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Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies

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 Choosing the best genetic strains of mice for developing a new knockout or transgenic mouse requires extensive knowledge of the endogenous traits of inbred strains. Background genes from the parental strains may interact with the mutated gene, in a
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  & p.1: Abstract Choosing the best genetic strains of mice fordeveloping a new knockout or transgenic mouse requiresextensive knowledge of the endogenous traits of inbredstrains. Background genes from the parental strains mayinteract with the mutated gene, in a manner which couldseverely compromise the interpretation of the mutantphenotype. The present overview summarizes the litera-ture on a wide variety of behavioral traits for the 129,C57BL/6, DBA/2, and many other inbred strains of mice. Strain distributions are described for open field ac-tivity, learning and memory tasks, aggression, sexual andparental behaviors, acoustic startle and prepulse inhibi-tion, and the behavioral actions of ethanol, nicotine, co-caine, opiates, antipsychotics, and anxiolytics. Using thereferenced information, molecular geneticists can chooseoptimal parental strains of mice, and perhaps developnew embryonic stem cell progenitors, for new knockoutsand transgenics to investigate gene function, and to serveas animal models in the development of novel therapeu-tics for human genetic diseases. & kwd: Key words Mouse · Inbred strains · Behavior · Genetics ·Locomotion · Open field activity · Learning · Memory ·Aggression · Parental behaviors · Acoustic startle · Prepulse inhibition · Alcohol · Nicotine · Cocaine · Opiates·Haloperidol · Diazepam · Breeding · Embryonic stemcell lines · Transgenic · Knockouts · Null mutation & bdy: Introduction Recent advances in molecular genetics have greatly ex-panded the search for genetic determinants of complexbehaviors (Lander and Schork 1994). Transgenic andknockout mice provide a powerful new tool for the eluci-dation of biological functions. Molecular geneticistshave been spectacularly successful in developing thetechnology for targeted germ line mutations. A greatmany new knockouts have been made for genes ex-pressed in the mammalian central nervous system. Be-havioral neuroscientists are now analyzing the behavioralphenotypes of these fascinating mice.The phenotype of a mutant mouse is not only the re-sult of the targeted gene, but it also reflects interactions J.N. Crawley ( ✉ ) · R. PaylorSection on Behavioral Neuropharmacology,Experimental Therapeutics Branch,National Institute of Mental Health, Building 10, Room 4D11,Bethesda, MD 20892-1375, USAJ.K. Belknap · J.C. CrabbePortland Alcohol Research Center,Department of Behavioral Neuroscience,Oregon Health Sciences University and VA Medical Center,Portland, OR 97201, USAA. Collins · J.M. WehnerInstitute for Behavioral Genetics, University of Colorado,Boulder, CO 80303, USAW. FrankelThe Jackson Laboratory, Bar Harbor, ME 04609, USAN. HendersonDepartment of Psychology, Oberlin College,Oberlin, OH 44074-1086, USAR.J. HitzemannDepartment of Psychiatry, State University of New York,Stony Brook, NY 11794–8101, USAS.C. MaxsonBiobehavioral Sciences Graduate Degree Program and Departmentof Psychology, University of Connecticut,Storrs, CT 06269-4154, USAL.L. MinerMolecular Neurobiology Laboratory,National Institute on Drug Abuse, Baltimore, MD 21224, USAA.J. SilvaCold Spring Harbor Laboratory,Cold Spring Harbor, NY 11724, USAA. Wynshaw-BorisLaboratory of Genetic Disease Research,National Institute, Human Genome Research Institute,Bethesda, MD 20892-4470, USA &  /fn-bloc k: Psychopharmacology (1997) 132:107–124© Springer-Verlag 1997 REVIEW & role s:& role s: Jacqueline N. Crawley · John K. BelknapAllan Collins · John C. Crabbe · Wayne FrankelNorman Henderson · Robert J. HitzemannStephen C. Maxson · Lucinda L. MinerAlcino J. Silva · Jeanne M. WehnerAnthony Wynshaw-Boris · Richard Paylor Behavioral phenotypes of inbred mouse strains:implications and recommendations for molecular studies & misc : Received: 8 November 1996 / Final version: 15 March 1997  with background genes, and other unknown mutations inthe genetic background. Proteins do not work alone butin large biochemical complexes and cascades, whereeach