FRANK A. BEACH AWARD Oxytocin and Vasopressin Receptors and Species-Typical Social Behaviors 1

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1 Hormones and Behavior 36, (1999) Article ID hbeh , available online at on FRANK A. BEACH AWARD Oxytocin and Vasopressin Receptors and Species-Typical Social Behaviors 1 Larry J. Young 2 Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia Received June 8, 1999; revised July 20, 1999; accepted July 23, This article is based on the 1998 Frank A. Beach Award lecture, presented at the 1998 Society for Neuroscience Meeting. 2 To whom correspondence and reprint requests should be addressed at Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta GA Fax: (404) lyoun03@emory.edu. Over the past 50 years, a great deal of behavioral endocrinological research has established a central role for gonadal steroid hormones in the control of sexual behaviors in a wide range of vertebrate taxa. The underlying mechanisms controlling these behaviors appear to be well conserved throughout evolution. Both the neural structures regulating male and female sexual behaviors and the distribution of sex steroid receptors within the brain are similar from reptiles to mammals (Meisel and Sachs, 1994; Pfaff and Schwartz-Giblin, 1994). More recently, the roles of the neuropeptides oxytocin (OT) and vasopressin (AVP) in modulating reproductive and social behaviors have begun to be elucidated. Comparative studies focusing on these hormones suggest that unlike gonadal steroids, the specific behavioral roles of these neuropeptides may be quite species specific. Here I review some of the behaviors associated with OT and AVP systems and discuss the idea that OT and AVP receptor systems are phylogenetically plastic, perhaps facilitating the evolution of species-typical social behavior patterns. Oxytocin and vasopressin are related nonapeptide hormones which differ in structure at only two amino acid positions (Gainer and Wray, 1994). Both peptides are synthesized in magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei of the hypothalamus, which project to the neurohypophysis and are released into circulation where they modulate peripheral physiology (Brownstein, Russell, and Gainer, 1980). The peptides are also synthesized in parvocelluar neurons of the PVN and other limbic nuclei which project throughout the brain where they influence behavior. Vasopressin synthesis by neurons in the amygdala and bed nucleus of the stria terminalis is sexually dimorphic, with males producing more than females (DeVries and Al-Shamma, 1990). Centrally released oxytocin plays a role in the induction of maternal behavior in rats (Pedersen, Caldwell, Walker, Ayers, and Mason, 1994; Pedersen and Prange, 1979), house mice (McCarthy, 1990), and sheep (Costa, Guevara-Guzman, Ohkura, Goode, and Kendrick, 1996; Kendrick, Keverne, and Baldwin, 1987). Infusion of OT into the brain of virgin animals facilitates maternal behavior, while blocking OT receptors prevents these behaviors. In rats, oxytocin is involved in the induction of sexual receptivity by estrogen and progesterone (Caldwell, Prange, and Pedersen, 1986; Schumacher, Coirini, Pfaff, and McEwen, 1990). In addition, OT appears to play an important role in the formation of long-lasting pair bonds in female monogamous prairie voles (Williams, Insel, Harbaugh, and Carter, 1994). While many of the roles of OT are associated with female-typical behaviors, most of the behaviors associated with AVP have been demonstrated in the male. Arginine vasotocin (AVT), the nonmammalian homolog of AVP, stimulates sexual behavior in newts (Moore and Miller, 1983) and vocalization in frogs (Boyd, 1994) and birds (Goodson, 1998a; Maney, Goode, and Wingfield, 1997). Vasopressin facilitates scentmarking behavior in hamsters (Ferris, Albers, Wesolowski, Goldman, and Leeman, 1984) and aggression in hamsters and voles (Ferris, Melloni, Koppel, Perry, Fuller, and Delville, 1997; Young, Winslow, X/99 $30.00 Copyright 1999 by Academic Press All rights of reproduction in any form reserved.

