58), same way tradition holds that there are as who insisted that the first task in any behav- many psychologies as there are psycholo- ioural study ought to be the observation and gists, there are probably as many plus-maze description of the full behavioural repertoire ‘paradigms’ as there are laboratories con- of the species under study. In a similar fash- ducting preclinical research on anxiety. ion, the British ethologist W.H. Thorpe (59) Major inter-laboratory differences there- cogently argued that the closer the initial fore exist in the use of the plus-maze para- rapport a behavioural scientist can establish digm, and these encompass a wide range of with the animal he is studying, the more organismic and procedural variables (Table successful his investigations are likely to be. 2; see also 5). This point is very firmly In the present context, the benefits of this emphasized in a recent survey of 65 ‘plus- general philosophy are clearly evident in the maze laboratories’, the results of which elegant work of the Blanchards (reviewed clearly show that pharmacological response above) on defensive patterns in rats and is heavily influenced by choice of strain, mice, work that has led to important devel- pretest manipulation of subjects and the opments in contemporary approaches to re- aversiveness of the test conditions (41). For search on the neurobiology of fear and anxi- example, it would appear that sensitivity to ety. potential (particularly non-benzodiazepine) Over the past 6 years, we have developed anxiolytics is enhanced by stressing animals a procedure that allows for the comprehen- prior to testing (e.g., by moving from hold- sive ‘profiling’ of behaviour in the murine ing to test room) and/or by using more aver- elevated plus-maze paradigm. This approach sive test conditions (e.g., high light), thereby was stimulated by advances in our under- suggesting the fundamental importance of standing of the rodent defensive repertoire, endogenous tone in key neurochemical sys- several years of experience in using conven- tems. It is therefore essential that laborato- tional plus-maze methodology (60), and pre- ries using, or planning to use, the plus-maze liminary evidence that rodents display at invest sufficient time and effort in defining least some defensive elements in this test optimal test conditions prior to drug studies. (e.g., freezing, defaecation, and stretched In view of these considerations, the impor- attend postures (SAP); 44,50). Detailed vid- tance of response baselines in behavioural pharmacology and the fact that conventional Table 2 - Some sources of inter-laboratory variation in use of the elevated plus-maze plus-maze scoring actually pays minimal at- paradigm. tention to actual behaviour, several research Species, strain, age and gender groups have argued that the utility/reliabil- Housing conditions and light cycle ity/sensitivity of this model might also be Prior handling and injection experience Prior maze experience improved by adopting a more ethological Prior exposure to holeboard/activity test approach to data collection (e.g., 50,53-56). Adaptation to test laboratory Time of testing and lighting level Presence of experimenter in test room Behavioural profiling in the plus-maze Maze construction, e.g., opaque/transparent walls, open arm ledges Method of scoring, e.g., live, manual/automated, videotape In considering alternate approaches to Definition of measures, e.g., arm entry the study of animal behaviour, the German Validation of measures Measures scored: conventional, ethological ethologist Konrad Lorenz (57) referred to

