Pergamon Preee

Lite 8aienoe~ Vol. 16, PP " 187-200 Printed is the D .S .7~.

MINIRBVLSiP WfiAT DOES IADIVIDUAL HO~IAG MEAR TO A MJUSEY Paul Brain Department of Zoology, University College of Swansea, SA2 ÔPP, South Wales, U .K . Summary Some recent studies relating the effects of individual housing (isolation) and group housing to behavior, physiology and neurochemistry in laboratory rats and mice are reviewed and theae~accounte related to comparable information derived from ezperiments employing "social stresses" e. g. subjecting the eaimal to defeat . The data is discussed in relation to the problem of whether individual housing constitutes a "stress" (in terms of adrenocortical and adrenal medullary rimctioning) in these species, as it appears to do in primates . In spite of the large number of papers which ascribe the behavioral and endocrine changes obtained in isolation versus grouping comparisons to the effects of "the isolation-induced stress syndrome", it ie concluded that, in terms of adrenal function, there is little evidence that isolation gar ae constitutes a stress in rate sad mice, slthough there is some evidence that adrenocortical reactivity is increased by housing eaimals in this manner . It should be noted that the wild progenitor of the laboratory strains of house mouse often appears to evidence territoriality . The view is adveaced that the isolated condition in male mice may result is changes characteristic of territorial dominance.. This may represent a mechanism for inducing social or territorial stability in this species . It appears that ezperiments inwlving physical isolation in laboratory indents are unlikely to provide good models for the effects of "social deprivation" in man. It is thoughtthat more studies employing measurement of hormone titers in biological samples obtained as a result of non-stressful pxrocedures, will lead to a clearer under ateading of the effects seen in isolation versus grouping studies . Organ weight studies often appear to be very misleading, particularlry in female rodents . It ie also hoped that other studies will concexa themselves with effects of isolation with respect to other social cues in rodents including odors and ultrasound .

~Thie work has been supported by grants from The Medical Research Council and the Royal Society . I would like to thanà Dr David Bentoa, Dr Catherine Wilson, Charles ihrana end Angela Poole for ~eiuable discussion sad advice . 187

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Differential H ouei~ end Aggressiveness Although rodent social behaviors necessarily depend on a variety of sensory modalities, mere physical isolation (the major form of isolation studied) has been shown to have many profound behavioral and physiological consequences in laboratory strains of house mice (Mus musculus L .) . For example, aggressiveness of the male mouse is increased by such isolation . Aggressiveness, as used here, refers to the type of motivation which tends to result in a higher incidence and intensity of overt intraspecific fighting in a neutral arena . It should be noted that such diverse behaviors as mouse killing in rats, intermale fighting in mice, shock elicited "aggression" in either mice or rats and attacking a pencil (all of which have been used as indicators of "aggressiveness"), are unlikely to have the same internal or situational determinants (1) . Physical isolation seems to have its most potent action oa intermale fighting behavior in mice (2,3,4,5,6,7,8,9,10,11, 12) . The effects of isolation on this behavior have been shown to be pro gressive (9,13,14) . A similar action on fighting behavior has been shown is other rodent species e .g . the gerbil (15), the hamster (16) and the rat (1,18 19,20) . Certain workers have indicated the importance of prior experience to agonistic encounters in rodents (13,21,22) and some reports have suggested that early social isolation cea reduce fighting behavior in mice (23,24) . Conversely, isolating male guinea pigs soon after weaning has been shown to induce more aggressiveness than isolation in adulthood (25) " However, it appeara,in general, that isolation does cause as augmentation of fighting behavior in the mouse and other species . Differential HOU91IIg and Adrenocortical Functioning Although the reported results and interpretations are often at variance, there is also much evidence that adrenocortical activities differ in isolated sad group-housed male end female mice (10,11,26,27,28,29,30,31,32,33,34,35,36, 37,38,39) " Similar results have been described in other species (40,41,42,43) " So far ae the mouse is concerned, there has been support for the view that physical isolation constitutes a "high stress" condition (e . g . 8) sad, also, that such housing constitutes a "low stress" condition (e .g . 39) " Measures of adrenocortical activity have been very different in diverse studies, ranging from relative adrenal weight, through leucocyte count and adrenal ascorbic acid levels, to the direct determination of circulating levels of adrenocortical hormones . Even direct measurement of hormone titer may be imperfect as such determinations rarely take account of circadian variations is adrenocortical activity, proportions of bound and free hormone, distribution in different physiological compartments etc . Biochemical methods have also been applied to blood sad tissue samples obtained by different methods which differ in their "streasfulness" e .g . blood sampling may be by decapitation or after anaesthesia and/or restraint . Variables, such as those reported above, could influence the results oP comparisons between isolated and group-housed mice . Although there is a comprehensive series of papers on "isolation stress" in male (8) and female mice (34 ,35,48 ,49,50 ), as well as a similar body of evidence in the rat (51,52,53), it would appear that there is a concenaus, as a result of the majority of studies in which hormones are measured, in favour of the view that isolation se is less "stressful" than group housing . However, isolated mice appear to show a greater response to additional stresses, for example, isolated mice are hypersensitive to different types of environmental stimuli including "handling" (54) noise (55,56) electroshock (57), restraint (58) or 15 wins attack by a "trained fighter" (59) " Hatch et al (52) have also shown that the

