Oxytocin, vasopressin, and autism: is there a connection?

Oxytocin, Vasopressin, and Autism: Is There a Connection? Thomas R. Insel, Derek J. O’Brien, and James F. Leckman Autism is a poorly understood developmental disorder characterized by social impairment, communication deficits, and compulsive behavior. The authors review evidence from animal studies demonstrating that the nonapeptides, oxytocin and vasopressin, have unique effects on the normal expression of species-typical social behavior, communication, and rituals. Based on this evidence, they hypothesize that an abnormality in oxytocin or vasopressin neurotransmission may account for several features of autism. As autism appears to be a genetic disorder, mutations in the various peptide, peptide receptor, or lineage-specific developmental genes could lead to altered oxytocin or vasopressin neurotransmission. Many of these genes have been cloned and sequenced, and several polymorphisms have been identified. Recent gene targeting studies that alter expression of either the peptides or their receptors in the rodent brain partially support the autism hypothesis. While previous experience suggests caution in hypothesizing a cause or suggesting a treatment for autism, the available preclinical evidence with oxytocin and vasopressin recommends the need for clinical studies using gene scanning, pharmacological and neurobiological approaches. Biol Psychiatry 1999;45: 145–157 © 1999 Society of Biological Psychiatry Key Words: Receptor, polymorphism, affiliation, social memory, stereotypy

Introduction

A

utism remains one of the most challenging frontiers of psychopathology. Although the syndrome’s incidence is generally reported at less than 0.1%, its emergence early in life, its profound impact on families, and its chronic, treatment-refractory course have resulted in enormous emotional and financial costs (Bristol et al 1996). The absence of a specific, reliable medical treatment for autistic children has proven especially vexing. Over the past two decades successful new pharmacotherapies have been developed for most other major psychiatric illnesses,

From the Yerkes Regional Primate Research Center and Department of Psychiatry and Behavioral Sciences, Emory University, Atlanta, Georgia (TRI, DJO); and Yale Child Study Center, Yale University School of Medicine, New Haven, Connecticut (JFL). Address reprint requests to Thomas R. Insel, MD, Yerkes Primate Center, Emory University, Atlanta, GA 30322. Received January 26, 1998; revised March 25, 1998; accepted April 1, 1998.

© 1999 Society of Biological Psychiatry

but for autism little significant medical progress has been made since the syndrome was first described by Kanner in 1943 (Bailey et al 1996; Kanner 1943). There is general agreement between DSM-IV and ICD-10 over the major diagnostic criteria for autism (American Psychiatric Association 1994; World Health Organization 1993). These criteria define autism as a disorder of early childhood with the following triad of behavioral signs: 1) social impairment (lack of social reciprocity, decreased eye contact, failure to recognize the uniqueness of others); 2) communication abnormalities (delayed or incomplete language acquisition, deficits in both prelinguistic and verbal expression, decreased play); and 3) stereotyped behaviors (unusual attachments to objects, rigid adherence to routines or rituals, simple motor mannerisms such as hand flapping). Several aspects of this syndrome may prove especially important for investigating the neurobiology of autism. First, this is a developmental syndrome. The onset of autism is virtually always observed before 3 years of age, and often parents report abnormalities in social interest even in the first months of life (Lord 1995). Affected children show a range of cognitive deficits, with about 75% functioning at a retarded level (reviewed in Gillberg and Coleman 1992). As with many neurodevelopmental syndromes, boys are affected about 4 –5 times more often than girls (Gillberg and Coleman 1992). Together these observations suggest that the relevant neurobiological events may occur relatively early in the course of central nervous system (CNS) development, involving a cascade of complex gene– environment interactions. Second, autism has an important genetic component. Siblings have an incidence of 2.9 –3.7% (Bolton et al 1994; Jorde et al 1990; Szatmari and Jones 1991), representing nearly a 100-fold increased risk relative to the general population. Twin studies have found a concordance of 36 –91% in monozygotic twins compared to a ,1% concordance rate in dizygotic twins (Bailey et al 1995; Folstein and Rutter 1977; Steffenberg et al 1989). After pooling the data from two British twin samples and using the sibling rate of nontwin probands to estimate the dizygotic rate, Bailey et al calculated a heritability of 91–93% for autism (Bailey et al 1995). In addition, autism or autistic behavior is associated with several specific genetic disorders, including fragile X disorder (Bailey et al 0006-3223/99/$19.00 PII S0006-3223(98)00142-5