2 Oxytocin and Vasopressin Receptors 213 Nilsen, and Insel, 1997) and plays a role in pair bonding and paternal care in male monogamous prairie voles (Wang, Ferris, and DeVries, 1994; Winslow, Hastings, Carter, Harbaugh, and Insel, 1993). Vasopressin modulates social behaviors in a species-specific manner. For example, Goodson recently demonstrated that AVT increases aggression in the colonial zebra finch (Goodson and Adkins-Regan, 1998) but inhibits aggression in the territorial field sparrow and violet eared waxbill (Goodson, 1998a; Goodson, 1998b). Similarly, AVP increases male male aggression and affiliative behavior in the male monogamous, nonterritorial prairie vole, but not in the nonmonogamous, territorial montane vole (Young, Nilsen, Waymire, MacGregor, and Insel, 1999; Young et al., 1997). These observations suggest that the role of vasopressin in regulating social behavior may vary across species with social organization. SPECIES DIFFERENCES IN OXYTOCIN AND VASOPRESSIN RECEPTORS The central effects of OT and AVP are mediated by G-protein coupled, seven transmembrane receptors, which, like the peptides, share similar structures (Barberis and Tribollet, 1996). A single oxytocin receptor (OTR) has been identified and is present in both peripheral and brain tissues. Three subtypes of AVP receptors, V1a, V1b, and V2, have been identified which differ both structurally and pharmacologically. Both V1a and V1b receptors are expressed in the brain (Barberis, Balestre, Jard, Tribollet, Arsenijevic, Dreifuss, Bankowski, Manning, Chan, Schlosser, Holsboer, and Elands, 1995; Vaccari, Lolait, and Ostrowski, 1998), although most of the behavioral effects of AVP have been attributed to the V1a subtype (Wang et al., 1994; Winslow et al., 1993). Comparisons of the neuroanatomical distribution of OTR and V1a receptors among species have revealed marked species differences in the pattern of receptor binding in the brain, although some generalities have been noted (Barberis and Tribollet, 1996). For example, rats (DeKloet, Rotteveel, Voorhuis, and Terlou, 1985; Tribollet, Barberis, Jard, Dubois-Dauphin, and Dreifuss, 1988), mice (Insel, Young, Witt, and Crews, 1993), and voles (Insel and Shapiro, 1992; Young, Huot, Nilsen, Wang, and Insel, 1996) each have a distinct pattern of OTR binding in the brain. Figure 1 illustrates the different patterns of vasopressin (V1a) receptor binding sites in the brains of several species. As can be seen in this illustration, the distribution and densities of V1a receptors vary quite dramatically even among closely related species. There is evidence that the pattern of receptor binding in the brain is associated with social organization. For example, among voles, species with similar social organizations share a common pattern of OTR and V1a receptor binding (Insel and Shapiro, 1992; Insel, Wang, and Ferris, 1994). The monogamous prairie (Microtus ochrogaster) and pine (Microtus pinetorum) voles have similar OTR and V1a receptor binding patterns which are distinct from the nonmonogamous montane (Microtus montanus) and meadow (Microtus pennsylvanicus) voles. In these species differences in receptor distribution and density may contribute to the species differences in the effects of OT and AVP on behavior. The association between receptor binding pattern and social organization is correlative, but does not prove a relationship between regional distribution of receptors and social behavior. In addition to species differences in distribution of OT and V1a receptors in the brain, the receptors are regulated differently by gonadal steroids in different species. This species-specific regulation of peptide receptors may have important implications for understanding the roles of the peptides in modulating behavior. In the female rat, the induction of lordosis behavior by estrogen involves the up-regulation of OTR in the VMN. Estrogen increases both the level of OTR binding and gene expression in the VMN (Bale and Dorsa, 1995; Johnson, Coirini, Insel, and McEwen, 1991; Tribollet, Audigier, Dubois-Dauphin, and Dreifuss, 1990) and OTR expression in the VMN varies with the ovarian cycle, with peak levels of expression found during proestrus (Bale, Dorsa, and Johnston, 1995). The ovulatory surge of progesterone then causes the OTR to spread to the ventrolateral regions of the VMN in close proximity to OT fibers (Schumacher, Coirini, Frankfurt, and McEwen, 1989; Schumacher et al., 1990). Blockade of the OT receptor with specific antagonists (Witt and Insel, 1991) or preventing the up-regulation of OTR using antisense oligonucleotides (McCarthy, Kleopoulos, Mobbs, and Pfaff, 1994) inhibits sexual receptivity in the female rat. In the female prairie vole, which is induced into estrus pheromonally and ovulates after mating (Sawrey and Dewsbury, 1985), estrogen does not influence OTR binding in the VMN (Witt, Carter, and Insel, 1991). In the mouse, gonadectomy increases and gonadal steroids decrease OTR binding in the VMN, a pattern opposite to that of the rat (Insel et al., 1993). Therefore, the relationship between gonadal steroids, OTR in the VMN, and sexual receptivity may differ among rodent