step is directly dependent on many other simulta-neous molecular events. Thus, genetic backgroundshould be as carefully controlled as any other experimen-tal variable. The simplest way to do this is to derive andmaintain mutations in an isogenic genetic background, astandard practice in other model organisms such asyeast,  Drosophila , and C. elegans . However, not all iso-genic backgrounds are appropriate for a given study,since the behavioral characteristics of certain isogenicstrains could overshadow the effects of the targeted mu-tation. Understanding of the behavioral phenotype of thestrain in which a mutation will be studied can avoidoverinterpretation of the mutant phenotype.Since natural strain differences exist for behavioraltraits, the genetic background of the inbred mouse strainsmust be carefully considered in the interpretation of be-havioral phenotypes of knockout mice (Crawley 1996;Gerlai 1996). Embryonic stem cells from substrains of 129 mice are commonly used in targeting experiments.C57BL/6 is the strain commonly used for breeding, andas the background strain for spontaneous mutations.However, mice from the various substrains of 129 andC57BL/6 are unusual on many standard behavioral para-digms. For example, 129/Sv mice are impaired on manylearning tasks (described below). The allelic contribu-tions from each parental strain can have profound inter-active effects with the mutated gene of interest. Theunique traits of 129 and C57BL/6 mice are examples of awidespread problem for the interpretation of behavioralphenotypes of null mutations, given the genetic diversitythat exists amongst the dozens of other commonly avail-able inbred mouse strains.A comprehensive database on behavioral phenotypesof inbred strains of mice would provide the informationneeded by molecular geneticists to make the optimalchoice of parental strains and breeding strategies for theexpected phenotype of each targeted mutation, and tointerpret the results appropriately. Towards this goal, aguide to some of the critical literature on strain distribu-tions for a variety of commonly used behavioral para-digms is assembled herein. The findings described be-low summarize the behavioral genetics literature and re-fer the reader to pertinent reviews. The 129 substrainsand the C57BL substrains are emphasized, includingsummaries assembled by the present authors at a recentmeeting of behavioral geneticists, behavioral neurosci-entists, and molecular geneticists (Workshop on Behav-ioral Phenotypes of Inbred Strains of Mice 1996). Thisfirst step towards conveying the rich diversity of inbredmouse strains available to the molecular genetics com-munity may also highlight the need for thorough behav-ioral testing of 129 substrains, and for further develop-ment and testing of new embryonic stem cell progenitorlines. Strain distributions of spontaneous behaviors Open field locomotionMotor activity underlies almost every mouse behavioralparadigm. Dysfunctions in physical movement can pro-duce false positives and false negatives on behaviors of interest for knockout and transgenic mice. Simple, auto-mated tests of spontaneous locomotion are routinely per-formed on homozygous mutants, heterozygotes, andwild-type littermates, before any further behavioral test-ing begins. Photocell beam measurements of open fieldlocomotion, in standard photocell-equipped automatedopen field equipment, can evaluate total amount of movement, rate of movement, and type of spontaneousactivity, over a 5- to 60-min test period.The open field test is also one of the oldest, most ex-tensively used, and simplest measures of mouse and ratemotional behavior. Over 60 years ago, Hall provided therationale for using decreased ambulation and increaseddefecation in a brightly lit open field as indices of heightened emotionality (Hall 1934, 1936). Subsequent-ly, over 20 additional behavioral measures have beenproposed as indices of emotionality/anxiety in the openfield, as well as suggested sequential analyses of multi-ple behaviors (Bindra and Thompson 1953; Archer 1973;Walsh and Cummins 1976; Hay 1985). Of these addi-tional measures, rearing behavior, which decreases in ananxiogenic environment, and thigmotaxis, the proportionof time the animal remains close to the walls of the openfield, are the additional behaviors most commonly as-sessed. The availability of open field apparatus