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1993; Gillberg and Wahlstro¨m 1985) and tuberous sclerosis (Gillberg et al 1994; Hunt and Shepherd 1993; Smalley et al 1992) as well as several rare chromosomal anomalies (reviewed in Gillberg and Coleman 1992). On the basis of the available evidence, Bailey et al (1996) have concluded that “genetic influences predominate in the aetiology of autism and, moreover, that this is likely to apply to the great majority of cases” (p 96). It should be noted, however, that the genetic component of etiology is likely to be complex, involving multiple genes that act additively or possibly act via genetic heterogeneity. In addition, the phenotype may extend beyond DSM-IV autism to include what Rutter has called the “lesser variant” (Rutter et al 1993). It seems likely that the putative genes do not cause autism; they increase the probability of developing one or more of the features of this complex disorder. Finally, in spite of broad recognition that autism is a neurodevelopmental syndrome, there is still no consistent neurochemical, neurophysiological, or neuroanatomical abnormality. Although several reports have noted hyperserotonemia in autism, there is little evidence for a specific abnormality of serotonin in the CNS, and treatments that target serotonin (e.g., fluvoxamine) have proven only modestly helpful (Gordon et al 1993; McBride et al 1996; McDougle et al 1996). Brain opiates have been implicated in social behavior, but there are no data demonstrating that these peptides are involved in autism, nor is there a clear therapeutic effect of opiate agonists or antagonists (Gillberg and Coleman 1992). Several neuroanatomic abnormalities have been noted by magnetic resonance imaging and by postmortem examination, but the significance of these findings and their relationship to the symptom complex remain to be demonstrated (Bailey et al 1996; Bauman 1991; Courchesne et al 1994). An etiologic theory of autism needs to account for the social, cognitive, and communication deficits, as well as the compulsive behavior of these children. In addition, this theory should address the early onset, predominance in boys, genetic loading, and subtle neuroanatomical anomalies observed in this disorder. In this paper, we suggest that abnormalities in the neural pathways for either oxytocin or vasopressin could account for many of these aspects of autism. This suggestion, which is based on animal studies of oxytocin and vasopressin effects on social and cognitive function (see, for instance, Insel 1992; Panksepp 1992), is enhanced by recent molecular studies of these neuropeptide systems in humans.

Oxytocin and Vasopressin as Candidate Neuropeptides Oxytocin (OT) and vasopressin (AVP), nine amino acid peptides synthesized in the hypothalamus, are released

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into the bloodstream via axon terminals in the posterior pituitary or neurohypophysis (hence their designation as neurohypophyseal peptides). The peptides are closely related structurally, differing at only two amino acids. Both are part of a family of nine amino acid peptides (nonapeptides) that can be traced phylogenetically to invertebrates (Gainer and Wray 1994). Ancestral nonapeptides have been implicated in various forms of nonmammalian reproductive behaviors such as nest building. Oxytocin and vasopressin are unique among this family in that they are found exclusively in mammals, probably evolving from the ancestral peptide arginine vasotocin, from which oxytocin and vasopressin each differ in only a single amino acid (Archer 1974). The traditional view of the neurohypophyseal peptides as endocrine hormones acting on peripheral organs has recently been revised to consider these peptides as neurotransmitters or neuromodulators, that is, peptides with central actions. Within the hypothalamus, oxytocin and vasopressin are synthesized in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). Not only do PVN cells synthesizing oxytocin and vasopressin project to diverse sites within the brain and brainstem (Sofroniew and Weindl 1981), receptors for both peptides have been found throughout the limbic system in the forebrain and autonomic centers in the brainstem (Barberis and Tribollet 1996). Furthermore, both peptides are released within the brain following chemical depolarization of the appropriate neurons, and fibers have been demonstrated at the ultrastructural level to make synaptic contacts in the CNS (Buijs and van Heerikhuize 1982). Thus, the evidence is quite strong that the brain is a target organ for these peptides. As vasopressin can bind to oxytocin receptors, and expression of oxytocin and vasopressin genes appears linked (see below), the most conservative approach to understanding how these peptides function in the brain is to discuss them together, rather than focusing on either one in isolation. Three aspects of the central pathways for oxytocin and vasopressin deserve special note. First, specific vasopressin pathways appear to be sexually dimorphic. In both rodents (De Vries and Buijs 1983) and primates (Wang et al 1997), extrahypothalamic vasopressin cells (e.g., cells in the bed nucleus of the stria terminalis) and their projections (e.g., lateral septum) are androgen-dependent and markedly more abundant in males. Second, neurotransmission for both peptides appears to depend largely on an unusual variability in their respective receptor. Oxytocin receptors are remarkably plastic, induced severalfold after gonadal steroid administration (Johnson et al 1990) and expressed in different brain regions even in closely related species (Insel and Shapiro 1992; Insel et al 1994a). In the hypothalamus, vasopressin receptors are

