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The social deficits of the oxytocin knockout mouse
Neuropeptides (2002) 36(2±3), 221±229 Special Issue on Transgenics and Knockouts with Mutations in Genes for Neuropeptides and their Receptors. ß 2002 Elsevier Science Ltd. All rights reserved. doi: 10.1054/npep.2002.0909, available online at http://www.idealibrary.com on
The social deficits of the oxytocin knockout mouse J. T. Winslow, T. R. Insel Center for Behavioral Neuroscience, Yerkes Regional Primate Center, Psychiatry and Behavioral Sciences, Emory University, Atlanta, GA
Summary Numerous studies have implicated oxytocin (OT) and oxytocin receptors in the central mediation of social cognition and social behavior. Much of our understanding of OT's central effects depends on pharmacological studies with OT agonists and antagonists. Recently, our knowledge of OT's effects has been extended by the development of oxytocin knockout (OTKO) mice. Mice with a null mutation of the OT gene manifest several interesting cognitive and behavioral changes, only some of which were predicted by pharmacological studies. Contrary to studies in rats, mice do not appear to require OT for normal sexual or maternal behavior, though OT is necessary for the milk ejection reflex during lactation. OTKO pups thrive if raised by a lactating female, but OTKO pups emit fewer ultrasonic vocalizations with maternal separation and OTKO adults are more aggressive than WT mice. Remarkably, OTKO mice fail to recognize familiar conspecifics after repeated social encounters, though olfactory and non-social memory functions appear to be intact. Central OT administration into the amygdala restores social recognition. The development of transgenic mice with specific deficits in social memory represents a promising approach to examine the cellular and neural systems of social cognition. These studies may provide valuable new perspectives on diseases characterized by social deficits, such as autism or reactive attachment disorder. ß 2002 Elsevier Science Ltd. All rights reserved.
Oxytocin (OT) belongs to a family of nine amino acid peptides that have been identi®ed in all classes of vertebrates and many invertebrate species (Hoyle, 1999). OT and the closely related nonapeptide arginine-vasopressin (AVP) are the only members of this family identi®ed in mammals. Both OT and AVP are classi®ed as neurohypophyseal peptides, synthesized in the hypothalamus and released into the bloodstream via axon terminals in the posterior pituitary or neurohypophysis. OT's classic physiological roles include stimulating smooth muscle contractions in the uterus during labor and in mammary myoepithelium during nursing (Zingg, 2001). OT also plays a role in a variety of other reproductive-related functions, such as modulation of estrous cycle length, follicle luteinization in the ovary, and ovarian steroidogenesis (Fuchs, 1988). In addition to these endocrine effects, OT appears to have a role as a neurotransmitter Received 2 April 2002 Accepted 20 April 2002 Correspondence to: James T. Winslow, 954 Gatewood Road, N.E. Yerkes Primate Center, Atlanta, GA 30322. Tel.: 404-727-7728; Fax: 404-727-7845; E-mail: [email protected]
or neuromodulator in the brain, in¯uencing socio-sexual behaviors (Insel et al., 1998; Insel and Young, 2000; Kendrick, 2000; Young et al., 2001) (Table 1). Our knowledge of OT's central effects is based largely on pharmacological studies with OT agonists and antagonists. As these agonists and antagonists are peptides that do not cross the blood-brain barrier, they must be given centrally to assess effects in the CNS. While pharmacological studies have been instructive, one never knows if agonists mimic physiological effects or antagonists fully and selectively inhibit endogenous peptide. Here we review the results of studies in oxytocin knockout (OTKO) mice. Mice with a null mutation of the OT gene manifest several interesting cognitive and behavioral changes, only some of which were predicted by pharmacological studies. ANATOMICAL SYNTHESIS AND DISTRIBUTION OF PEPTIDE The OT gene is expressed primarily in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus (Gainer, 1998). In the PVN two populations of OT-staining cells have been identi®ed: the magnocellular 221
222 Winslow and Insel
Table 1 Oxytocin and Social Behavior
Sexual Behavior Female Male Autogrooming Yawning Nociception Anxiety Stress Feeding Salt-Appetite Memory and Learning
Mouse OT ICV
OTKO
Rat OT ICV
? ? ?
nc nc nc nc ?
?
? ?
, Increase; , Decrease; ?, Unknown; nc, No Change; refers generally to modulated behavior associated with OT administration or gene mutation. (Amico et al., 2001; Ferguson et al., 2000; Gimpl and Fahrenholz, 2001; Johnson and Thunhorst, 1997; Kendrick, 2000; Puryear et al., 2001; Winslow et al., 2000).
neurons with projections that terminate in the neurohypophysis and the parvocellular neurons with projections to various areas of the CNS (Gainer and Wray, 1994). In the rat, OT terminals and ®bers have been localized in the thalamic nuclei, dorsal and ventral hippocampus, subiculum, entorhinal cortex, medial and septal nuclei, amygdala, olfactory bulbs, mesencephalic gray nucleus, substantia nigra, locus coeruleus, raphe nucleus, the nucleus of the solitary tract, and dorsal motor nucleus of the vagus nerve (Argiolas and Gessa, 1991). In all species, OT and vasopressin genes are located on the same chromosomal locus but are transcribed in opposite directions (Hoyle, 1999). The OT gene is located on mouse chromosome 2 (Hara et al., 1990) and human chromosome 20 (Summar et al., 1990). The genes for AVP and OXT are separated by 12 kb in humans and 3 kb in the mouse (Hoyle 1999). The human and mouse genes consist of three exons (Richter, 1983; Ivell and Richter, 1984; Sausville et al., 1985): the ®rst exon encodes a signal peptide, the nonapeptide, a tripeptide processing peptide (GKR), and the ®rst nine amino acids of neurophysin, a carrier polypeptide important for transport. The second exon encodes the central part of neurophysin (residues 10-76) while the third encodes the COOH-terminal region of neurophysin (residues 77-93/95) (Sausville et al., 1985). Further modi®cation of the OT prepropeptide occurs as it is transported down the axon to terminals located in the posterior pituitary. OT and its carrier neurophysin are then stored in the axon terminals until neural activity stimulates release.
RECEPTOR AND DISTRIBUTION IN BRAIN OT's effects appear to be mediated by a rhodopsin-type G-protein coupled receptor with seven transmembrane Neuropeptides (2002) 36(2^3), 221^229
domains linked to phosphoinositol for signal transduction (Kimura et al., 1992). This OT receptor is only relatively selective for OT compared to AVP, with a 10-fold higher af®nity for OT. (Chini et al., 1996; Postina et al., 1996). OT receptors are found not only in uterus and mammary tissue, but also in kidney, thymus, and several regions of the brain. The brain receptor appears identical to the receptor found in peripheral organs (Gimpl and Fahrenholz, 2001). Within the brain, OT receptors are abundant in limbic and autonomic areas; however, the speci®c distribution in substructures is quite variable across species (Insel and Young, 2000). For instance, in the rat brain, OT receptors are abundant in the ventromedial nucleus of the hypothalamus and sparse in the hippocampus, whereas in the mouse brain there is relatively little binding in the hypothalamus and abundant binding in the hippocampus (Gimpl and Fahrenholz, 2001). Indeed, nearly every species tested to date has a unique pattern of expression of OT receptors, suggesting a role in species-typical behaviors (Insel and Young, 2000). In addition to the marked species variation in distribution, there is also variation in regulation of these receptors. In several regions of the rat brain, OTRs are induced by gonadal steroids (Breton and Zingg, 1997), whereas in select regions of the mouse brain gonadal steroids appear to inhibit expression (Insel et al., 1993). THE OXYTOCIN KNOCKOUT MOUSE Three groups have created OT knockout (OTKO) mice: Nishimori et al. (1996) deleted the ®rst exon of the OT gene by homologous combination in embryonic stem cells (Figure 1), Young et al. (1996) deleted a segment of the second exon to eliminate neurophysin and Gross et al. (Gross et al., 1998) replaced all three exons, so there are no preproOT/neurophysin coding sequences present (L. J. Muglia, personal communication). All of these mutations eliminated OT, although there were subtle differences in collateral effects. The exon 1 deletion did not affect arginine vasopressin mRNA, but the exon 2 deletion resulted in a slight decrease in AVP and dynorphin mRNA. Neither the distribution nor the concentration of OT receptors was measurably affected in the OTKO compared to wild-type mice. Thus, presynaptic OT is not necessary for the normal neuroanatomical distribution of OT receptors during development, and consequently does not account for developmental or species differences in receptor distribution. With each of these mutations, OT-KO mice show de®cits in lactation, due to an absent milk ejection re¯ex. Parturition appears normal, as measured by duration of pregnancy and length of parturition. Although OT increases during parturition, OT is not necessary for delivery. ß 2002 Elsevier Science Ltd. All rights reserved.