whichcan record gross activity in three dimensions allowscomplete automation of activity scoring. High levels of ambulation and rearing are positively correlated witheach other and negatively correlated with defecation(Henderson 1967; Van Abeelen 1977; De Fries etal.1978). Increasing the stressful properties of the openfield, by increasing illumination level or backgroundnoise, generally results in decreased activity. Environ-mental conditions and prior treatments, such as handling,stress, surgery, and drug treatments, will affect perfor-mance on open field testing.No single behavior commonly measured in the openfield appears to reflect only anxiety or emotional reactiv-ity. The open field parameters all appear to reflect multi-ple underlying traits. Thus, genes linked to open fieldperformance may be involved in the regulation of gener-al locomotor activity, exploratory activity, olfaction andvision, as well as fear and anxiety. In low stress test envi-ronments, general activity probably dominates observedscore variance, whereas in stressful test environments,e.g., high levels of illumination which are stressful fornocturnal rodents, anxiety-based factors are likely to be alarge component of observed variance in activity. Newerbehavioral models which are more specific for anxietyare discussed below, in the section on anxiolytics.Reviews using several types of open field equipmentand test conditions have described strain distributions of  108  inbred mice on open field activity (Crabbe 1986; DeFriesetal. 1978; Henderson 1986; Marks etal. 1989a; Mathisetal. 1994). There are many different types of apparatusfor measuring open field behavior, and for measuringother types of spontaneous activity, and strain distribu-tions vary with type of test and equipment. In general,the C57 inbred strains of mice, including C57BL/6,C57BL/10, C57BR, and C57L, consistently show highlevels of open field locomotion and low levels of anxi-ety-related measures in the open field. Intermediatestrains include the DBA/2, CBA, AKR, and LP. Strainstypically exhibiting low locomotor activity and high lev-els of emotional reactivity include DBA/1, BALB/c andA/J. Other strains show greater inconsistencies acrossstudies. In general, albino strains are overrepresented atthe high anxiety end of the distribution. In part, this maybe a function of the added stress of high illumination toalbino mice. Recent quantitative trait loci analyses(QTL) have identified six chromosomal loci which arestrongly linked to open field activity (Flint etal. 1995).Three significant QTLs accounted for most of the genet-ic variance in open field activity and defecation, and didnot show linkage to a separate measure of general activi-ty, closed arm entries in the elevated plus maze, suggest-ing that sites on chromosomes 1, 12, and 15 may repres-ent facets of the genetic basis of emotionality.The best choice of an inbred background on which toexplore the impact of a null mutation on locomotor activ-ity and emotional reactivity can be taken from these ref-erenced strain distributions. Highly active strains are thebest choice when the mutation is predicted to decreaselocomotion or increase anxiety; low activity strains arethe best choice when the mutation is predicted to in-crease activity (e.g. hyperlocomotion predicted by thedopamine transporter knockout; Giros etal. 1996).Learning and memoryMouse inbred strain differences for performance onlearning and memory tasks are well documented. A widevariety of paradigms have been examined which measurecomplex learning, simple associative learning and avoid-ance learning. Further, the important distinction betweentrue learning differences versus sensory impairments thatlead to poor performance is evaluated for inbred mousestrains. Visual acuity is important for spatial learningtasks. Some mouse strains are albino and demonstratepoor vision under bright lights, while others have the ret-inal degeneration gene which leads to blindness in adultmice. Auditory function is important for paradigms in-volving conditioning to the presentation of a tone; somestrains show deafness as a function of age. Responses toelectrical shock may also vary across strains; jumpthresholds should be determined in those paradigmswhich employ such aversive stimuli. Analogously, therole of strain differences in motivation, whether appeti-tive or aversive, needs to be