3 214 Larry J. Young FIG. 1. Vasopressin (V1a) receptor binding patterns in (A) prairie and (B) montane voles; (C) Peromyscus californicus and (D) Peromyscus leucopus. (Bester-Meredith, Young, and Marler, 1998); and (E) common marmoset (Wang, Toloczko, Young, Moody, Newman, and Insel, 1997) and (F) rhesus monkey (Young, Toloczko, and Insel, 1999). Abbreviations: ac, anterior commissure; BST, bed nucleus of the stria terminalis; Ctx, cortex; DB, diagonal band; LS, lateral septum. species. This may explain the lack of an obvious deficit in female sexual behavior in oxytocin knockout mice (Nishimori, Young, Guo, Wang, Insel, and Matzuk, 1996). Vasopressin receptors are also regulated in a species-specific manner. In hamsters, AVP injected into the preoptic-anterior hypothalamic area is a potent inducer of flank marking as well as intermale aggression (Ferris et al., 1984, 1997). The ability of exogenous AVP to facilitate flankmarking is androgen dependent, with testosterone-treated castrated males requiring less AVP to stimulate the behavior than untreated castrates (Albers, Liou, and Ferris, 1988). In hamsters, castration reduces V1a binding (Johnson, Barberis, and Albers, 1995) and testosterone treatment increases V1a receptor binding and gene expression in the preoptic area (L. J. Young, T. Cooper and H. E. Albers, unpublished data). In contrast, V1a receptor binding in the rat brain is independent of gonadal steroids (Tribollet et al., 1990). Given the remarkable species differences in OTR and V1a receptor distribution, it should not be surprising to find significant individual variation in receptor densities within a species. We have recently noted that among our colony of prairie voles, there is significant individual variation in both OTR and V1a receptor binding in specific brain regions (L. J. Young, B. Gingrich, and T. R. Insel, unpublished data). This variability is most pronounced for the OTR in the nucleus accumbens (NAcc) (Fig. 2). There appears to be a bimodal distribution of OT receptor density in the NAcc, with OTR binding being very intense in the NAcc in some animals, while binding in this area is just above background in others. Little individual variability is found in other brain regions. This variation is particularly interesting since recent pharmaco-

4 Oxytocin and Vasopressin Receptors 215 UNDERLYING MECHANISMS OF RECEPTOR EXPRESSION FIG. 2. Individual variation in oxytocin receptor binding in the nucleus accumbens (NAcc) of female prairie voles. In some individual voles, oxytocin receptor binding in the nucleus accumbens is one of the most intense of all brain areas, while in other individuals oxytocin receptor binding in the same region is barely above background. logical experiments have demonstrated that OTR in the NAcc is involved in pairbond formation in female prairie voles (B. Gingrich and T. R. Insel, submitted). Oxytocin receptors are not detected in the NAcc of the nonmonogamous montane vole. A similar variability among individual prairie voles is found for V1a receptor binding in the cingulate cortex. We are currently investigating whether individual differences in receptor density are correlated with social behavioral phenotypes. Intraspecies variability in receptor densities could be due to genetic, hormonal, or environmental/social influences. Treatment with gonadal steroids does not reduce the individual variation in OTR density in the NAcc, and the variability is similar in males and females. One month of social isolation also does not reduce the variability. The mechanisms underlying these individual differences are a mystery and currently the focus of investigation. We have begun to investigate the molecular mechanisms underlying the species differences in OTR and V1a receptor patterns in the brain. In prairie and montane voles, there is a good correlation between ligand binding pattern and receptor mrna distribution (Young et al., 1996, 1997). Therefore, the species differences in receptor binding pattern are due to the fact that the OTR and V1a receptor genes are expressed