The social deficits of the oxytocin knockout mouse
Development of an OT-KO Mouse Exon 1
223
WT Exon 2
Exon 3
5'
3'
WT gene
Recombination
Non-OT
5'
Non-OT
Exon 2
Exon 3
Exon 2
Exon 3
5'
3'
Deletion Vector
Recombinant OT-KO 3' Allele
HET
Targeting vector in 129sSvEv ES cells Implant into C57BL/6J
Nishimori et al., PNAS, 1996
HOM
Fig. 1 Left Panel. The first-exon null mutation for OT was produced by homologous recombination in embryonic stem cells. Right Panel. OT immunoreactivity in the paraventricular and the supraoptic nuclei was reduced in heterozygous (HET) and absent in homozygous (HOM) mice compared to wild type (WT) siblings (Adapted from (Nishimori et al. 1996)).
BEHAVIORAL PHENOTYPE Our examination of the behavioral phenotype of the OTKO mouse created by the ®rst exon deletion strategy (Nishimori et al., 1996) was guided by two decades of pharmacological studies of central OT and OT receptor antagonist administration. These studies, as well as a convergence of neurochemical data suggest a key role for OT in the development and maintenance of social cognition and social behavior. For this reason, we examined the behavior of OTKO mice in maternal behavior, infant ultrasonic vocalizations, intermale aggression, and social recognition.
Maternal behavior Much of what is known about central OT has been derived from studies of OT's role in the onset of maternal behavior by female rats. The laboratory rat has been an ideal subject for studies of maternal care (Numan, 1988). Unlike many ß 2002 Elsevier Science Ltd. All rights reserved.
mammals, female rats show little interest in infants of their own species until shortly before parturition. About a day prior to delivery they shift from avoiding pups from other females' litters to showing intense interest with vigorous nest building, retrieval, grooming, and defense of young. These behaviors persist through lactation, and then abate with weaning. Rats, therefore, provide an opportunity to study two quite distinct aspects of maternal care: its onset and maintenance. Several investigators have reported that OT given centrally to estrogen-primed, nulliparous female rats facilitates the onset of maternal behavior. Pedersen and Prange noted that the effect was speci®c to OT (the only other neuropeptide with signi®cant albeit weaker effects was vasopressin) and resulted in rapid onset (within 30 minutes) of full maternal behavior (Pedersen and Prange, 1979; Pedersen et al., 1982; Fahrbach et al., 1984). Blockade of OT neurotransmission with central injection of an antagonist or antiserum or by lesions of OT-producing cells in the hypothalamus, results in a signi®cant inhibition of maternal behavior (Fahrbach Neuropeptides (2002) 36(2^3), 221^229
224 Winslow and Insel
et al. 1985; Pedersen et al., 1985; van Leengoed et al., 1987; Insel and Harbaugh 1989). These various interventions appear to inhibit the onset but not the maintenance of maternal behavior ± an OT antagonist or a lesion has no effect on maternal care once a female begins to show interest in pups (Fahrbach et al., 1985; Insel and Harbaugh, 1989). These results support the notion that OT is necessary for the transition to maternal attachment to pups and that a central increase in OT given under the appropriate gonadal steroid conditions might facilitate the onset of maternal care. Given the strong evidence that OT is critical for the onset of maternal behavior in rats, one might predict that OT-KO mice would not be maternal. Surprisingly we and others failed to detect any differences in pup retrieval, nest building or time spent grooming pups between OTKO and wild-type mice (Nishimori et al., 1996; Young et al., 1996). Of course, pups from OTKO mothers fail to thrive unless cross-fostered, due to the OTKO mother's inability to eject milk. However, once treated with systemic OT to re-instate milk ejection, full maternal behavior ensues. Indeed, even virgin OTKO females show full maternal behavior. Because OT receptors are unaffected by the mutation, compensatory mechanisms involving OT receptors may account for the expression of maternal behavior. To test this possibility, adult virgin OTKO mice were infused centrally with an OT antagonist for 5 days using Alzet pumps (Insel et al., 2001). At the end of 5 days, these animals still showed a full repertoire of maternal behaviors. A similar experiment in pregnant females has not been done, so the possible contribution of OT receptors to normal gestation or the development of maternal behavior has not been examined. How can OTKO mice show full maternal behavior when OT appears necessary for maternal behavior in rats? Unlike rats, in which maternal behavior has an abrupt onset at parturition, in inbred strains of mice including the C57BL/ 6J strain of mouse used as background for the OTKO mutation, maternal behavior emerges spontaneously in virgin mice (Numan, 1994). That is, mice do not require pregnancy or parturition to show full maternal behavior. For this reason, it seems likely that OT may not be essential for maternal behavior in mice (Russell and Leng, 1998).
generally are powerful stimuli for maternal retrieval. We have previously reported that exogenous administration of OT reduces the rate of ultrasonic vocalizations expressed by rat pups during separations from mother and peers (Insel and Winslow, 1991). However, an OT antagonist did not increase calling, so it is not clear that endogenous OT is important for this behavior. Vasopressin and OT are both found early in fetal development, although in the rat OT does not show a fully processed transcript until the postnatal period (Whitnall et al., 1985). Receptors for both peptides are found in the developing brain (Shapiro and Insel, 1989; Tribollet et al., 1989). Indeed, there is a transient, but marked ``over-production'' (relative to the adult) of both OT and vasopressin receptors in limbic brain areas during infancy (in both rodents and primates). Although these binding sites are particularly enriched in the cingulate cortex (a region implicated in separation distress), we do not know if these putative receptors are involved in infant behavior nor do we know why they disappear by the time of weaning. OTKO pups emit fewer ultrasonic calls than wild-type pups (Figure 2 Left Panel (Winslow et al., 2000)). These differences do not appear to be associated with differences in gross developmental features since OTKO pups do not differ in body weight, thermoregulation or motor activity. It is unlikely that the reduced calling rate in OTKO mice is a consequence of ¯anking genes carried over with the targeting vector from the 129SvEv strain as mice of this strain actually give more calls than C57BL/6J mice (Winslow et al., 2000). Reduced vocal behavior also did not appear to vary signi®cantly with prenatal environments, since OTKO pups derived from both homozygote and heterozygote mothers had fewer vocalizations than wild-type mice. Taken together, these data suggest that OT may modulate calling in mouse pups. Note however, that the direction of effect is in the opposite direction from what we would have predicted based on pharmacological studies with rat pups in which OT decreased calling and an OT antagonist did not alter calling. One intriguing explanation for decreased calling by OTKO pups is that they may be less sensitive to maternal separation than WT pups, however this possibility has not been tested directly. Aggressive behavior
Infant ultrasonic vocalizations Rodents have a surprising repertoire of audiovocal communication, mostly in the ultrasonic range, Mouse pups emit calls in the 60±70 kHz range in response to social separation (Noirot, 1966). Several studies have suggested that these separation calls are similar to distress calls in other mammals. For instance, infants of highly social species give more calls than infants of less social species (Shapiro and Insel, 1990) and these pup ultrasonic calls Neuropeptides (2002) 36(2^3), 221^229
Previous studies of the central administration of OT indicate that aggressive behaviors may be in¯uenced by OT, although the effects appear to vary depending upon the species studied. OT appears to decrease aggression in male prairie voles (Witt et al., 1990; Winslow et al., 1993b), while facilitating maternal aggression (Ferris et al., 1992) and dominance-related behaviors in squirrel monkeys (Insel and Winslow, 1991). A recent study of OT depletion produced by a different null mutation of the OTß 2002 Elsevier Science Ltd. All rights reserved.