rigorously dissociated fromtrue learning and memory differences. Complex learning tasks These are behavioral tasks which require an animal touse multiple pieces of information simultaneously tolearn. The Morris water task is frequently used to exam-ine spatial learning in rats (Morris 1981), and has beenmodified slightly for use in mice (Owen etal. 1997; Up-church and Wehner 1989). Animals are trained to locatea hidden platform in a circular pool filled with opaquewater, using distal room cues. Latencies to locate theplatform are recorded on each trial, but in mice do notprovide robust measures of spatial learning. Measure-ments of spatial learning require analysis of spatial se-lectivity by examining performance on a probe trial, inwhich the platform is removed and the search pattern of the mouse is evaluated. An animal that has learned theposition of the platform will demonstrate greater plat-form crosses over the trained site versus other possiblesites, and spend more time in the area of the pool whichpreviously contained the platform versus other areas of the pool. Visual acuity is examined by measuring the la-tencies to locate a visibly marked platform that is movedto various positions throughout training. The spatial se-lectivity of inbred mouse strains and F1 hybrids on Mor-ris water task performance is shown in Table 1.In another form of complex learning, contextual fear-conditioning, animals are trained to associate a shock paired with a tone, in a particular contextual environ-ment. The methods of Fanselow (1990) and LeDoux(Phillips and LeDoux 1992) were adapted to mice byPaylor etal. (1994). Bouts of behavioral immobility,termed “freezing”, are used as a measure of perfor-mance. Adequate performance is defined as learning todiscriminate a pairing of shock and tone in a particularcontext versus an altered context. The performance of in-bred strains and F1 hybrids on contextual fear-condition-ing is shown in Table 1.Spatial and/or working memory has been examined ineight-way radial arm maze tasks, in which animals mustremember the arm of a maze in which they previouslyobtained a food reward. Rank orders of performance asdefined by the number of correct choices using appetitiverewards have been determined for several inbred mousestrains (Ammassari-Teule etal. 1985, 1993; Reinstein etal. 1983). “Good” and “poor” strains are defined in Table1. Conditional spatial alternation in a water filled T-mazeis also sensitive for detection of strain differences, asshown in Table 1. In contrast, a spatial open-field test(Roullet and Lassalle 1990) did not allow detection of significant differences across genotypes, when nine in-bred strains and three F1 hybrids were examined, be-cause variation within individual strains was great.  Avoidance tasks In avoidance paradigms, animals do not enter or willquickly leave a location where they previously received afootshock. One-way avoidance tasks can require either 109  an active (moving) or passive (resting) response, and per-formance must evaluate the possible role of general ac-tivity (see previous section). Two-way and avoidance-avoidance paradigms introduce a potentially confound-ing contribution of anxiety-related behaviors (see be-low). Literature published before 1972 was previouslyreviewed and evaluated by Wahlsten (1972). Some of themore recent literature is summarized in Table 2.The best choice of an inbred background on which toexplore the impact of a null mutation on learning appearsto be C57BL/6. C57BL/6 are moderate learners, suchthat either an impairment or an improvement could theo-retically be observed. However, breeding characteristicsand the possibility of lethality of a particular null muta-tion on an inbred background must also be examined foreach specific mutation. The data summarized here sup-port the view that many inbred mouse strains performpoorly on complex learning tasks and several also per-form poorly on avoidance tasks.Aggressive behaviorsHere, aggressive behavior is defined for mice as thatwith the potential for attack bites on another animal, andit is suggested that predation, infanticide, defense, andoffense are types of aggressive behaviors in mice (Brain1979; Maxson 1992a). This section concerns strain dif-ferences in offense; the literature on predation, infanti-cide, and defense is summarized by Maxson (1992a).  