in different brain regions in each species. In rather simple terms, a gene can be considered to be composed of two functional units: (1) the coding sequence, which determines the structure of the protein, and (2) the promoter, which contains transcriptional regulatory elements that control the expression of the gene. Several studies have demonstrated that sequences surrounding the coding region are responsible for determining the tissue-specific expression pattern of genes. As an example, transgenic studies have shown that the DNA sequences surrounding the OT gene are responsible for the expression of oxytocin in the magnocellular neurons of the paraventricular and supraoptic nuclei of the hypothalamus (Ho, Carter, Ang, and Murphy, 1995; Young III, Reynolds, Shepard, Gainer, and Castel, 1990). Typically, but not always, the sequences upstream, i.e., in the 5 -flanking region, of a gene contain the transcriptional elements that confer region-specific expression. We have examined the 5 flanking region of the OTR and V1a receptor genes of prairie and montane voles in order to gain insights into the evolution of receptor expression patterns. Initial experiments focused on the OTR. Oxytocin receptor clones were isolated from genomic libraries derived from prairie and montane vole tissues. The structure of the vole OTR coding sequence was similar to that reported for other species with 93% sequence homology in the coding region with the rat sequence (Young et al., 1996). Comparison of both the 5 -flanking region and coding sequences of the prairie and montane vole genes revealed differences in potential regulatory element sequences, a consensus IFN- site in the prairie sequence not found in the montane sequence, and an IL-6 site in the montane sequence not found in the prairie sequence; however, no dramatic differences in promoter structure were found (Young et al., 1996). Although IFN- and IL-6 elements are thought to modulate gene expression, neither has been shown to influence region-specific gene expression in the brain. It should be noted that only the first 1500 basepairs were examined in detail. In order to

5 216 Larry J. Young determine whether the 5 -flanking region of the OTR gene contained sequences responsible for tissue-specific expression in the brain, we created a reporter gene in which 5000 basepairs of the prairie vole OTR 5 -flanking region were spliced upstream to the bacterial lac-z gene. The lac-z gene encodes the enzyme -galactosidase, which, when expressed, produces a blue precipitate under the appropriate conditions. This transgene was injected into mouse embryos where it incorporated into the mouse genome. Of the four transgenic lines produced, one line expressed -galactosidase in several regions of the mouse brain, which, in the prairie vole brain, expresses the OTR, such as the cortex, lateral septum, amygdala, and VMN (Young, Waymire, Nilsen, Macgregor, Wang, and Insel, 1996; Young, Winslow, Wang, Gingrich, Guo, Matzuk, and Insel, 1997). The expression pattern was not identical to that of the prairie vole OTR since little -galactosidase was detected in the prelimbic cortex and some ectopic expression was found in the thalamus. Caution must be taken in interpreting transgenic experiments such as this because reporter gene constructs often result in ectopic expression and independently created lines using identical DNA constructs can result in different expression patterns. This is likely due to the fact that distant regulatory elements upstream of the promoter or located in introns or in the 3 -flanking region, which are missing in the construct, may also contribute to gene expression. In addition, transgene expression is influenced by the site of integration of the transgene in the genome. However, these results do suggest that DNA sequences upstream of the OTR coding region are capable of conferring tissue-specific expression of the OTR gene and therefore could potentially contribute to the species-specific pattern of gene expression. A more extensive comparison of the 5 - and 3 -flanking region of the OTR gene from montane and prairie voles may reveal more extensive differences in gene structure which could contribute to species differences in expression patterns. Nonetheless, this experiment demonstrates the ability of transgenic technology to manipulate receptor gene expression in a targeted manner. Comparison of the V1a receptor genes of prairie and montane voles has been more informative. The V1a receptor coding sequence is 98% identical between the vole species (Young et al., 1999). However, two major differences were found between