The social deficits of the oxytocin knockout mouse
Resident-intruder aggression
Infant mouse calls
40 Attacks per 5 min session
Cals per 2 min session
140 120 100 80 60
*
40 20 0
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OTKO 30
*
WT
* 20
*
10 0
KO
WT
1
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3
Repeated confrontations Fig. 2 Left Panel portrays the number of ultrasonic vocalizations expressed by WT (closed symbol) and OTKO (open symbol) mouse pups during brief separations from mother and littermates. Right Panel represents the frequency of attack biting by adult, resident male WT (closed symbol) and OTKO (open symbol) mice during repeated 5 min confrontations with a male outbred intruder. Each confrontation was separated by 48 hrs. (Adapted from (Winslow et al. 2000)).
neurophysin gene in mice disrupted intermale aggression; however, these mice showed several unintended neuroendocrine changes (Young et al., 1996) and the behavioral effects were subtle (DeVries et al., 1997). In particular, OT de®cient mice showed decreased attack bout duration in a resident±intruder paradigm and decreased bout duration and total attack time in a neutral arena test. The frequency of aggressive episodes in each of these tests was relatively low compared to aggression expressed in outbred strains (Miczek and Winslow, 1987). This mutation also reduced vasopressin which appears to be necessary for aggression in many species (Ferris et al., 1989; Koolhaas et al., 1990; Winslow et al., 1993a) Our studies of aggressive behavior in OTKO mice revealed increased aggression (Figure 2, Right Panel) and a complex interaction between pup genotype and maternal genotype. In general, OTKO mice were signi®cantly more aggressive than WT mice tested both in isolation-induced and resident-intruder paradigms, though OTKO animals derived from heterozygote mothers appeared to be somewhat less aggressive compared to those derived from homozygous mothers. We interpreted these results to suggest that the genotype of the test animal did not fully account for differences in aggressive behavior with additional prenatal or postnatal rearing differences also in¯uential (Winslow et al., 2000). It is possible that some behavioral phenotypes may be in¯uenced by contrasting postnatal environments produced by differing mothering styles or litter compositions associated with the production and rearing by homozygote or heterozygote mothers. However, it is appears unlikely that postnatal environments account for phenotypic differences detected for offspring of homozygote mothers since both OTKO and WT pups were crossfostered to WT females. The moderation of differences ß 2002 Elsevier Science Ltd. All rights reserved.
in the isolation induced aggression of OTKO mice derived from heterozygous litters suggests that some aspect of the phenotype of these mice may be rescued by intrauterine exposure to OT. Indeed, there are several examples of maternal or sibling ``rescue'' of a phenotype as a consequence of intrauterine exposure derived from heterozygous or WT mothers or from neighboring siblings (see for example (Letterio et al., 1994)). However, the differences described here are rather subtle and we currently do not know of evidence of maternal or sibling contributions of embryonic OT. Earlier studies in rats (Shapiro and Insel 1989) suggest little prenatal OT receptor binding to support a neuronal mechanism for this in¯uence. However, the effects may be indirect: maternal OT may in¯uence other systems that modify fetal development. Social recognition The development of social familiarity in rodents depends predominantly on olfactory or pheromonal cues. Social memory can be assessed reliably by a decrease in olfactory investigation in repeated or prolonged encounters with a conspeci®c (Gheusi et al., 1994; Winslow and Camacho, 1995). AVP has been reliably found to facilitate social recognition across a range of doses and systemic and central routes of administration (Dantzer et al., 1987; Popik et al., 1991; Popik et al., 1992a). On the other hand, OT appears to have more complex dose effects in the rat. High doses of OT administered subcutaneously appear to inhibit while lower doses appear to facilitate social recognition (Popik et al., 1992a, 1996). Similarly, the effects of the intraventricular administration OT or OT fragments are also complex with both amnestic and facilitatory effects detected depending on doses (Benelli et al., 1995). Site speci®c injections of both AVP and OT into the lateral Neuropeptides (2002) 36(2^3), 221^229
226 Winslow and Insel
OT antagonist was effective in inhibiting species typical recognition in female but not male rats (Engelmann et al., 1998), implying perhaps that there are sexual dimorphisms in the sensitivity to the memory enhancing properties of both AVP and OT. Male OTKO mice completely fail to recognize familiar conspeci®cs even after 4 1-min encounters with the same female followed by an encounter with a new female (Figure 3 (Ferguson et al., 2000)). This de®cit does not represent an abnormality in sensory processing or a generalized impairment of learning or memory, because OTKO mice do not differ in their ability to locate hidden food, learn spatial cues for the Morris water maze, or habituate to an acoustic startle. Indeed, when scented cotton balls were used for the stimulus instead of a conspeci®c, subjects of both genotypes rapidly habituated to non-social odors, suggesting that male OTKO mice are able to process olfactory cues. OTKO mice even appear to recognize cotton balls with social scents, such as urine, when the stimulus was presented in isolation. However, they cannot use the same social cues to recognize previously encountered individuals.
septum of rats enhance social recognition (Popik et al., 1992b). On the other hand, injections of very low doses of OT but not AVP into the medial preoptic area were effective (Popik and van Ree, 1991). Both OT and AVP were able to enhance social recognition when injected into the olfactory bulbs (Dluzen et al., 1998a). The effects of both peptides were then subsequently shown to be dependent on an intact noradrenergic projection from the locus coeruleus to the olfactory bulbs (Dluzen et al., 1998b). Further pharmacological investigation then established that OT's effect on social recognition in the bulbs was mediated through the activation of the alpha-adrenergic receptors (Dluzen et al., 2000). In neither the olfactory bulb, the lateral septum nor the medial preoptic area were injections of a selective OT antagonist able to inhibit normal recognition (Dluzen et al., 1998a; Popik and van Ree, 1991; Popik et al., 1992b). Disparities between agonist and antagonist effects are worth noting also because agonists are much less selective for the OT receptor. It is possible that OT effects in the olfactory bulbs are mediated by V1A rather than OT receptors. Interestingly, the central administration of an
1
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1 min encounter−10 min ITI
Recall/Dishabituation Non-social olfactory stimulus
Social stimulus 60
40
50 Duration (sec)
30 40
*
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*
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*
KO WT
10
10
0
0 1
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1
2
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Repeated 1-minute trials (10-min ITI) Fig. 3 Time allocated to olfactory investigation of an ovariectomized mouse (A) or a scented object (B) during repeated 1 min encounters. Male WT (open symbol) mice show a rapid decline in investigation with renewed interest in a new female or scented object. OTKO mice (closed symbol) showed no decline in interest in a female but a rapid decline in interest in a scented object. (Adapted from (Ferguson et al. 2000)). Neuropeptides (2002) 36(2^3), 221^229
ß 2002 Elsevier Science Ltd. All rights reserved.