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increase in plasma corticosterone tbllowing ACTH administration is greater in isolated rather then grouped mice . Such differences mayy ezplein conflicting results obtained in isolation versus grouping comparisons . It has also been reported that isolates are more active in a novel situation (60,61) then group housed animals but it has been reported that such animals are less active in their home cages without disturbance (10) . Much of the data indicating ea "isolation streae" is based on relative adrenal weights, which are increased in the isolated female mouse compared to its group-housed counterpart, and it has been shown (36,38,62) that enhanced estrogen titans in the former category mavy account für the noted adrenocortical hypertrophy, in the absence of markedly increased steroidogeaesis . It should be noted that sex steroids may have profound influences on adrenocortical size sad ftimetioning in a variety of rodent species including the rat (63) and the hamster (64) . Androgens sad estrogeae have also been shown to influence adrenal (35) and neural (8) levels of catecholaminee . As it is likely that differential housing influences production sad release of se: steroids (see later), it seems important to quantify such actions before one can relate changes to "stress" er se . In many cassa the changea obtained may not b~ directly related to altered production sad release of ACTH as in a classical "stress" response . The recent account of Schwartz et el (50), which reviews much of the literature concerning isolation "stress" in rodents, and claims that plasma glucocorticoid levels are higher in isolated as compared to paired female CFFI mice, mqy, perhaps, be explained by the complex and eztended battery of behavioral sad physiological taste to which e11 animals were subjected before plasma sampling sad the fact that only pairs of mice in which no fighting was apparent were used is the comparisons . Differential Housing and Oonadal Faction An increase is goaadal activity, ea a coneequence of isolation, has also been indicated in many studies of male mice . Pbr ezemple, studies contrasting isolated sad group-housed males of this species have indicated that the former animals have heavier sex accessories they the latter (e .g . 11,26,2T,29,39) " Such effects also seem present in female mice (e . g . 3!8) which may account for the relative adrenal hypertrophy in isolated females . Similar effects have been noted with respect to gland size sad/or hormone production in the males of other species including the gerbil, (65), the hamster (16), and the rat (66) . Differential Houeia~and Adrenal Medullar~ l~nçtioning Isolation has also been reported to result is a decline in stored medullary catecholamiaes with respect to grouped mice (14,6T,68) . The basal activity of the adrenal medulla has been reported to be lower is isolated mice . By inhibiting catecholamine synthesis (using the tyrosine hydrozylase inhibitor, armethyltyroeine) it has been shown that the turnover rate for epinephrine is lower in isolated animals (69), although the concentration of norepinep~hrine (AE) is not different in isolated sad grouped male mice . A contrary indication has been provided by a study which reported, that there are reduced plasma free fatty acids in isolated mice, indicating that adrenal epinephrine activity is high (5,6) . Diffe rential Housing and AeurochemistrY Isolation has also been shown to ezert marked effects on the levels sad turnovers of many of the putative neurotraasmittere, their precursors and metabolites in the central nervous system, which m4Y be related to some of the behavioral and endocrine changes reported previously (i0) . Uiiortunately, the correlation of aeurochemical changes with housing density has also been