the prairie and montane V1a receptor loci. First, while the montane vole genome has a single V1a receptor gene as expected, the prairie vole genome has two distinct loci, likely the result of a gene duplication (Fig. 3A). One of the prairie vole genes has a LINE element located in the 5 -flanking region. LINE elements are transposable DNA elements, making up 5% of the mammalian genome, which encode a reverse transcriptase similar to that of retroviruses (Evans and Palmiter, 1991). Recently, it has been shown that LINEs are capable of causing surrounding DNA sequences to be duplicated and translocated to different chromosomal regions, providing a mechanism for the evolution of the genome (Moran, Deberardinis, and Kazazian, 1999). The prairie vole V1a receptor gene may be an example of the evolutionary power of LINEs at the genomic level. One of the prairie vole V1a receptor loci has a single base mutation, resulting in a premature stop codon, and therefore a truncated receptor protein which may not be functional. Both prairie vole loci have a sequence stretch of approximately 460 basepairs located just 723 basepairs upstream of the transcription start site which is absent in the montane vole gene. This sequence appears to be an expansion of a short repetitive sequence which is found in the montane vole locus in this area. These data suggest that the ancestral prairie vole V1a receptor gene first accumulated the expansion in the 5 -flanking region and then duplicated, perhaps via a LINE dependent mechanism. Either the duplication or the accumulation of the additional promoter sequence near the transcription start site could dramatically impact the neuroanatomical pattern of receptor gene expression. Although at first it may seem that the duplication of the prairie vole V1a gene is not significant since one copy contains a premature stop, this process would result in one copy of the gene translocating to a new chromosomal environment. Since both the regulatory element surrounding the gene and the position of the gene in the genome influence expression, gene duplication could dramatically alter expression if the remaining functional gene is the translocated locus. Despite the differences in gene structure, we cannot rule out the possibility that species differences in the distribution of the transcription factors which interact with promoter elements could alone account for the species differences in V1a receptor expression patterns. We have not investigated this possibility in the voles. Since the monogamous pine vole has a pattern of V1a receptor binding that is similar to that of prairie voles, while the meadow vole pattern is similar to that of the montane vole, we also examined the 5 -flanking region of these species. Using PCR to amplify the 5 -flanking region in the pine and meadow vole V1a receptor genes we found that pine voles, like prairie

6 Oxytocin and Vasopressin Receptors 217 FIG. 3. (A) Structure of the montane and prairie vole vasopressin receptor genes. The prairie vole has two V1a receptor genes. Note the presence of the repetitive expansion in both prairie voles genes which is absent from the montane vole gene. The upper prairie vole gene was used to create transgenic mice. (B D) V1a receptor binding pattern in (B) prairie vole, (C) mouse, and (D) a mouse transgenic for the prairie vole V1a receptor gene. Note the similarity between the binding pattern of the prairie vole and the transgenic mouse (Young et al., 1999). voles, also have a similar 460-bp expansion sequence upstream of the transcription start site, while meadow voles do not (Young et al., 1999). These studies provide important clues as to the genetic mechanisms which may lead to the species differences in behaviorally relevant gene expression. If these changes in receptor expression result in differences in behavior, it would provide a potential molecular mechanism for the evolution of social behaviors. V1A RECEPTOR EXPRESSION AND SOCIAL BEHAVIOR Are the species differences in neuropeptide receptor expression behaviorally relevant? Pharmacological studies suggest that they may be. Prairie and montane voles differ in their behavioral response to exogenous AVP. Vasopressin injected icv increases male male aggression in a resident intruder paradigm of prairies, but not in montane voles (Young et al., 1997). Similarly, AVP increases social contact time in male prairie voles, but not in male montane voles (Young et al., 1999). Therefore, male prairie and montane voles exhibit different behavioral responses to exogenous AVP. This difference in response to AVP could be due to the