The social deficits of the oxytocin knockout mouse
Pharmacological studies in OTKO mice have demonstrated that intracerebroventricular injections of very small doses of OT (at least 1 pg) can completely rescue the social recognition de®cit (Ferguson et al., 2001). Moreover, the effect appears to be speci®c for OT since AVP is ineffective. Contrary to the effects of AVP in rats, OT is only effective when administered prior to the initial encounter. Administered 10 minute after the ®rst encounter, OT failed to restore recognition. Also contrasted with rats, administration of an OT receptor antagonist interfered with social recognition in WT mice. OTKO and WT mice also appear to process social information differently at the neural systems level (Ferguson et al., 2001). Following a 90 second social encounter, WT and OTKO mice showed different patterns of FOS induction in the brain. Both genotypes activated Fos in the olfactory bulbs, lateral septum, pyriform cortex, and dorsal lateral septum, OTKO males failed to induce Fos in the medial amygdala, and in several downstream projections of that nucleus, including the bed nucleus of the stria terminalis and the medial preoptic area. In addition to these areas of reduced activation in the OTKO males, increased activation was detected in several regions of the hippocampus (CA1 and CA2), somatosensory cortex, and the dentate gyrus. Although speculative, these males may be compensating for olfactory processing de®cits by recruiting alternative neural pathways. The medial amygdala, which failed to become activated in the OTKO males, is enriched with OT receptors. Injections of low doses of OT (0.1 pg) restored recognition in OTKO animals while administration of OTA into WT mice disrupted recognition (Ferguson et al., 2001). OT and OTA administration into the olfactory bulbs failed to modulate social recognition in either genotype. Taken together these data demonstrate that OTKO mice have a de®cit in social recognition associated with altered processing of social olfactory stimuli. Fos activation and pharmacological studies indicate that the medial amygdala may be a key locus of the defective olfactory processing. Future studies will need to investigate what OT is doing in this region to support the formation of a social memory. Based on previous studies in the olfactory bulb, it seems likely that OT is in¯uencing NE release or activation of adrenergic receptors.
CONCLUSION OTKO mice have provided some surprising new insights about the potential role of OT in mouse behavior. Findings related to OT's role in the maternal behavior of inbred mice inform us about strain differences and the complexity of the neuroendocrine control of this critically important function (Leckman and Herman, 2002). Interestingly, ß 2002 Elsevier Science Ltd. All rights reserved.
227
the importance of the OT gene in species for whom maternal behavior must be ``primed'' remains to be determined, as does the relevance of the OTKO mouse ®ndings to human maternal behavior. Social recognition is a key element of complex social relationships. Our data demonstrate a critical role for OT in the processing of the olfactory or pheromonal cues which form the basis of mouse recognition. While social recognition may be important for some types of social behavior in mice, it does not appear to be crucial for the capacity of mice to reproduce and thrive (at least not in the laboratory). One might speculate that this capacity might be under somewhat less selection pressure than sexual or maternal behavior, and consequently less apt to be supported by redundant or compensatory mechanisms. The apparently discrete nature of the de®cit and its selective restoration by OT but not AVP support this notion. The neural systems which support social recognition are complex and will continue to be a topic of vigorous inquiry. Based on our and other's ®ndings in rats and mice, it appears that AVP is more important for memory consolidation, while OT may in¯uence memory acquisition. The development of transgenic mice with speci®c de®cits in social memory provides new tools to examine the cellular and neural systems necessary for social recognition. These in turn may provide valuable new perspectives on diseases characterized by social de®cit syndromes, such as Autism or Reactive Attachment disorder.
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Insel TR, Young LJ (2000) Neuropeptides and the evolution of social behavior. Curr Opin Neurobiol 10: 784±789. Ivell R, Richter D (1984) Structure and comparison of the oxytocin and vasopressin genes from rat. Proc Natl Acad Sci USA 81: 2006±2010. Johnson AK, Thunhorst RL (1997) The neuroendocrinology of thirst and salt appetite: visceral sensory signals and mechanisms of central integration. Front Neuroendocrinol 18: 292±353. Kendrick KM (2000) Oxytocin, motherhood and bonding. Exp Physiol 85 Spec No: 111S±124S. Kimura T, Tanizawa O, Mori K, Brownstein MJ, Okayama H (1992) Structure and expression of a human oxytocin receptor. Nature 356: 526±529. Koolhaas JM, van den Brink THC, Roozendaal B, Boorsma F (1990) Medial amygdala and aggressive behavior: Interaction between testosterone and vasopressin. Aggressive Behavior 16: 223±229. Leckman JF, Herman AE (2002) Maternal behavior and developmental psychopathology. Biol Psychiatry 51: 27±43. Letterio JJ, Geiser AG, Kulkarni AB, Roche NS, Sporn MB, Roberts AB (1994) Maternal Rescue of Transforming Growth Factor-betal Null Mice. Science 264: 1936±1938. Miczek KA, Winslow JT (1987) Psychopharmacological research on aggressive behavior. In: Greenshaw AJ, Dourish CT (eds) Experimental Psychopharmacology. Humana Press, Clifton, New Jersey, pp. 27±114. Nishimori K, Young LJ, Guo Q, Wang Z, Insel TR, Matzuk MM (1996) Oxytocin is required for nursing but is not essential for parturition or reproductive behavior. Proc Natl Acad Sci USA 93: 11699±11704. Noirot E (1966) Ultrasounds In Young Rodents: I. Animal Behaviour 14: 459±462. Numan M (1988) Neural basis of maternal behavior in the rat. Psychoneuroendocrinology 13: 47±62. Numan M (1994) Maternal Behavior. In: Knobil E, Neil J (eds) The Physiology of Reproduction. Raven Press, New York. Pedersen CA, Ascher JA, Monroe YL, Prange AJ Jr. (1982) Oxytocin induces maternal behavior in virgin female rats. Science 216: 648±650. Pedersen CA, Caldwell JD, Johnson MF, Fort SA, Prange AJ Jr. (1985) Oxytocin antiserum delays onset of ovarian steroid-induced maternal behavior. Neuropeptides 6: 175±182. Pedersen CA, Prange AJ Jr. (1979) Induction of maternal behavior in virgin rats after intracerebroventricular administration of oxytocin. Proc Natl Acad Sci USA 76: 6661±6665. Popik P, van Ree JM (1991) Oxytocin but not vasopressin facilitates social recognition following injection into the medial preoptic area of the rat brain. European Journal of Neuropsychopharmacology 1: 555±560. Popik P, Vetulani J, Van Ree JM (1992a) Low doses of oxytocin facilitate social recognition in rats. Psychopharmacology 106: 71±74. Popik P, Vetulani J, Van Ree JM (1996) Facilitation and attenuation of social recognition in rats by different oxytocin-related peptides. European Journal of Pharmacology 308: 113±116. Popik P, Vos PE, Van Ree JM (1992b) Neurohypophyseal hormone receptors in the septum are implicated in social recognition in the rat. Behavioural Pharmacology 3: 351±358. Popik P, Wolterink G, De Brabander H, Van Ree JM (1991) Neuropeptides related to [Arg8] vasopressin facilitates social recognition in rats. Physiology & Behavior 49: 1031±1035. Postina R, Kojro E, Fahrenholz F (1996) Separate agonist and peptide antagonist binding sites of the oxytocin receptor defined by their transfer into the V2 vasopressin receptor. J Biol Chem 271: 31593±31601. Puryear R, Rigatto KV, Amico JA, Morris M (2001) Enhanced salt intake in oxytocin deficient mice. Exp Neurol 171: 323±328. Richter D (1983) Synthesis, processing, and gene structure of vasopressin and oxytocin. Prog Nucleic Acid Res Mol Biol 30: 245±266.