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eotape analysis of the behaviour patterns of error and technical innovation. On the trial untreated mice on the maze, initially con- and error side, we have found that by re- ducted using manual event recorders/timers, positioning the videocamera (from directly yielded a catalogue of readily identifiable overhead to an angle of ca. 50o), our ability behaviours including rearing, head-dipping, to discriminate non-exploratory behaviours stretched attend postures and closed arm (i.e., immobility, grooming) was markedly returns (i.e., doubling back, rather than leav- improved as was our ability to accurately ing, a closed arm) as well as several non- score additional behavioural elements (e.g., exploratory behaviours (61). In addition, it sniffing, flatback approach). This work has was noted that the spatial distribution of been greatly facilitated by the adoption of certain acts and postures (i.e., head-dipping, ethological analysis software (‘Hindsight’, a SAP) appeared to be dependent upon con- package developed by Dr. Scott Weiss) which tact with the maze walls, leading us to differ- permits direct keyboard entry to a PC using entiate ‘protected’ (wall contact in closed separate keys for location and behaviour. arms and/or central platform) and ‘unpro- The datafiles created cannot only be easily tected’ (no wall contact, i.e., on open arms) downloaded for standard statistical analyses forms of these behaviours. This differentia- (i.e., group differences) but are also ame- tion agrees well with subsequent studies by nable to reconfiguration thereby permitting Treit et al. (62) in Canada which confirmed the execution of more detailed analysis, e.g., that the most important maze feature pro- minute-by-minute behavioural changes. ducing open arm avoidance is the absence of Table 3 summarizes the various spatiotem- walls (i.e., the absence of thigmotactic cues) poral and behavioural measures now rou- and not, as might have been suspected, the tinely recorded in our laboratory, grouped height of the maze above floor level. according to the outcome of a recent factor Most research techniques evolve over analytic study on a large cohort of undrugged time, usually through a combination of trial/ DBA/2 mice. This analysis identified a 6- factor structure accounting for >75% of the Table 3 - Behaviours recorded in the ethological plus-maze, grouped according to total variance (63). factor analysis (subjects, male DBA/2 mice). In close agreement with earlier factor

All factor loadings ≥0.40, with highest loading elements emboldened. Total variance analyses in both mice (45) and rats (1), the accounted for = 76.1%. For full details, see Ref. 63. *% open entries = (open entries/ primary indices of anxiety and locomotor total entries) x 100; % time = (time in area/session length in s) x 100; % protected activity loaded on separate factors (Factors 1 scores = (protected/total) x 100. and 2, respectively). In this context, the ‘pro- Factor Interpretation Behavioural elements* tected’ forms (%p values) of head-dipping, SAP, sniffing, and flatback approach also 1 ‘Anxiety’ Total arm entries, open arm entries, % open entries, loaded highly on Factor 1, confirming their % open time, % closed time (-), % centre time (-), closed arm returns (-), % protected head-dipping (-), close relationship to open arm avoidance. In % protected SAP (-), % protected sniffing (-) and % contrast, closed arm entries loaded highly protected flatback approach (-) and exclusively on Factor 2, confirming the 2 ‘Locomotion’ Total arm entries, closed arm entries, total superiority of this measure (vs total entries) flatback approach as an index of locomotor activity (see also 3 ‘Risk assessment’ Total SAP, total sniffing 1,45,64-66). However, unlike previous fac- 4 ‘Decision-making’ Closed arm returns (-), grooming (-), % centre tor analyses based solely on conventional time, % closed time (-) measures, the incorporation of specific be- 5 ‘Vertical activity’ Total arm entries, closed arm entries, rear havioural acts and postures revealed four frequency, rear duration, grooming (-) additional dimensions to behaviour displayed 6 ‘Exploration’ Total head-dips, total SAP in the maze. It is particularly interesting to