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Table 1. Central Effects of Oxytocin and Vasopressin Effects Social behaviors Initiate maternal care (rat) Paternal care (vole) Onset sexual receptivity (rat) Male sexual behavior (rat) Female pair bonding (vole) Male pair bonding (vole) Aggression (vole, hamster) Decrease isolation calls (rat) Cognition Active avoidance (rat) Passive avoidance (rat) Social memory (rat) Pup’s memory (rat) Stereotypies Grooming (rat, monkey) Flank-marking (hamster)

OT

OTA

AVP

V1A

111 ? 111

— ? —

1 111 —

? — 111

111 111

— —

? 0

? 0

0 0

0 0

111 111

— —

111

0

111

0

— — 111/— 111

0 0 ? —

111 111 111 ?

— — — ?

111 ?

— ?

111 111

0 —

References are provided in text. OT, oxytocin; OTA, oxytocin antagonist; AVP, vasopressin; V1A, antagonist at V1a receptor. 111 indicates facilitation of behavior; — indicates inhibition of behavior or, in some cases, blockage of agonist effect; 0 indicates no effect; ? indicates no data available.

sexually dimorphic and dependent on both steroids and photoperiod (Dubois-Dauphin et al 1991; Johnson et al 1995). Finally, both oxytocin and vasopressin receptors are developmentally regulated and expressed more in the immature brain (Shapiro and Insel 1989; Tribollet et al 1989, 1991), and both peptides have been shown to have effects on neural development (Boer 1991, 1993). Vasopressin can be detected by embryonic day 16 in the developing rat brain and increases to high levels prior to birth. Oxytocin is also found in the fetal brain, although in the rat it is not processed to its mature, amidated form until after birth (Whitnall et al 1985). It has long been known that vasopressin has mitogenic effects on various cell lines and can alter neurite outgrowth (reviewed in Boer and Swaab 1985). Chronic perinatal administration of both vasopressin and oxytocin has been associated with altered adult brain weight, with the most significant and persistent changes observed in the cerebellum (Boer 1991). More than three decades of research indicates that both peptides have important cognitive and behavioral effects (Argiolas and Gessa 1991; de Wied et al 1993) (Table 1). In 1965, De Wied reported that rats trained to avoid one side of a shuttle box appeared to “forget” this training following removal of the posterior–intermediate lobes of the pituitary unless treated with a vasopressin analogue (de Wied 1965). Several subsequent studies have demonstrated that vasopressin and oxytocin modulate learning and memory processes (reviewed in Kovacs and Telegdy

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1985). In general, vasopressin facilitates learning of both active and passive avoidance behavior in a dose-dependent fashion. The peptide appears to influence both the consolidation and retrieval phases of learning, possibly with different metabolites affecting consolidation and retrieval independently. Although many of the early studies were based on peripheral administration of vasopressin, it is now clear from studies using central administration of the peptide that vasopressin effects on learning are mediated by a central receptor, probably in the hippocampus (reviewed in de Wied et al 1993). Oxytocin’s effects on learning are also dose-dependent, but generally opposite to vasopressin, with an inhibition of extinction in both passive and active avoidance paradigms. Although the majority of patients diagnosed with autism also show mental retardation, the nature of the cognitive deficit is complex and not simply a defect in consolidation or retrieval (Rutter 1983). Paradoxically, although vasopressin facilitates learning in adults, vasopressin given during gestation has been reported to confer long-term deficits in memory (in male subjects) (Tinius et al 1987). The mechanism of these effects is unclear, as the increase in vasopressin undoubtedly alters maternal physiology with a number of potential effects on fetal perfusion and nutrition.