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Russell JA, Leng G (1998) Sex, parturition and motherhood without oxytocin? J Endocrinol 157: 343±359. Sausville E, Carney D, Battey J (1985) The human vasopressin gene is linked to the oxytocin gene and is selectively expressed in a cultured lung cancer cell line. J Biol Chem 260: 10236±10241. Shapiro LE, Insel TR (1989) Ontogeny of oxytocin receptors in rat forebrain: a quantitative study. Synapse 4: 259±266. Shapiro LE, Insel TR (1990) Infant's response to social separation reflects adult differences in affiliative behavior: a comparative developmental study in prairie and montane voles. Dev Psychobiol 23: 375±393. Summar ML, Phillips JA 3rd, Battey J, Castiglione CM, Kidd KK, Maness KJ, Weiffenbach B, Gravius TC (1990) Linkage relationships of human arginine vasopressin-neurophysin-II and oxytocin-neurophysin-I to prodynorphin and other loci on chromosome 20. Mol Endocrinol 4: 947±950. Tribollet E, Charpak S, Schmidt A, Dubois-Dauphin M, Dreifuss JJ (1989) Appearance and transient expression of oxytocin receptors in fetal, infant, and peripubertal rat brain studied by autoradiography and electrophysiology. J Neurosci 9: 1764±1773. van Leengoed E, Kerker E, Swanson HH (1987) Inhibition of post-partum maternal behaviour in the rat by injecting an oxytocin antagonist into the cerebral ventricles. J Endocrinol 112: 275±282. Whitnall M, Key S, Ben-Barak Y, Ozato K, Gainer H (1985) Neurophysin in the hypothalamo-neurohypophysial system.
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II. Immunocytochemical studies of the ontogeny of oxytocinergic and vasopressinergic neurons. Journal of Neuroscienceh 5: 98±109. Winslow JT, Camacho F (1995) Cholinergic modulation of a decrement in social investigation following repeated contacts between mice. Psychopharmacology (Berl) 121: 164±172. Winslow JT, Hastings N, Carter CS, Harbaugh CR, Insel TR (1993a) A role for central vasopressin in pair bonding in monogamous prairie voles. Nature 365: 545±548. Winslow JT, Hearn EF, Ferguson J, Young LJ, Matzuk MM, Insel TR (2000) Infant vocalization, adult aggression, and fear behavior of an oxytocin null mutant mouse. Horm Behav 37: 145±155. Winslow JT, Shapiro L, Carter CS, Insel TR (1993b) Oxytocin and complex social behavior: Species comparisons. Psychopharmacology Bulletin 29: 409±414. Witt DM, Carter CS, Walton DM (1990) Central and peripheral effects of oxytocin administration in prairie voles (Microtus ochrogaster). Pharmacology, Biochemistry & Behavior 37: 63±69. Young LJ, Lim MM, Gingrich B, Insel TR (2001) Cellular mechanisms of social attachment. Horm Behav 40: 133±138. Young WS, Shepard E, Amico J, Hennighausen L, Wagner KU, LaMarca ME, McKinney C, Ginns EI (1996) Deficiency in mouse oxytocin prevents milk ejection, but not fertility or parturition. Journal of Neuroendocrinology 8: 847±853. Zingg HH (2001) Oxytocin and uterine activity. Front Horm Res 27: 57±65.
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The social deficits of the oxytocin knockout mouse J. T. Winslow, T. R. Insel Center for Behavioral Neuroscience, Yerkes Regional Primate Center, Psychiatry and Behavioral Sciences, Emory University, Atlanta, GA
Summary Numerous studies have implicated oxytocin (OT) and oxytocin receptors in the central mediation of social cognition and social behavior. Much of our understanding of OT's central effects depends on pharmacological studies with OT agonists and antagonists. Recently, our knowledge of OT's effects has been extended by the development of oxytocin knockout (OTKO) mice. Mice with a null mutation of the OT gene manifest several interesting cognitive and behavioral changes, only some of which were predicted by pharmacological studies. Contrary to studies in rats, mice do not appear to require OT for normal sexual or maternal behavior, though OT is necessary for the milk ejection reflex during lactation. OTKO pups thrive if raised by a lactating female, but OTKO pups emit fewer ultrasonic vocalizations with maternal separation and OTKO adults are more aggressive than WT mice. Remarkably, OTKO mice fail to recognize familiar conspecifics after repeated social encounters, though olfactory and non-social memory functions appear to be intact. Central OT administration into the amygdala restores social recognition. The development of transgenic mice with specific deficits in social memory represents a promising approach to examine the cellular and neural systems of social cognition. These studies may provide valuable new perspectives on diseases characterized by social deficits, such as autism or reactive attachment disorder. ß 2002 Elsevier Science Ltd. All rights reserved.
Oxytocin (OT) belongs to a family of nine amino acid peptides that have been identi®ed in all classes of vertebrates and many invertebrate species (Hoyle, 1999). OT and the closely related nonapeptide arginine-vasopressin (AVP) are the only members of this family identi®ed in mammals. Both OT and AVP are classi®ed as neurohypophyseal peptides, synthesized in the hypothalamus and released into the bloodstream via axon terminals in the posterior pituitary or neurohypophysis. OT's classic physiological roles include stimulating smooth muscle contractions in the uterus during labor and in mammary myoepithelium during nursing (Zingg, 2001). OT also plays a role in a variety of other reproductive-related functions, such as modulation of estrous cycle length, follicle luteinization in the ovary, and ovarian steroidogenesis (Fuchs, 1988). In addition to these endocrine effects, OT appears to have a role as a neurotransmitter Received 2 April 2002 Accepted 20 April 2002 Correspondence to: James T. Winslow, 954 Gatewood Road, N.E. Yerkes Primate Center, Atlanta, GA 30322. Tel.: 404-727-7728; Fax: 404-727-7845; E-mail: [email protected]
or neuromodulator in the brain, in¯uencing socio-sexual behaviors (Insel et al., 1998; Insel and Young, 2000; Kendrick, 2000; Young et al., 2001) (Table 1). Our knowledge of OT's central effects is based largely on pharmacological studies with OT agonists and antagonists. As these agonists and antagonists are peptides that do not cross the blood-brain barrier, they must be given centrally to assess effects in the CNS. While pharmacological studies have been instructive, one never knows if agonists mimic physiological effects or antagonists fully and selectively inhibit endogenous peptide. Here we review the results of studies in oxytocin knockout (OTKO) mice. Mice with a null mutation of the OT gene manifest several interesting cognitive and behavioral changes, only some of which were predicted by pharmacological studies. ANATOMICAL SYNTHESIS AND DISTRIBUTION OF PEPTIDE The OT gene is expressed primarily in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus (Gainer, 1998). In the PVN two populations of OT-staining cells have been identi®ed: the magnocellular 221
222 Winslow and Insel
Table 1 Oxytocin and Social Behavior
Sexual Behavior Female Male Autogrooming Yawning Nociception Anxiety Stress Feeding Salt-Appetite Memory and Learning
Mouse OT ICV
OTKO
Rat OT ICV
? ? ?
nc nc nc nc ?