different distribution of V1a receptors, although we cannot rule out other species differences in neural circuitry downstream of the receptor effects. However, this observation presents the hypothesis that the species-typical pattern of neuropeptide receptor expression is associated with a specific behavioral response to vasopressin. To test this hypothesis we again used a transgenic approach. Transgenic mice were created using a prairie vole V1a receptor minigene containing 2.2 kb of the 5 flanking region, both exons with the 2.5-kb intron, and 2.4 kb of the 3 -flanking region. Mice transgenic for the prairie vole V1a receptor gene exhibited a pattern of V1a receptor binding sites with many similarities to that found in the prairie vole, but different from that found in nontransgenic mice (Figs. 3B 3D). The density of V1a receptor binding in several brain regions was similar to that found in the prairie vole brain. The expression of the V1a receptor transgene did not alter oxytocin receptor expression since the distribution

7 218 Larry J. Young and density of OTR binding was identical in transgenic and nontransgenic mice (Young et al., 1999). Although the distribution of V1a binding in the transgenic mouse was similar to the prairie vole, there were some notable differences. For example, V1a binding in the hypothalamus and amygdala of the transgenic mouse was less intense than that of the prairie vole. In addition, since the transgenic mouse continued to express its endogenous V1a receptor gene, binding in the lateral septum of the transgenic mouse was higher than in the prairie vole, but similar to that of the wildtype mouse. Interestingly, the V1a binding in the hypothalamus was decreased in the transgenic mouse compared to that of the wildtype mouse. With in situ hybridization, we have detected antisense V1a transcripts in the lateral hypothalamus, indicating the transgene is being transcribed in the opposite direction in this region. Antisense mrna could potentially result in decreased translation of the endogenous mrna and hence a decrease in V1a binding. To determine whether the altered V1a receptor expression pattern altered the functional response to vasopressin, both transgenic and nontransgenic mice were cannulated and injected intraventricularly with 2 ng AVP. In a social behavioral testing paradigm similar to that used in the vole studies mentioned above, AVP increased the time that transgenic male mice spent in social contact with a tethered, ovariectomized female mouse (Fig. 4) (Young et al., 1999). The increased social contact consisted of an increase in time spent investigating and grooming the female. Vasopressin had no effect on the social interactions with the nontransgenic male mice. The increase in social contact appeared to be a specific increase in social interest rather than a general increase in olfactory investigation since AVP did not increase the time spent investigating a cotton ball scented with soiled bedding from the ovariectomized female cage or with lemon extract. Since AVP elicited increased social contact in both prairie voles and the transgenic mice, but not in montane voles or wildtype mice, and this behavior is likely influenced by olfactory processing, we examined V1a receptor binding in the olfactory bulbs of each group (Fig. 5). V1a receptor binding was detected in the mitrial and granular cell layers, but not in the external plexiform layers of the montane vole olfactory bulb. No V1a receptor binding was detected in any layer of the olfactory bulb of the wildtype mice. In contrast, high levels of V1a receptor binding were detected in all layers of both the prairie vole and transgenic mice. It is important to note that the transgenic mice do not form pair bonds or provide extensive paternal care FIG. 4. (A) Vasopressin increases the duration of affiliative behaviors directed toward an ovariectomized female in male transgenic mice with a prairie vole pattern of V1a receptor expression, but not in wildtype mice. (B) Vasopressin has no effect on olfactory investigation of cotton balls soiled with bedding from an ovariectomized female s cage in either transgenic or wildtype males. (Reprinted from Young et al., 1999). as do male prairie voles. This suggests that although the species-specific pattern of neuropeptide receptor may contribute to species typical behaviors, other factors are involved in complex social behaviors such as parental care and monogamy. For example, the neural mechanisms underlying social memory, the proper neuropeptide innervation pattern, and control of central release of peptide must complement the neuroanatomical distribution of peptide receptors. For example, differences in AVP fiber densities and release have also been noted among vole species with different social organization (Bamshad, Novak, and DeVries, 1994; Wang, Zhou, Hulihan, and Insel, 1996). Although the transgenic mouse approach is a pow-