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complicated by differences in interpretation, methodology etc . but a few findings of general application may be indicated . Isolation in the male mouse has been reported to result, for example, in decreased levels of neural N-acetyl-L-aspartic acid (71), as well as changes in many other neurochemicals (14), including variable effects pa brain serotonin (5HT) levels (5,6,69,72 .73) . The moat consistent finding concerning putative neurotransmitters in the brain appears to be that in isolated mice the turnover rate for 5HT and NE is lower than that found in group-housed mice . This has been indicated by the presence of lower levels of 5-hydroxyindQleacetic acid (the chief degradation product of 5HT) and NE in the brain of mice isolated for at least 14 weeks (58) . In addition, in individually housed mice there is a relative decrease in the rate of accumulation of both amines after treatment with monoamine oxidase inhibitors (tranylcypromine or pargyline) (5,6,14, 68,73) when contrasted with group-housed mice . Not all reports agree with these general findings . Some suggest that there are no differences in the monoamine activities of isolated or grouped mice (3,35 .75) " Welch and Welch (74) even found that norepinephrine levels were higher in acutely isolated mice, a result which was probably due to the stressful effects of a sudden change of conditions . Changes in both 5HT and NE may be related to the earlier reported isolational effects on aggressiveness, as Welch and Welch (14) have concluded, that the tendency to fight is diminished by impairing the biosynthesis of neural catecholamines and serotonin . It is also been recently reported that neural changes in 5HT may exert profound influences on the functioning of the rodent pituitary-adrenocorticel aais (143),iadicating a relationship between these changes is putative neurotransmitter concentrations and the production of glucocorticoids . Differential Housing and Drug Sensitivity Grouped mice are more sensitive to effects of d-amphetamine than isolated mice (76) . Similar results have been reported concerning a variety of sympathomimetic drugs by Greenblatt and Osterberg (77) . Hoxever,this also appears to be an acute effect because when similar experiments on drug toxicity are carried out on animals which have been isolated or grouped for several weeks rather than just differentially housed immediately post treatment, amphetamine proves to be more toxic to the pre-isolated animals (3,67,78) whereas tranquilizers, such as reserpine and chlorpromazine, have been reported to have greater actions on pre-grouped animals (79,80) . Defeat and Biochemical Changes As well as isolation having been shown to influence behavior sad physiology in the mouse, a series of related studies have been reported in which the effects of the "stress" of defeat on such variables have been des cribed . Subjecting mice to defeat increases adrenocortical activity (13,81,82), as does grouping, an effect which may occur, given a history of defeat, even when physical attack is precluded (83) " Defeat also alters whole brain, hypothalamic, eaygdaloid and frontal cortical levels of the putative neurotransmitters, such as serotonin and nor-epinephrine (14,84) . Such ezposure has also been shown to alter 5-$Ydroxytryptophan decarbo~grlase activity in the frontal cortex and the amygdele (85)~as well as changing levels of RNA bases in various regions of the brain (86) . The most striking finding in this limited aeries of studies is that serotonin shows ea initial decline in the hypothalamic and aa~gdaloid areas of the brain followed by an increase after prolonged subjection to defeat, whereas levels of this putative neurotransmitter show a