?
? ?
, Increase; , Decrease; ?, Unknown; nc, No Change; refers generally to modulated behavior associated with OT administration or gene mutation. (Amico et al., 2001; Ferguson et al., 2000; Gimpl and Fahrenholz, 2001; Johnson and Thunhorst, 1997; Kendrick, 2000; Puryear et al., 2001; Winslow et al., 2000).
neurons with projections that terminate in the neurohypophysis and the parvocellular neurons with projections to various areas of the CNS (Gainer and Wray, 1994). In the rat, OT terminals and ®bers have been localized in the thalamic nuclei, dorsal and ventral hippocampus, subiculum, entorhinal cortex, medial and septal nuclei, amygdala, olfactory bulbs, mesencephalic gray nucleus, substantia nigra, locus coeruleus, raphe nucleus, the nucleus of the solitary tract, and dorsal motor nucleus of the vagus nerve (Argiolas and Gessa, 1991). In all species, OT and vasopressin genes are located on the same chromosomal locus but are transcribed in opposite directions (Hoyle, 1999). The OT gene is located on mouse chromosome 2 (Hara et al., 1990) and human chromosome 20 (Summar et al., 1990). The genes for AVP and OXT are separated by 12 kb in humans and 3 kb in the mouse (Hoyle 1999). The human and mouse genes consist of three exons (Richter, 1983; Ivell and Richter, 1984; Sausville et al., 1985): the ®rst exon encodes a signal peptide, the nonapeptide, a tripeptide processing peptide (GKR), and the ®rst nine amino acids of neurophysin, a carrier polypeptide important for transport. The second exon encodes the central part of neurophysin (residues 10-76) while the third encodes the COOH-terminal region of neurophysin (residues 77-93/95) (Sausville et al., 1985). Further modi®cation of the OT prepropeptide occurs as it is transported down the axon to terminals located in the posterior pituitary. OT and its carrier neurophysin are then stored in the axon terminals until neural activity stimulates release.
RECEPTOR AND DISTRIBUTION IN BRAIN OT's effects appear to be mediated by a rhodopsin-type G-protein coupled receptor with seven transmembrane Neuropeptides (2002) 36(2^3), 221^229
domains linked to phosphoinositol for signal transduction (Kimura et al., 1992). This OT receptor is only relatively selective for OT compared to AVP, with a 10-fold higher af®nity for OT. (Chini et al., 1996; Postina et al., 1996). OT receptors are found not only in uterus and mammary tissue, but also in kidney, thymus, and several regions of the brain. The brain receptor appears identical to the receptor found in peripheral organs (Gimpl and Fahrenholz, 2001). Within the brain, OT receptors are abundant in limbic and autonomic areas; however, the speci®c distribution in substructures is quite variable across species (Insel and Young, 2000). For instance, in the rat brain, OT receptors are abundant in the ventromedial nucleus of the hypothalamus and sparse in the hippocampus, whereas in the mouse brain there is relatively little binding in the hypothalamus and abundant binding in the hippocampus (Gimpl and Fahrenholz, 2001). Indeed, nearly every species tested to date has a unique pattern of expression of OT receptors, suggesting a role in species-typical behaviors (Insel and Young, 2000). In addition to the marked species variation in distribution, there is also variation in regulation of these receptors. In several regions of the rat brain, OTRs are induced by gonadal steroids (Breton and Zingg, 1997), whereas in select regions of the mouse brain gonadal steroids appear to inhibit expression (Insel et al., 1993). THE OXYTOCIN KNOCKOUT MOUSE Three groups have created OT knockout (OTKO) mice: Nishimori et al. (1996) deleted the ®rst exon of the OT gene by homologous combination in embryonic stem cells (Figure 1), Young et al. (1996) deleted a segment of the second exon to eliminate neurophysin and Gross et al. (Gross et al., 1998) replaced all three exons, so there are no preproOT/neurophysin coding sequences present (L. J. Muglia, personal communication). All of these mutations eliminated OT, although there were subtle differences in collateral effects. The exon 1 deletion did not affect arginine vasopressin mRNA, but the exon 2 deletion resulted in a slight decrease in AVP and dynorphin mRNA. Neither the distribution nor the concentration of OT receptors was measurably affected in the OTKO compared to wild-type mice. Thus, presynaptic OT is not necessary for the normal neuroanatomical distribution of OT receptors during development, and consequently does not account for developmental or species differences in receptor distribution. With each of these mutations, OT-KO mice show de®cits in lactation, due to an absent milk ejection re¯ex. Parturition appears normal, as measured by duration of pregnancy and length of parturition. Although OT increases during parturition, OT is not necessary for delivery. ß 2002 Elsevier Science Ltd. All rights reserved.
The social deficits of the oxytocin knockout mouse
Development of an OT-KO Mouse Exon 1
223
WT Exon 2
Exon 3
5'
3'
WT gene
Recombination
Non-OT
5'
Non-OT
Exon 2
Exon 3
Exon 2
Exon 3
5'
3'
Deletion Vector
Recombinant OT-KO 3' Allele
HET
Targeting vector in 129sSvEv ES cells Implant into C57BL/6J
Nishimori et al., PNAS, 1996
HOM
Fig. 1 Left Panel. The first-exon null mutation for OT was produced by homologous recombination in embryonic stem cells. Right Panel. OT immunoreactivity in the paraventricular and the supraoptic nuclei was reduced in heterozygous (HET) and absent in homozygous (HOM) mice compared to wild type (WT) siblings (Adapted from (Nishimori et al. 1996)).
BEHAVIORAL PHENOTYPE Our examination of the behavioral phenotype of the OTKO mouse created by the ®rst exon deletion strategy (Nishimori et al., 1996) was guided by two decades of pharmacological studies of central OT and OT receptor antagonist administration. These studies, as well as a convergence of neurochemical data suggest a key role for OT in the development and maintenance of social cognition and social behavior. For this reason, we examined the behavior of OTKO mice in maternal behavior, infant ultrasonic vocalizations, intermale aggression, and social recognition.
Maternal behavior Much of what is known about central OT has been derived from studies of OT's role in the onset of maternal behavior by female rats. The laboratory rat has been an ideal subject for studies of maternal care (Numan, 1988). Unlike many ß 2002 Elsevier Science Ltd. All rights reserved.