Braz J Med Biol Res 30(3) 1997 Ethology and anxiety 295 note that % time spent on the centre platform closed arm entries and rearing. It might be (Factor 4, ‘decision-making’) and total SAP inferred from this pattern of behavioural (Factor 3, ‘risk assessment’) loaded on fac- change that drug ‘X’ has had a non-selective tors other than the main ‘anxiety’ factor behavioural (e.g., sedative) action. However, (Factor 1), and that rearing (Factor 5, ‘verti- if we add into the equation that total arm cal activity’) and head-dipping (Factor 6, entries remained unchanged and knowledge ‘directed exploration’) loaded separately that virtually all rearing occurs in the closed from locomotor activity (Factor 2). Although arms, this hypothetical drug profile might differing somewhat in specific details, simi- instead suggest a treatment-induced redistri- lar multidimensionality has been revealed in bution of exploratory behaviour (i.e., in- recent ethological studies of rat behaviour in creased open arm entries plus decreased the plus-maze (64-66). However, as strain closed entries = no change in total; decreased comparisons in mice have not only sug- closed arm entries = reduced opportunity for gested that basal plus-maze profiles differ rearing). Furthermore, should the hypotheti- quantitatively (67,68) but also qualitatively cal profile also indicate an apparent increase (i.e., factor structure; 69), caution should be in head-dipping and no change in immobility exercised in too readily applying knowledge scores, an interpretation in terms of behav- of behavioural structure from one strain/ ioural non-selectivity becomes even more species to another. untenable. The following section provides This new conceptualization of response some concrete examples of the advantages patterns in the plus-maze offers several po- of behavioural profiling in the murine plus- tential advantages in the interpretation of maze. treatment effects in this model. For example, in DBA/2 mice, the apparent independence Research findings of measures related to avoidance (Factor 1), risk assessment (Factor 3) and decision-mak- Psychosocial stress. Social stress, in the ing (Factor 4) would clearly suggest the pos- form of defeat experience, is a potent stimu- sibility of dissecting treatment effects on lus in activating non-opioid analgesia mecha- different components of the affective state nisms in mice (71), with both behavioural induced by exposure to the maze. Further- (72) and pharmacological (73-77) evidence more, the important issue of behavioural pointing to anxiety as a key factor in this specificity, normally addressed by examin- anticipatory defence reaction. In confirma- ing treatment effects on closed arm entries tion of this hypothesis, immediate prior so- (Factor 2), may also be approached by exam- cial defeat (and, to a lesser extent, exposure ining treatment effects on other active to the scent of an aggressive male conspe- behaviours such as rearing (Factor 5) and cific) was found to produce a significant head-dipping (Factor 6). As in other areas of anxiogenic-like effect in the murine plus- ethopharmacology, e.g., research on agonis- maze (78). This enhancement of anxiety was tic behaviour (70), such interpretation should evident on both conventional open arm avoid- be guided by the overall behavioural profile ance measures as well as several ethological and not by dogmatic adherence to precon- measures, including SAP and closed arm ceptions regarding the importance of any returns. Not only is this finding consistent single variable. A hypothetical example may with the anxiety hypothesis of non-opioid help to convey this point more clearly. As- defeat analgesia but it also shows that the sume that drug ‘X’ produces an anxiolytic- plus-maze is sensitive to biologically mean- like increase in the frequency and duration ingful, as well as artificial ‘stressors’ (5). of open arm visits yet significantly reduces Importantly, the observed stress-induced

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changes in measures related to anxiety were fects in the plus-maze though, interestingly, accompanied by significant behavioural sup- changes in risk assessment are less apparent pression indicating that care should be taken (ceiling effect?) than changes in open arm in the interpretation of certain drug profiles. avoidance (85).

In particular, when considering the action of GABAA receptor ligands. Although it is anxiogenic drugs, reduced behavioural out- widely held that benzodiazepine anxiolytics put should not automatically be taken as exert their behavioural effects through a fa-

evidence of behavioural non-selectivity. cilitation of GABAA receptor function, the Afterall, movement inhibition is a character- effects of direct GABAergic manipulations istic of very frightened animals (28). in animal models of anxiety are actually Benzodiazepines. Studies from our labo- highly variable. However, recent studies from ratory have confirmed that full and partial our laboratory (86,87) have shown that, in benzodiazepine receptor agonists produce contrast to the lack of specific effect of a

behavioural changes in the maze consistent GABAB receptor agonist (R(+)baclofen) and with anxiety reduction (79-81). Typically, a GABAB (CGP35348), these agents reduce open arm avoidance and indirect facilitation of GABA function (so-

risk assessment (e.g., SAP) measures while dium valproate) or direct GABAA receptor enhancing exploratory head-dipping. Such stimulation (muscimol) produces a behav- changes generally occur at doses which do iourally selective and diazepam-like not suppress general activity, and are appar- anxiolytic profile in the murine plus-maze.