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gradual decline in the fYontal cortical region . Changes in regional concentrations of nor-epinephrine are generally mirror images of changes is serotonin levels . Changes in hypothalamic levels of luteinizing hormone also occur following subjection to defeat (87,88) . Prolonged aggressive interaction has been ahrnm to cause suppression of the weights of sex accessory glands (89) and declines in the production of testicular androgens (90) is subordinate but not dominant mice . Subjecting male mice to defeat has also been reported to elevate adrenal medullary tyrosine hydrorylase and phenyl-ethaaolamine- N-methyl transferase activity (91) and plasma levels of epinephrine and nor-epinephrine (92) . Welch sad Welch (14) have also reported that the stress of defeat progressively elevates the levels of adrenal epinephrine sad norepinephrine . Defeat also appears to influence pituitary-thyroidal activity in the mouse (93) indicating that thin gland also mavy function differently in isolated and grouped members of this species . Correlat ion of Differ ential Houaiag sad Defeat Experiments In spite of , or perhaps because of, this plethora of biochemical and behavioral information, there still remains considerable doubt about the exact role of isolation in the mouse. The traditional view ie that isolation in rodents, ae in primates (96) constitutes a "stress" in terms of "social deprivation" and that it has its action oa behavior sad endocrinology by increasing the "irritability" of these animals . Such a position is frequently taken by Psychopharmacologists (e .g. 9) and, in addition to the studies on female mice indicating "isolation stress", there have been a number of reports which indicate that early isolation may lead to deficiencies in behavior in some rodents . For example, early isolation has been reported to cause partial impairment of sexual behavior is male guinea pigs (97,98) and male rata (99) . Such treatment has been shown to result in a lowered brain weight in rata (100). However, it should be noted that the effects of early isolation are not necessarily related to the effects of such housing in later life . It could also be that some of these detriments in sezuel behavior are related to increased aggressiveness . Although there are many reported exceptions sad the information is scanty in places, the changes in behavior sad biochemistry ascribed to chronically isolated,as opposed to the majority of long-term group-housed mice are generally opposite in direction to those changes seen following prolonged subjection to defeat . Most studies indicate that chronically isolated male mice are more aggressive, show lower adrenocortical sad adrenal medullary function, increased goaedal finctioa, depressed levels of neural nor-epinephrine and lowered turnover of neural serotoain with respect to group-housed mice . Conversely, chronically defeated mice show declines in aggressiveness, increases in adrenocortical sad adrenal medullary functioning, and declines in gonadal function . So far as the rntea~ of synthesis of brain catecholamines are concerned, strong support for isolation induced changes being opposite in direction to the effects of defeat are provided by the studies reported by Modigh (14h), who found that synthesis of catecholamines is lower in isolated than in grouped mice sad that intensive fighting rapidl3r accelerated the synthesis of these compounds . Taken ae a whole, the spectrum of changes seen in isolated male mice are largely the reverse of those seen following the obvious stress of defeat and it seems apparent that individual housing is more related to a low rather than a high stress condition . Many of the differences is mesa functioning of endocrine glands in mice

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housed in different numbers per cage may be a consequence of there being increasing numbers of defeated (subordinate) mice in these cages as group size increases (see below end 94,95) . The Pact that there are a number of reported differences in the literature concerning the responses of rats and mice to isolation and crowding, may be partially explained by the observation that mice are potentially individually territorial animals . In this species one dominant male may defend an area (101,102,103,104), when population densities are low sad non-linear hierarchies are often formed in small cages . The rat appears to be much more "social" generally displaying a type of group-territoriality (41, 105,106,107) and rarely leading a solitary existence . It could consequently be argued that species with different basic social organizations would be unlikely to show similar responses to differential housing . It has been claimed that laboratory strains of mice may be developed from two different types of natural precursor with very different social organizations (123) and consequently the strain employed may drastically alter the results one obtains when attempting to correllate hormonal changes and behavior in this species . Merry of the physiological and behavioral variables described in this paper have also been shown to be profoundly influenced by the strain of mouse employed (e . g . 44,45,46) . Differing early experience (reviewed by Brain, 47) is also likely to influence these responses, as well as differing cage sizes, variàble pretreatment housing conditions and differing durationa of isolation or grouping at different stages oP development . It also often appears difficult even to extrapolate between wild animals and animals derived from wild progenitors which have been reared in the laboratory for a few generations, as pronounced behavioral changes are induced by such procedures (108) . Meay of the behavioral end physiological variables consequent upon isolation and grouping interact in a complex way . It has been noted, also, that there are similarities with the change in direction oP these variables seen when dominant and subordinate individuals are contrasted (39,89,90,92,109) . Ng et e1 (110) have also shown that dominant sad "socially withdrawn" mice also differ with respect to neural and adrenal metabolism of catecholamines . It has consequently been argued that the changes seen on isolation may represent a device whereby a territorial animal many be produced . As early as 1944, Collies (111) related much of aggressive behavior in vertebrates to the concept of territoriality . Conversely, territorial individuals tend to be "behaviorally isolated" in that they do not tolerate mature male conspecifics on their territory and are consequently rarely subjected to the stress of defeat . As a corrollary it mavy be argued that housing male mice in small cages in groups, is a highly artificial situation in that defeated subordinates are unable to escape from the "territory" of the dominant . Stable social organizations in mice tend to be despotic dominances (95,112) in which one mouse is dominant and the rest are equally subordinate . This, and the Pact that it has been noted that paired aggressive mice often kill their partners by repeated attack (95) may both be consequences of the artificiality of housing mice in small groups in restricted cages . It is interesting to note that dominance or territoriality may be a prerequisite for good reproductive success in this species (89,90, 113) " It does appear that a number of mechanisms can be identified whereby the mouse may stabilise despotic dominance in small groups or maintain territoriality . Chronic elevations of adrenocorticotrophic hormone, such as those genera ted in Subordinate group-housed or in mice defeated on another's territory, seem likely to predispose the animal towards showing low levels of aggressiveness and demonstrating a potentiality for subordinate behavior (114,115,116, 117) . This eßfect may have similarities with the action of this compound on conditioned awidance reactions, in which the animal also learns to avoid a noxious stimulus (reviewed in 118) . The acute effects of elevated