mammals, female rats show little interest in infants of their own species until shortly before parturition. About a day prior to delivery they shift from avoiding pups from other females' litters to showing intense interest with vigorous nest building, retrieval, grooming, and defense of young. These behaviors persist through lactation, and then abate with weaning. Rats, therefore, provide an opportunity to study two quite distinct aspects of maternal care: its onset and maintenance. Several investigators have reported that OT given centrally to estrogen-primed, nulliparous female rats facilitates the onset of maternal behavior. Pedersen and Prange noted that the effect was speci®c to OT (the only other neuropeptide with signi®cant albeit weaker effects was vasopressin) and resulted in rapid onset (within 30 minutes) of full maternal behavior (Pedersen and Prange, 1979; Pedersen et al., 1982; Fahrbach et al., 1984). Blockade of OT neurotransmission with central injection of an antagonist or antiserum or by lesions of OT-producing cells in the hypothalamus, results in a signi®cant inhibition of maternal behavior (Fahrbach Neuropeptides (2002) 36(2^3), 221^229
224 Winslow and Insel
et al. 1985; Pedersen et al., 1985; van Leengoed et al., 1987; Insel and Harbaugh 1989). These various interventions appear to inhibit the onset but not the maintenance of maternal behavior ± an OT antagonist or a lesion has no effect on maternal care once a female begins to show interest in pups (Fahrbach et al., 1985; Insel and Harbaugh, 1989). These results support the notion that OT is necessary for the transition to maternal attachment to pups and that a central increase in OT given under the appropriate gonadal steroid conditions might facilitate the onset of maternal care. Given the strong evidence that OT is critical for the onset of maternal behavior in rats, one might predict that OT-KO mice would not be maternal. Surprisingly we and others failed to detect any differences in pup retrieval, nest building or time spent grooming pups between OTKO and wild-type mice (Nishimori et al., 1996; Young et al., 1996). Of course, pups from OTKO mothers fail to thrive unless cross-fostered, due to the OTKO mother's inability to eject milk. However, once treated with systemic OT to re-instate milk ejection, full maternal behavior ensues. Indeed, even virgin OTKO females show full maternal behavior. Because OT receptors are unaffected by the mutation, compensatory mechanisms involving OT receptors may account for the expression of maternal behavior. To test this possibility, adult virgin OTKO mice were infused centrally with an OT antagonist for 5 days using Alzet pumps (Insel et al., 2001). At the end of 5 days, these animals still showed a full repertoire of maternal behaviors. A similar experiment in pregnant females has not been done, so the possible contribution of OT receptors to normal gestation or the development of maternal behavior has not been examined. How can OTKO mice show full maternal behavior when OT appears necessary for maternal behavior in rats? Unlike rats, in which maternal behavior has an abrupt onset at parturition, in inbred strains of mice including the C57BL/ 6J strain of mouse used as background for the OTKO mutation, maternal behavior emerges spontaneously in virgin mice (Numan, 1994). That is, mice do not require pregnancy or parturition to show full maternal behavior. For this reason, it seems likely that OT may not be essential for maternal behavior in mice (Russell and Leng, 1998).
generally are powerful stimuli for maternal retrieval. We have previously reported that exogenous administration of OT reduces the rate of ultrasonic vocalizations expressed by rat pups during separations from mother and peers (Insel and Winslow, 1991). However, an OT antagonist did not increase calling, so it is not clear that endogenous OT is important for this behavior. Vasopressin and OT are both found early in fetal development, although in the rat OT does not show a fully processed transcript until the postnatal period (Whitnall et al., 1985). Receptors for both peptides are found in the developing brain (Shapiro and Insel, 1989; Tribollet et al., 1989). Indeed, there is a transient, but marked ``over-production'' (relative to the adult) of both OT and vasopressin receptors in limbic brain areas during infancy (in both rodents and primates). Although these binding sites are particularly enriched in the cingulate cortex (a region implicated in separation distress), we do not know if these putative receptors are involved in infant behavior nor do we know why they disappear by the time of weaning. OTKO pups emit fewer ultrasonic calls than wild-type pups (Figure 2 Left Panel (Winslow et al., 2000)). These differences do not appear to be associated with differences in gross developmental features since OTKO pups do not differ in body weight, thermoregulation or motor activity. It is unlikely that the reduced calling rate in OTKO mice is a consequence of ¯anking genes carried over with the targeting vector from the 129SvEv strain as mice of this strain actually give more calls than C57BL/6J mice (Winslow et al., 2000). Reduced vocal behavior also did not appear to vary signi®cantly with prenatal environments, since OTKO pups derived from both homozygote and heterozygote mothers had fewer vocalizations than wild-type mice. Taken together, these data suggest that OT may modulate calling in mouse pups. Note however, that the direction of effect is in the opposite direction from what we would have predicted based on pharmacological studies with rat pups in which OT decreased calling and an OT antagonist did not alter calling. One intriguing explanation for decreased calling by OTKO pups is that they may be less sensitive to maternal separation than WT pups, however this possibility has not been tested directly. Aggressive behavior
Infant ultrasonic vocalizations Rodents have a surprising repertoire of audiovocal communication, mostly in the ultrasonic range, Mouse pups emit calls in the 60±70 kHz range in response to social separation (Noirot, 1966). Several studies have suggested that these separation calls are similar to distress calls in other mammals. For instance, infants of highly social species give more calls than infants of less social species (Shapiro and Insel, 1990) and these pup ultrasonic calls Neuropeptides (2002) 36(2^3), 221^229
Previous studies of the central administration of OT indicate that aggressive behaviors may be in¯uenced by OT, although the effects appear to vary depending upon the species studied. OT appears to decrease aggression in male prairie voles (Witt et al., 1990; Winslow et al., 1993b), while facilitating maternal aggression (Ferris et al., 1992) and dominance-related behaviors in squirrel monkeys (Insel and Winslow, 1991). A recent study of OT depletion produced by a different null mutation of the OTß 2002 Elsevier Science Ltd. All rights reserved.
The social deficits of the oxytocin knockout mouse
Resident-intruder aggression
Infant mouse calls
40 Attacks per 5 min session
Cals per 2 min session
140 120 100 80 60
*
40 20 0
225
OTKO 30
*
WT
* 20
*
10 0
KO
WT
1
2
3
Repeated confrontations Fig. 2 Left Panel portrays the number of ultrasonic vocalizations expressed by WT (closed symbol) and OTKO (open symbol) mouse pups during brief separations from mother and littermates. Right Panel represents the frequency of attack biting by adult, resident male WT (closed symbol) and OTKO (open symbol) mice during repeated 5 min confrontations with a male outbred intruder. Each confrontation was separated by 48 hrs. (Adapted from (Winslow et al. 2000)).
neurophysin gene in mice disrupted intermale aggression; however, these mice showed several unintended neuroendocrine changes (Young et al., 1996) and the behavioral effects were subtle (DeVries et al., 1997). In particular, OT de®cient mice showed decreased attack bout duration in a resident±intruder paradigm and decreased bout duration and total attack time in a neutral arena test. The frequency of aggressive episodes in each of these tests was relatively low compared to aggression expressed in outbred strains (Miczek and Winslow, 1987). This mutation also reduced vasopressin which appears to be necessary for aggression in many species (Ferris et al., 1989; Koolhaas et al., 1990; Winslow et al., 1993a) Our studies of aggressive behavior in OTKO mice revealed increased aggression (Figure 2, Right Panel) and a complex interaction between pup genotype and maternal genotype. In general, OTKO mice were signi®cantly more aggressive than WT mice tested both in isolation-induced and resident-intruder paradigms, though OTKO animals derived from heterozygote mothers appeared to be somewhat less aggressive compared to those derived from homozygous mothers. We interpreted these results to suggest that the genotype of the test animal did not fully account for differences in aggressive behavior with additional prenatal or postnatal rearing differences also in¯uential (Winslow et al., 2000). It is possible that some behavioral phenotypes may be in¯uenced by contrasting postnatal environments produced by differing mothering styles or litter compositions associated with the production and rearing by homozygote or heterozygote mothers. However, it is appears unlikely that postnatal environments account for phenotypic differences detected for offspring of homozygote mothers since both OTKO and WT pups were crossfostered to WT females. The moderation of differences ß 2002 Elsevier Science Ltd. All rights reserved.