Social Behavior One form of memory that may be especially relevant to autism is the recognition of a familiar conspecific. A rodent test of this form of recognition, termed “social memory” was developed by Thor and Holloway (1982) and has been used by Dantzer et al (1987) as well as others (Popik et al 1992) to determine the role of vasopressin and oxytocin. After a brief exposure to a novel juvenile, a male rat may recognize this juvenile after 60 min but not after a 120-min interval. Vasopressin appears to facilitate and a vasopressin (V1a) receptor antagonist inhibits the consolidation of a social memory (Dantzer et al 1987). A rat treated with vasopressin after being briefly exposed to a novel juvenile appears to recognize this juvenile 120 min later, whereas treatment with a vasopressin antagonist leads to no evidence of recognition when tested 30 min postexposure. Vasopressin’s effects on social memory have been described only in male rats and appear to be androgen-dependent (Bluthe et al 1990). In contrast to vasopressin’s effects on avoidance behavior, which are believed to be mediated via the hippocampus, effects on social memory involve the lateral septum (Dantzer et al 1988), which receives more vasopressinergic innervation in males. Oxytocin, although initially reported to inhibit social memory (Dantzer et al 1987), has more recently been shown to facilitate social memory at low doses

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(Popik et al 1992). Oxytocin knockout mice show a remarkable deficit in social memory, although performance on spatial memory and olfactory discrimination tasks is unimpaired (author’s unpublished data). In a recent experiment with rat pups, oxytocin was shown to facilitate conditioning to olfactory cues associated with the mother (Nelson and Panksepp 1996). These results suggested that oxytocin may not be essential for recognizing the mother, but that the peptide was critical for learning about factors associated with the mother. Curiously, as with the selective social memory deficits of oxytocin knockout mice, other forms of learning in rat pups, such as conditioning to odors associated with nonsocial stimuli, did not appear to be oxytocin-dependent. A growing body of evidence has implicated oxytocin and vasopressin in the central mediation of sociosexual behaviors (Carter 1992; Insel 1992; Witt 1995). Oxytocin, given chronically by ICV minipump, increases affiliation, as measured by the amount of time rats spend in side-byside contact (Witt et al 1992). Oxytocin has been demonstrated to facilitate both maternal and sexual behaviors in rats (and maternal behavior in sheep). Both rats and sheep shift from avoiding to approaching neonates just prior to parturition. Oxytocin appears to be important for this transition to maternal behavior, but is not essential once this behavior has been initiated (Insel 1990). Vasopressin can also facilitate the emergence of maternal behavior (Pedersen et al 1982), but it appears weaker than oxytocin and its effects on female sexual behavior may be opposite to oxytocin (So¨dersten et al 1983). Vasopressin has also been implicated in paternal behavior (Wang et al 1993) and, consistent with nest-defense, appears to facilitate certain forms of aggression (Ferris 1992; Winslow et al 1993a). Perhaps most relevant to autism, both peptides have been implicated in the development of social attachments. In monogamous prairie voles that form pair bonds, oxytocin has been shown to be both necessary and sufficient for the normal development of a partner preference in females (Insel and Hulihan 1995). In this species, pair bonds normally develop as a consequence of mating. For females, central administration of an oxytocin antagonist prevents pair bonding without inhibiting mating, while central administration of oxytocin facilitates pair bonding even in the absence of mating. Vasopressin appears to influence an analogous process of pair bond formation in males (Winslow et al 1993a).