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glucocorticoida and ACTS appear to be the reverse, in that such treatments iacreese fighting behavior . The fact, that levels of circulating eadrogene appear to be elevated is dominent or isolated mice, means that the aggressiveness of these animals may be higher than that of defeated mice or the majority of group-housed animals due to the stimulatory effects of such hormones on this behavior (109) . It hen been also reported that dominent or isolated mice produce a eubsteace in their urine which on "ageing" is aversive to introduced male mice (119,120,121, 122) . This also could be a mecheaism, making it likely that the integrity of a territory is maintained without expenditure of large amounts of energy . If mice cén indeed evidence territoriality, even small groups of mice mey show evidence of adrenocortical "stress" a factor which ~Y influence the response to actual sad ppteatiel infective agents etc. (124,125,126,127,128) . If isolation is not markedly "stressful" in adult male mice, it fbllowa that these animals do not provide a good model für the effects of comparable situations in men sad primates, and the influences of drugs on agonistic behavior may be open to other interpretations than those which have predominated is the literature in recent veers . Suggested Approach To "Isolation" Studien Attempts to resolve whether individual housing constitutes a "stress" in laboratory rodents, will be probably aided by studies in which direct assessment of circulating titers of glucocorticoida are made, both 'basal" (unstressed) sad "stressed" (following superimposition of steadard stresses e . g. ether, defeat etc .) values being obtained . It would also seem to be desirable to attempt to assess the dynamics of hormone production and degradation, rather than relying on absolute values obtained at a fized point in time . It seems evident that, in many cases, studies employing organ weights have led to misleading results . Obviously, studies involving deprivation or restriction of other sensory inputs, normally little influenced by individual housing, could be moat fruitful . Animals could be restricted with respect to visual, olfactory and ultrasonic cues from members of their own species . There is much indirect evidence implicating the importance of these sensory modalities with respect to behaviors . Für ezample, in many rodents e sense of smell appears to be of paramount importance to the appeareace of social behaviors . Olfactory bulb ablation appears to suppress intermale fighting in the rat (129) and in the mouse (130), although this may be related to a neural rather they a sensory deficit . There is also much evidence that behaviors are influenced by sex eteroiddependent olfactory cues (pheromones) in many rodent species . "Aggression modifying cues" for ezample, have been identified is the urine of laboratory mice (131,132) . Although it has been reported that blinded (133) and congenitally blind (134) mice are capable of showing organized fighting behavior, there have been a number of indications in the literature that fighting may be influenced by visual cues . White et al (135) recorded that fighting in mice appeared to be influenced by the size of the opponent but this is, of course, not necessarily related to vision . It has been reported that ultrasonic cues mayr influence social behaviors is a variety of rodent apeciea e .g . maternal retrieving behavior in mice (136),

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Prior mouse courtship 0137) and fighting (138) and mating (139) is rata . auditory ezposure has also been shown to influence susceptibility to audiogenic seizure in mice (140) and there have been indications that short end long-term isolated mice behave differently in a situation with a disturbing noise (61) . An extension of this approach, employing a detailed investigation of sensory modalities on responses to differential housing, could help to unravel the complex interrelationships between endocrine, neural and behavioral res ponses in rodents sad explain some of the differences obtained in apparently comparable experiments . Some tentative results concerning the effects of olfaction on differential housing have already been made . Archer (141) has shown that isolated male mice exposed to male odors from urine show an increase in relative adrenal weight end a depression of adrenal ascorbic acid compared with non-exposed animals . These factors show a similar pattern of chengee, in male mice housed in proximity without tactile contact when contrasted with isolated mice (142) . Nbre studies employing these considerations would seem desirable if one is really It would seem going to determine the effects of isolation on rodent species . teat a resolution of this problem is of considerable theoretical and practical importance . References 1.

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