in the isolation induced aggression of OTKO mice derived from heterozygous litters suggests that some aspect of the phenotype of these mice may be rescued by intrauterine exposure to OT. Indeed, there are several examples of maternal or sibling ``rescue'' of a phenotype as a consequence of intrauterine exposure derived from heterozygous or WT mothers or from neighboring siblings (see for example (Letterio et al., 1994)). However, the differences described here are rather subtle and we currently do not know of evidence of maternal or sibling contributions of embryonic OT. Earlier studies in rats (Shapiro and Insel 1989) suggest little prenatal OT receptor binding to support a neuronal mechanism for this in¯uence. However, the effects may be indirect: maternal OT may in¯uence other systems that modify fetal development. Social recognition The development of social familiarity in rodents depends predominantly on olfactory or pheromonal cues. Social memory can be assessed reliably by a decrease in olfactory investigation in repeated or prolonged encounters with a conspeci®c (Gheusi et al., 1994; Winslow and Camacho, 1995). AVP has been reliably found to facilitate social recognition across a range of doses and systemic and central routes of administration (Dantzer et al., 1987; Popik et al., 1991; Popik et al., 1992a). On the other hand, OT appears to have more complex dose effects in the rat. High doses of OT administered subcutaneously appear to inhibit while lower doses appear to facilitate social recognition (Popik et al., 1992a, 1996). Similarly, the effects of the intraventricular administration OT or OT fragments are also complex with both amnestic and facilitatory effects detected depending on doses (Benelli et al., 1995). Site speci®c injections of both AVP and OT into the lateral Neuropeptides (2002) 36(2^3), 221^229
226 Winslow and Insel
OT antagonist was effective in inhibiting species typical recognition in female but not male rats (Engelmann et al., 1998), implying perhaps that there are sexual dimorphisms in the sensitivity to the memory enhancing properties of both AVP and OT. Male OTKO mice completely fail to recognize familiar conspeci®cs even after 4 1-min encounters with the same female followed by an encounter with a new female (Figure 3 (Ferguson et al., 2000)). This de®cit does not represent an abnormality in sensory processing or a generalized impairment of learning or memory, because OTKO mice do not differ in their ability to locate hidden food, learn spatial cues for the Morris water maze, or habituate to an acoustic startle. Indeed, when scented cotton balls were used for the stimulus instead of a conspeci®c, subjects of both genotypes rapidly habituated to non-social odors, suggesting that male OTKO mice are able to process olfactory cues. OTKO mice even appear to recognize cotton balls with social scents, such as urine, when the stimulus was presented in isolation. However, they cannot use the same social cues to recognize previously encountered individuals.
septum of rats enhance social recognition (Popik et al., 1992b). On the other hand, injections of very low doses of OT but not AVP into the medial preoptic area were effective (Popik and van Ree, 1991). Both OT and AVP were able to enhance social recognition when injected into the olfactory bulbs (Dluzen et al., 1998a). The effects of both peptides were then subsequently shown to be dependent on an intact noradrenergic projection from the locus coeruleus to the olfactory bulbs (Dluzen et al., 1998b). Further pharmacological investigation then established that OT's effect on social recognition in the bulbs was mediated through the activation of the alpha-adrenergic receptors (Dluzen et al., 2000). In neither the olfactory bulb, the lateral septum nor the medial preoptic area were injections of a selective OT antagonist able to inhibit normal recognition (Dluzen et al., 1998a; Popik and van Ree, 1991; Popik et al., 1992b). Disparities between agonist and antagonist effects are worth noting also because agonists are much less selective for the OT receptor. It is possible that OT effects in the olfactory bulbs are mediated by V1A rather than OT receptors. Interestingly, the central administration of an
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Repeated 1-minute trials (10-min ITI) Fig. 3 Time allocated to olfactory investigation of an ovariectomized mouse (A) or a scented object (B) during repeated 1 min encounters. Male WT (open symbol) mice show a rapid decline in investigation with renewed interest in a new female or scented object. OTKO mice (closed symbol) showed no decline in interest in a female but a rapid decline in interest in a scented object. (Adapted from (Ferguson et al. 2000)). Neuropeptides (2002) 36(2^3), 221^229
ß 2002 Elsevier Science Ltd. All rights reserved.
The social deficits of the oxytocin knockout mouse
Pharmacological studies in OTKO mice have demonstrated that intracerebroventricular injections of very small doses of OT (at least 1 pg) can completely rescue the social recognition de®cit (Ferguson et al., 2001). Moreover, the effect appears to be speci®c for OT since AVP is ineffective. Contrary to the effects of AVP in rats, OT is only effective when administered prior to the initial encounter. Administered 10 minute after the ®rst encounter, OT failed to restore recognition. Also contrasted with rats, administration of an OT receptor antagonist interfered with social recognition in WT mice. OTKO and WT mice also appear to process social information differently at the neural systems level (Ferguson et al., 2001). Following a 90 second social encounter, WT and OTKO mice showed different patterns of FOS induction in the brain. Both genotypes activated Fos in the olfactory bulbs, lateral septum, pyriform cortex, and dorsal lateral septum, OTKO males failed to induce Fos in the medial amygdala, and in several downstream projections of that nucleus, including the bed nucleus of the stria terminalis and the medial preoptic area. In addition to these areas of reduced activation in the OTKO males, increased activation was detected in several regions of the hippocampus (CA1 and CA2), somatosensory cortex, and the dentate gyrus. Although speculative, these males may be compensating for olfactory processing de®cits by recruiting alternative neural pathways. The medial amygdala, which failed to become activated in the OTKO males, is enriched with OT receptors. Injections of low doses of OT (0.1 pg) restored recognition in OTKO animals while administration of OTA into WT mice disrupted recognition (Ferguson et al., 2001). OT and OTA administration into the olfactory bulbs failed to modulate social recognition in either genotype. Taken together these data demonstrate that OTKO mice have a de®cit in social recognition associated with altered processing of social olfactory stimuli. Fos activation and pharmacological studies indicate that the medial amygdala may be a key locus of the defective olfactory processing. Future studies will need to investigate what OT is doing in this region to support the formation of a social memory. Based on previous studies in the olfactory bulb, it seems likely that OT is in¯uencing NE release or activation of adrenergic receptors.
CONCLUSION OTKO mice have provided some surprising new insights about the potential role of OT in mouse behavior. Findings related to OT's role in the maternal behavior of inbred mice inform us about strain differences and the complexity of the neuroendocrine control of this critically important function (Leckman and Herman, 2002). Interestingly, ß 2002 Elsevier Science Ltd. All rights reserved.
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the importance of the OT gene in species for whom maternal behavior must be ``primed'' remains to be determined, as does the relevance of the OTKO mouse ®ndings to human maternal behavior. Social recognition is a key element of complex social relationships. Our data demonstrate a critical role for OT in the processing of the olfactory or pheromonal cues which form the basis of mouse recognition. While social recognition may be important for some types of social behavior in mice, it does not appear to be crucial for the capacity of mice to reproduce and thrive (at least not in the laboratory). One might speculate that this capacity might be under somewhat less selection pressure than sexual or maternal behavior, and consequently less apt to be supported by redundant or compensatory mechanisms. The apparently discrete nature of the de®cit and its selective restoration by OT but not AVP support this notion. The neural systems which support social recognition are complex and will continue to be a topic of vigorous inquiry. Based on our and other's ®ndings in rats and mice, it appears that AVP is more important for memory consolidation, while OT may in¯uence memory acquisition. The development of transgenic mice with speci®c de®cits in social memory provides new tools to examine the cellular and neural systems necessary for social recognition. These in turn may provide valuable new perspectives on diseases characterized by social de®cit syndromes, such as Autism or Reactive Attachment disorder.
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