ent following both acute (79-81) and chronic Furthermore, GABAA antagonists, such as (82) treatment. However, while higher doses picrotoxin (86) and pentylenetetrazole (85), of chlordiazepoxide and diazepam may in- produced clear evidence of anxiogenic-like crease immobility scores, no such changes activity accompanied, at high doses, by be- were observed with the partial agonist, havioural suppression. Intriguingly, compara- bretazenil. This comparison indicates that tively low doses (2-4 mg/kg) of pentylenetet- current methods are able to discriminate com- razole (vs an anxiogenic dose of 20 mg/kg) pounds from the same general class (see also were associated with an anxiolytic-like pro-

section on 5-HT1A receptor ligands), and that file, a finding that clearly merits further re- partial agonists may indeed be behaviourally search. more selective than full agonists. Further- Neurosteroids. Certain steroids, includ- more, and as has since been reported for the ing 5α- and 5ß-reduced metabolites of pro- rat ‘zero-maze’ (an annular variant of the gesterone and deoxycorticosterone, are plus-maze with alternating open/closed quad- known to exert rapid, non-genomic effects in rants; 83) and rat plus-maze (84), benzodiaz- the CNS through allosteric modulation of

epine-induced reductions in risk assessment GABAA-mediated chloride influx (88). In tend to occur at doses below those required this context, very recent work in our labora- to significantly increase open arm activity. tory (89) has revealed significant anxiolytic- This finding suggests that, as for enhanced like activity for the deoxycorticosterone head-dipping, reductions in risk assessment metabolite, 3α-tetrahydrodeoxycorticoste- may be a particularly sensitive marker for rone (THDOC) as well as the progesterone benzodiazepine-like behavioural activity. metabolites, pregnanolone and 3α- (but not Finally, and as would be predicted on the 3ß-) allopregnanolone. Further work has basis of bidirectional control of anxiety via shown that an intermediate metabolite of

the benzodiazepine/GABAA complex, com- progesterone, 5ß-dihydroprogesterone pounds acting as inverse agonists at this site (DHP), also produces an anxiolytic profile (e.g. FG 7142) produce anxiogenic-like ef- whereas 5α-DHP does not (90). The behav-

Braz J Med Biol Res 30(3) 1997 Ethology and anxiety 297 ioural profiles of the active steroids are very effects from anxiolysis through neutrality to similar to those seen with benzodiazepine anxiogenesis have been reported (5,8). How- anxiolytics, valproate and muscimol (see ever, ethological analysis has revealed an above), providing some support for media- anxiolytic-like effect with both acute and tion through the GABAA receptor complex. chronic buspirone in the murine plus-maze However, further studies are required to con- (93). At doses of 2.5-5.0 mg/kg, acutely firm this presumed mechanism of action. administered buspirone induces an anxio- Serenics. The term ‘serenic’ has been lytic-like effect on conventional open arm coined to describe the effects of a group of 5- avoidance measures coupled with a profound HT receptor ligands which, it is argued, se- suppression of most active behaviours (both lectively inhibit offensive aggression (91). actions more evident after chronic treatment). Although apparently producing this effect in However, at lower doses, significant anxio- the absence of sedation or psychomotor lytic-like effects were observed on several stimulation, evidence from tests not involv- ethological measures, including reductions ing aggression (92) suggests that these agents in risk assessment (see also 94 for compa- may increase defence/fear. To further test rable findings in rat plus-maze). Although this hypothesis, we examined the effects of we have also observed a generally similar two serenic compounds, and profile with another 5-HT1A receptor ago- , in the murine plus-maze. For nist, (95), some potentially im- comparative purposes, and particularly in portant within-class differences were appar- view of similarities in affinity for and action ent. For example, whereas the behavioural at 5-HT receptor subtypes (5-HT1B and 5- suppression induced by high-dose buspirone HT2C), parallel studies with the well-known treatment was associated with prolonged 5-HT anxiogenic agents, mCPP and TFMPP, bouts of immobility (a dopaminergic effect?), were also conducted (61). Although none of this was not observed with high-dose the test compounds produced convincing flesinoxan treatment which, instead, appeared effects on conventional plus-maze anxiety to reduce the overall rate of behaviour. Al- indices (i.e., % open entries, and % open though some weak anxiolytic-like activity time), all markedly increased SAP, percent has also been seen with (±)-8-OH-DPAT, protected forms of SAP and head-dipping, the predominant effect of this compound is and closed arm returns. These effects were also to reduce behavioural output (61). Very strongly dose-dependent and apparent at dose recent work from our laboratory has shown levels below those producing significant be- that the motoric effects of this prototypical havioural suppression. Apart from confirm- 5-HT1A receptor ligand reside in its R(+)- ing that serenics can indeed enhance anxi- enantiomer (full agonist), whereas any weak ety/fear-related behaviours, this study im- anxiolytic-like action seems to be due to the portantly emphasizes that the current meth- action of the S(-)-enantiomer (partial ago- odology is sensitive to changes in affective nist) (96). state that are not necessarily revealed by Considerable debate continues regarding conventional measures. the mechanism whereby buspirone achieves