Male offense The first research on strain differences for male mouseoffensive behavior was reported more than 50 years ago(Ginsburg and Allee 1942; Scott 1942). Since then, manyarticles provide extensive information on strain distribu-tions and male offense (Scott 1966; Lagerspetz and La-gerspetz 1974; Simon 1979; Maxson 1981; Hewitt andBroadhurst 1983; Maxson etal. 1983; Michard and Car-lier 1985; Jones and Brain 1987; Guillot etal. 1994; Ku-likov and Popova 1996). For example, in either homoge-nous set/novel cage tests or standard opponent/novelcage tests, it has been reported than DBA/1 and DBA/2are more aggressive (offense) than C57BL/6 andC57BL/10 males (Selmanoff etal. 1976, 1977; Guillottetal. 1994). However, these strain differences and othersin offense depend on life history, test situation, and op-ponent type (Maxson 1992b). For example, they are re- 110 Table1 Performance of inbred mouse strains and F1 hybrids forcomplex learning &  /t bl.c :& t bl.b: GoodPoorVisually impaired  A. Morris water task  a C57BL/6J129Sv/JA/JC57BL/10JDBA/2SJL/J129/SvevTacfBrLP/JC3H/IbgBALB129F1BALB/cByJFVB/NJB6D2F1Bub/BNJB10C3F1129B6F1  B. Contextual fear conditioning b C57BL/6JFVB/NJC57BL/10JDBA/2129/SvJBub/BNJ129/SvevTacfBrC3H/IbgSJL/JBALB/cByJLP/JBALB129F1FVB129F1B6D2F1B6SJLF1B10C3F1129B6F1 C. Eight-way radial arm maze c C57BL/6NZBDBA/2CBACB6F1C3H/HeB6D2F1BALB/C  D. Conditional spatial alternation d  C57BL/6IbgDBA/2Ibg a Based on data and conclusions from Owen etal. (1997) and Up-church and Wehner (1989), in which good-learning strains showedsignificantly greater crosses at the trained platform site comparedto three other sites during a probe trial after 36 training trials.Poor-learning strains did not show greater crosses over the trainedsite compared to other sites. Visually impaired animals could notreliably locate a visibly marked platform in less than 30s aftereight trials b Based on data and conclusions from Owen etal. (1997). Goodcontextual learners were those strains that exhibited greater freez-ing in the training context than in the altered context as measuredby bouts of freezing during a defined period of time. Poor learningstrains did not show greater freezing in the context versus the al-tered context c Based on data and conclusions from Ammassari-Teule etal.(1993), in which strains were compared on the number of correct,i.e., unrepeated arm, choices in a fully baited eight-way radial armmaze and data and conclusions from Roullet and Lassalle (1995),in which strains were compared for the number of errors in aneight-way baited radial arm maze over five training sessions. Noperformance criterion was established but good learning strainswere significantly better than poor learning strains in both studies d Based on data and conclusions from Paylor etal. (1993), inwhich nine out of ten correct trials were used as the criterion of learning. The poor learning strain required at least twice the num-ber of training trials &  /t bl.b: Table2 Performance of inbred mouse strains and F1 hybrids onavoidance learning &  /t bl.c :& t bl.b: A. One-way avoidance a 5 days of training DBA/2 >C57BL/6 >C3H/HeJ13 days of training DBA/2=C57BL/6=C3H/HeJB. Two-way avoidance b DBA/2 >BALB/CJ >NMRI >C57BL/6J >SM/J >C3HC. Avoidance – avoidance c CBA/CaJ >BALB/CJ=DBA/1J >SEC/1REJ >RF/J >LP/J >A/J>C57BL/10J a Weinberger etal. (1992) b Buselmaier etal. (1981) and Lipp etal. (1989) c Henderson (1989) &  /t bl.b:  versed (Ogawa etal. 1996) or eliminated (Jones andBrain 1987) with anosmic standard opponents. Regard-less, it would seem to be best to select life history, testsituation, and opponent type which give an intermediatelevel of offense in a strain to be used in knockout re-search. This would permit detection of increases or de-creases in offense as a consequence of the knockout mu-tation. Because the control animals had a low level of of-fense, the studies with 5-HT 1B (Saudou etal. 1994),MAO-A (Cases etal. 1995), and Nosl (Nelson etal.1995) knockouts could only have detected an incremen-tal effect on the mutant. Female offense Female aggression, which may be of the offense type, isdependent on reproductive state. Offense in female miceis assessed in three reproductive conditions: neither preg-nant or lactating, pregnant but not lactating, or lactatingbut not pregnant. Strain differences in offense for one ormore of these reproductive states have been described