8 Oxytocin and Vasopressin Receptors 219 CONCLUSION FIG. 5. V1a receptor binding in the main olfactory bulb of male montane voles, prairie voles, wildtype (Wt) mice, and mice transgenic for the prairie vole V1a receptor gene (Tg). Note the absence of binding in the olfactory bulb of wildtype mice and the similarity in binding between the prairie vole and the transgenic mice. Abbreviations: Epi, external plexiform layer; Mi, mitrial cell layer; IGr, granular cell layer. erful tool for manipulating gene expression, there are some limitations. First, it is difficult, if not impossible, to accurately predict the expression pattern of the transgene due to the complexity of the mechanisms controlling gene expression. It is difficult to control for the effects of ectopic expression, and expression may be lacking in crucial areas of interest. Second, in our mice, the contributions of both the endogenous expression and that of the prairie vole transgene must be considered. Also, most transgenic experiments are conducted with mice, which may limit the application of this technique for behavioral studies. For example, it is impossible to draw conclusions regarding the mechanisms underlying the differences in behavior between prairie voles and montane voles based on our comparisons with mice. Finally, since one cannot limit expression to individual areas, the olfactory bulbs, for example, it is of limited use for identifying the contribution of gene expression in specific brain regions. To address these issues, we have now developed an adeno-associated viral vector which allow us to transfer the V1a receptor gene into any brain region of an adult animal. This technology is not limited to mice and should extend the use of transgenic approaches to comparative studies. A growing number of studies are establishing that OT and AVP play important roles in the control of social and reproductive behaviors. However, the specific roles of these peptides in regulating behaviors may differ even among closely related species. The remarkable species differences in OTR and V1a receptor distribution and regulation likely contribute to the diversity in social behaviors of vertebrates. Perhaps there is something inherent in the genes of OTR and V1a receptors which facilitate this diversity in expression, leading to the evolution of new behavioral phenotypes. Further comparative studies of the behavioral roles of OT and AVP and the relationship with receptor distribution may reveal generalizations of neurohypophysial peptide function across species and aid in the elucidation of the roles of these peptides in human social and sexual behavior. ACKNOWLEDGMENTS Special thanks to David Crews and Thomas Insel for guidance and encouragement over the course of my career. Thanks to Zuoxin Wang for many discussions and collaborations in my work with the voles and to Roger Nilsen and Catherine Murphy for technical assistance. I also acknowledge the NIMH for support of this research. REFERENCES Albers, H. E., Liou, S. Y., and Ferris, C. F. (1988). Testosterone alters the behavioral response of the medial preoptic-anterior hypothalamaus to microinjection of arginine vasopressin in the hamster. Brain Res. 456, Bale, T. L., and Dorsa, D. M. (1995). Sex differences in and effects of estrogen on oxytocin receptor messenger ribonucleic acid expression in the ventromedial hypothalamus. Endocrinology 136, Bale, T. L., Dorsa, D. M., and Johnston, C. A. (1995). Oxytocin receptor mrna expression in the ventromedial hypothalamus during the estrous cycle. J. Neurosci. 15, Bamshad, M., Novak, M., and De Vries, G. J. (1994). Cohabitation alters vasopressin innervation and paternal behavior in prairie voles. Physiol. Behav. 56, Barberis, C., Balestre, M. N., Jard, S., Tribollet, S., Arsenijevic, Y., Dreifuss, J. J., Bankowski, K., Manning, M., Chan, W. Y., Schlosser, S. S., Holsboer, F., and Elands, J. (1995). Characterization of a novel, linear radioiodinated vasopressin antagonist: An excellent radioligand for vasopressin V1a receptors. Neuroendocrinology 62, Barberis, C., and Tribollet, E. (1996). Vasopressin and oxytocin receptors in the central nervous system. Crit. Rev. Neurobiol. 10, Bester-Meredith, J. K., Young, L. J., and Marler, C. A. (1999). Species

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