5-HT1A receptor ligands. As reviewed its anti-anxiety action although it is gener- above, the vast majority of animal models of ally agreed that it relates to a reduction in 5- anxiety have yielded inconsistent and often HT neurotransmission (97). Suggestions as contradictory profiles for 5-HT1A receptor to how such an effect might be achieved agonists/partial agonists such as 8-OH-DPAT range from an agonist action at somatoden- and buspirone. The plus-maze is no excep- dritic 5-HT1A autoreceptors, through an an- tion to this ‘rule’ in that the full range of tagonist action at post-synaptic 5-HT1A re-

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lytic activity in these studies, largely sup- Table 4 - Compounds with 5-HT1A antagonist ac- tivity, dose ranges studied, and control com- pressing active behaviours at the highest pounds tested. doses used. The dose-response profiles of

the 5-HT1A antagonists were invariably bell- For details, see text and Refs. 100-102. shaped, with either loss of activity or behav- Compound Dose range (mg/kg) ioural non-specificity evident at higher doses. Together, these results are very encourag- (-) 0.1-6.4 ing, particularly in view of the wide dose Pindobind 5-HT1A 0.1-2.5 WAY 100135 2.5-20.0 separation for anxiolytic and motoric effects WAY 100635 0.03-9.0 (a pattern very different to that observed pMPPI 0.5-13.5 with full and partial 5-HT1A receptor ago- SDZ 216-525 0.05-3.2 NAN-190 0.1-10.0 nists). However, although our findings sug- gest therapeutic potential for 5-HT1A antago- (+)Pindolol 0.1-6.4 nists in the management of anxiety disor- (ß1) 2.0-18.0 ders, the issue of a pre- versus post-synaptic ICI-118,551 (ß2) 1.0-9.0 (α1) 0.02-2.5 action remains open. Thus, while traditional views on 5-HT and anxiety would suggest a ceptors, to a combination of both effects post-synaptic locus (i.e., reduction in 5-HT (98). Following preliminary and positive transmission), empirical support must be observations with (S)-WAY 100135 (99), obtained through studies on the effects of we have recently explored further the possi- direct antagonist application to sites such as

bility that 5-HT1A receptor blockade would the raphe nuclei and hippocampus. In this produce an anti-anxiety profile in the murine context, it is worth noting that anxiolytic- plus-maze model. To this end, we have stud- like effects have been reported in several rat ied a range of compounds which (with vary- models following intrahippocampal injec-

ing degrees of selectivity) exert antagonistic tion of the mixed 5-HT1A antagonist/ß- effects at these receptors. Table 4 lists those blocker (103), while hippocampal agents/doses studied to date, together with and amygdaloid injections of 8-OH-DPAT compounds used as controls for additional have been found to enhance anxiety

pharmacological actions. (104,105). However, the fact that 5-HT1A Our results, some of which are ‘in press’, receptor manipulations would appear to have have revealed that, despite variation in chem- more consistent effects in mouse (vs rat) ical structure and degree of selectivity for 5- models of anxiety (e.g., 5,8,100-102) and