forDBA/2, C57BL/6, C57BL/10, AKR, BALB/c, C3H,CBA, and NZW inbred strains (Ogawa and Makino1981, 1984; Broida and Svare 1982; Jones and Brain1987; Svare 1988). Again, strain differences for femaleoffense depend on life history, test situation, and type of opponent. For example, non-pregnant and non-lactatingDBA/2 and C57BL/6 females do not differ in offenseagainst an intruder male (Ogawa and Makino 1981,1984), whereas C57BL/6 females are more aggressive(offense) than DBA/2 females against a lactating intruderfemale (Haug etal. 1992). Similarly, there is a differencein offense between DBA/2 and C57BL/6 females for ananosmic R-S male intruder (Svare 1988) but not for ananosmic TO male intruder (Jones and Brain 1987). Re-gardless, it would be best, as with male offense, to selectlife history, test situation, and opponent type which givean intermediate level of offense in females for the strainto be used for mutant testing.No general recommendation can be given for a singlebest strain for transgenic and knockout studies of aggres-sive behaviors, since aggressive behavior depends onmany developmental and experiential factors. The strain,test paradigm, and experience should be selected suchthat increases and decreases in an aggressive behaviorcan be detected for the null mutation.Reproductive behaviors Sexual behaviors The focus for strain differences in sexual behaviors hasprimarily been on male copulatory mounts, intromis-sions, and ejaculations. Latency, frequency, duration, andother measures have been used as indices for these be-haviors (McGill 1962, 1970). The stimulus female isusually artificially brought into estrus with hormonetreatment. Male copulatory behaviors have been de-scribed for DBA/2, C57BL, C57BL/6, BALB/c, AKR,A/J, C3H, and CBA inbred strains of mice (McGill 1962;McGill and Ransom 1968; Vale and Ray 1972; Mosigand Dewsbury 1976; Batty 1978; Ogawa etal. 1996,1997). High copulatory behaviors were reported forC57BL and C57BL/6, lower copulatory behaviors werereported for DBA/2 and AKR, and the strains BALB/cand A/J showed the lowest copulatory behaviors. Thesame strain differences were obtained for the most partwith estrus females of different strains or F1s. Parental behaviors Parental behaviors include pup retrieval, nesting withpup, nursing of pup, and licking pup. The most extensivestrain distributions for maternal pup care are by Carlieretal. (1982) and Cohen-Salmon etal. (1982, 1985).When mothers and infants are of the same strain, thenCBA/H, C4H/Ico, C57BL/6, and CBA/J are better pupretrievers than BALB/c, NZB, DBA/2, XLII, A/J andAKR. These data may be useful in selecting strains forknockout research on maternal behaviors where the wildtype or knockout mother is paired with pups of the wildtype. For example, DBA/2 mothers have intermediatevalues for pup retrieving and pup nesting, and they maytherefore be a good background strain to detect incre-mental or decremental effects of knockout mutants. Itmust be recognized that such differences may be due topup as well as to mother’s strain (Cohen-Salmon etal.1985).Acoustic startle and prepulse inhibitionAcoustic startle is the reflex response to a sudden, loudnoise. Prepulse inhibition (PPI) is the suppression of thenormal response to a startling stimulus when that stimu-lus is immediately preceded by a weak prestimulus orprepulse (Graham 1975). In the standard paradigm, thestartle response is measured by presenting a loud sound(acoustic startle stimulus) or puff of air (tactile startlestimulus) to a subject and measuring the reflexive startleresponse. A low level of acoustic stimulus that itself does not evoke a startle response is then presented, lessthan 100ms before the startle stimulus, and the reflexiveresponse to the startle stimulus is measured, using eyeblink in humans and muscle twitch in rodents. The re-duction in the startle response when the same startlestimulus is immediately preceded by the weak prepulseis used as the measure of prepulse inhibition.A number of studies have shown that schizophrenicpatients have an impaired prepulse inhibition response(Braff etal. 1978; Franks etal. 1983; Freedman etal.1983). The impairment observed in schizophrenics isthought to reflect an underlying problem with inhibitorymechanisms similar to those used for sensorimotor gat-ing (Braff etal. 1978; Freedman etal. 1987). Rats and 111
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