HT1A receptors, these agents share in com- that a clear species difference exists in 5- mon an ability to reduce anxiety in Swiss- HT1A receptor-mediated physiological re- Webster mice (100-102). In brief, anxiolytic- sponses (e.g., 8-OH-DPAT-induced hypo- like effects on both conventional and etho- thermia: pre-synaptic in mouse but post- logical indices have been observed with synaptic in rat, 106) would caution against

(-)pindolol, pindobind 5-HT1A, SDZ 216- over-interpretation of these findings. 525, WAY 100635 and pMPPI. Importantly, /antipanic agents. It has in view of pharmacological specificity, no been known for some time that panic disor- such effects were observed with wide dose der responds much better to certain antide-

ranges of (+)pindolol, metoprolol (ß1-antago- pressants (phenelzine and ) than nist), ICI-118,551 (ß2-antagonist) or prazosin to traditional anxiolytic agents (107,108). α ( 1-antagonist). Furthermore, both NAN-190 More recent research has suggested that sec- α (mixed 5-HT1A/ 1) and buspirone (partial ond-generation , in particu- agonist) were devoid of convincing anxio- lar the -selective reuptake inhibi-

Braz J Med Biol Res 30(3) 1997 Ethology and anxiety 299 tors (SSRI), are also effective in this regard ety modulation, but also allow a firmer char- and may actually have therapeutic advan- acterization of the procedure as one perhaps tage over conventional tricyclics (109,110). more suited to screening of compounds for However, neither acute nor chronic imi- clinical potential in the treatment of general- pramine treatment was found to alter anxiety ized anxiety disorder and to related mecha- indices in the murine plus-maze (81), with nistic studies. However, it should be noted negative results also reported for chronic that this conclusion may well only apply to fluvoxamine (SSRI) treatment (82). In con- use of the model with test-naive animals in trast, low doses of the noradrenaline-selec- that we have recently obtained evidence (69) tive reuptake blocker, , did pro- that, as for rats (112), the nature of the duce a selective reduction in open arm avoid- anxiety response displayed in test-experi- ance (82), suggesting possible therapeutic enced mice differs qualitatively from that application in anxiety and/or anxious-de- seen in test-naive subjects. In view of the pression. Surprisingly, the atypical antide- possible implications of this finding, behav- pressant, tianeptine, was seen to produce ioural and pharmacological studies on ‘re- anxiogenic-like changes in behaviour fol- test anxiety’ are eagerly awaited. lowing subchronic treatment in the plus- Miscellaneous. The utility of an animal maze and social interaction paradigms (111). model of anxiety rests not only in its ability Although this finding would not be inconsis- to detect bidirectional changes in anxiety tent with its presumed mechanism of action with established and putative anxiolytics/ (enhancement of 5-HT reuptake), it is puz- anxiogenics, but also in i) excluding com- zling in view of its use (at least in France) as pounds that are either inactive or non-specif- a prescription medication. Nevertheless, the ic in behavioural action, and ii) highlighting lack of efficacy of imipramine and fluvoxa- novel (perhaps unexpected) drug effects that mine under present test conditions would may provide fresh insights into mechanisms suggest that the murine plus-maze is not involved in the modulation of affective be- sensitive to compounds with established clini- haviour. Inactive compounds in the murine cal efficacy in the management of panic plus-maze include: the 5-HT2A/C ligands, disorder. This conclusion is further supported and DOI (113); the 5-HT3 receptor by our recent observation that several CCKB antagonists, and WAY 100289 receptor antagonists (L-365260, PD135158), (111,114); the peripherally acting antimus- thought to have potential as antipanic agents, carinic, methyl scopolamine (115); and the are also ineffective over wide dose ranges D1 receptor agonist, SKF38393 (80). (116). Compounds found to produce behav- Pro-anxiety/panicogenic agents. Refer- iourally non-selective effects include the ence has already been made to the anxiogenic- GABAB agonist (+)baclofen and the GABA α like effects of 5-HT1B/ agonists (TFMPP, reuptake blocker, No-711 (86); the 2- mCPP) (61), benzodiazepine receptor inverse adrenoceptor agonist, (117); the agonists (FG 7142) (85) and GABAA recep- neuroleptic, (94); the D1 recep- tor antagonists (picrotoxin, PTZ) (85,86) in tor antagonist, SCH 23390 (116); and the D3/ the present model. However, we have failed 2 receptor agonists, quinpirole (116) and 7- to observe any consistent signs of anxiety OH-DPAT (118). enhancement with the putative panicogenic The present model has also produced agents, CCK-4 and CCK-8 (80), as well as some unexpected findings. For example, the isoproterenol and sodium lactate (85). These centrally acting antimuscarinic, scopolamine findings not only confirm the differential hydrobromide, was found to produce an sensitivity of the murine plus-maze to anxi- anxiogenic-like profile (116). As the major

Braz J Med Biol Res 30(3) 1997 300 R.J. Rodgers et al.

change induced by this agent was a marked full behavioural profiling of the type de- increase in risk assessment, an action on scribed above (based on real-time, and com- more cognitively related, information-pro- puter-assisted videotape scoring) actually cessing mechanisms seems probable. We only requires a total of 10 min per subject (5 have also found that, unlike haloperidol, the min test + 5 min scoring). Less comprehen- sulpiride has quite pro- sive ethological analysis (e.g., scoring only nounced anti-anxiety effects in our model head-dipping and SAP in addition to con- (116). This finding confirms a similar report ventional measures) has been successfully from Costall’s group (119) based on the adopted for the rat ‘zero-maze’ paradigm mouse light/dark exploration model, and cer- (83) and has been reported not to involve any tainly deserves further research attention. significant additional time (120). Finally, in direct contrast to expectation, the Whatever additional time investment may ‘well-known anxiogenic agent’, , be needed for ethological analysis, this is actually produces a plus-maze profile con- more than adequately compensated by the sistent with an unambiguous anxiolytic ac- multiple advantages of this approach. These tion (117). Although it might be argued that may be summarised as i) increased face and this finding is problematic for the present construct validity, ii) comprehensive ‘profil- methodology, it should be noted that a) simi- ing’ of compounds, thereby greatly facilitat- lar anti-anxiety effects have been seen with ing conclusions regarding behavioural speci- yohimbine in other animal models, b) the ficity and permitting fine-grain comparisons effects in the plus-maze are seen in 3 mouse within as well as between drug classes, iii) strains, c) yohimbine has high affinity for reducing or eliminating the need for addi- α sites (e.g., 5-HT1A) other than 2-adrenocep- tional tests (resource implications) to con- tors, and d) more selective α2-antagonists trol for treatment effects on general activity, (e.g., ) are relatively ineffective in appetite, thirst and/or nociception, and iv) the present model (117). This analysis sug- enhanced sensitivity to drug action as evi- gests that, under certain test conditions (e.g., denced by the utility of risk assessment meas- high background stress?), yohimbine may ures. Although obvious progress has recently preferentially influence non-adrenergic been made in extending the principle of ‘be- mechanisms to achieve its apparent anti- havioural profiling’ to the rat zero-maze and anxiety action. plus-maze paradigms, it is particularly im- portant to note the demonstrable utility of Conclusion ethological measures in more clearly charac- terizing drug effects in the defensive burying It is hoped that this brief review has paradigm (37). In view of these findings, is it convinced the reader of the value of an etho- not now time to abandon the ‘quick fix’ logical approach to the elevated plus-maze approach to modelling, and extend ethologi- test. Although it may appear that our analytic cal analysis to other established animal tests technique demands substantially greater time of anxiety? investment than conventional scoring (51),

Braz J Med Biol Res 30(3) 1997 Ethology and anxiety 301

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