Communication As language is restricted to humans, there are obvious limitations to the study of linguistic abnormalities in nonhuman animals. Nevertheless, other species communicate using visual, olfactory, and audiovocal cues. Some of

these species-typical forms of communication appear to be influenced by the nonapeptides. For instance, flank-marking, a form of social communication observed in golden hamsters, is used by a dominant male to mark his territory. Flank marking can be elicited by microinjection of vasopressin into the lateral hypothalamus and is inhibited by administration of a vasopressin (V1a) receptor antagonist (Ferris et al 1984, 1988, 1993). Similar effects, including an increase in scent marking and auto-grooming, have been observed in squirrel monkeys but only when socially isolated (Winslow and Insel 1991a, 1991b). In most infant mammals, social separation results in a high-frequency distress call. In the infant rat, these calls, which are ultrasonic, are a potent stimulus for maternal retrieval. Central administration of oxytocin and vasopressin reduces the isolation calls of infant rats in a dosedependent fashion (Insel and Winslow 1991; Winslow and Insel 1993). This potent central effect of these peptides may be related to the transient, exuberant expression of oxytocin and vasopressin receptors in the developing cortex (especially in cingulate). In adult male canaries, the related nonapeptide vasotocin induces singing behavior (Voorhuis et al 1991). This effect is complex, as vasotocin’s effects on singing depend on the season and apparently on the gonadal status of the male bird.

Rituals Both peptides have been shown to induce stereotypic behaviors such as stretching, repetitive grooming, startle, and squeaking responses when administered ICV to mice (reviewed in Meisenberg and Simmons 1983). Grooming, particularly grooming of the genital regions, is consistently observed following ICV administration of oxytocin in rats (Drago et al 1986; van Wimersma Greidanus et al 1990). In chicks, oxytocin induces wing-flapping (Panksepp 1992). Repeated central administration of vasopressin induces seizures in rats, including so-called “barrelrolling” seizures that may be fatal. Although seizures have been reported in 25% of patients with autism, there appear to be no distinctive features of these seizures that make them unique to autism (reviewed in Gillberg and Coleman 1992). Self-injurious behavior is also a common feature of autism. It is certainly possible that the increase in aggression and self-directed behaviors observed in rodents after central administration of vasopressin could be related to the self-injurious behavior, often stereotyped, observed in autistic children.

Summary In summary, results from nonhuman animal studies suggest that these neuropeptide systems influence behaviors

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that are abnormal in autism, including deficits in social, cognitive, and communicative behaviors as well as motor stereotypies. In addition, the early abundance of oxytocin and vasopressin receptors is consistent with a developmental role, and the sexual dimorphism of vasopressin pathways might be related to the male preponderance of this disorder; however, there are problems extrapolating the animal data to autism. The direction of the effect is not always consistent; these peptides facilitate social behavior and learning (suggesting a nonapeptide deficit in autism) but also induce grooming and stereotypic behavior (suggesting an excess). Although vasopressin induces flankmarking, the neurobiological relationship of olfactory communication to human language is highly speculative. In addition, the developmental effects are confusing. Vasopressin administered during gestation has been reported to inhibit rather than facilitate subsequent cognitive performance. Perhaps some resolution of these apparent contradictions resides in the transition from adult pharmacology (administering microgram quantities of peptide) to developmental pathophysiology (selective deficits rather than excesses through early development). If autism is a genetic disorder, then one might expect that some alteration in one of these two neuropeptide systems throughout development would contribute to this disorder. Over the past decade, we have gained a greater understanding of the molecular biology of both these peptides and their receptors, providing several potential genetic mechanisms for autism.

Potential Genetic Mechanisms The search for a genetic mechanism involving oxytocin or vasopressin neurotransmission logically breaks down into studies of the genes for these peptides, genes for their receptors, and developmental genes that specify for oxytocin and vasopressin pathways (see Table 2).

Peptide Genes The genes for oxytocin and vasopressin have been among the most intensively studied in neuroendocrinology. First cloned and sequenced in 1984, these genes were localized to chromosome 20 in the human genome, where they are arranged in antiparallel orientation with a long interspersed repeated DNA element (LINE) (reviewed in Gainer and Wray 1994). The genes for both peptides consist of three exons (and two introns) with little change in structure across species from mouse to human. Transgenic studies with the rat oxytocin gene demonstrate that oxytocin expression is dependent upon sequences within or near the vasopressin gene, as only transgenes containing both oxytocin and vasopressin direct cell-specific expres-

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Table 2. Human Genes for OT, AVP, and Their Receptors Gene

Chromosomal location

Polymorphism

OT

20

?

AVP

20

Yes

OTR

3p25–26

Yes

V1a

12

Yes

V1b

?

?

V2

Xq28

Yes

Comments OT knockout mice show social deficits Brattleboro rats show cognitive deficits, brain anomalies Receptor distribution associated with affiliative behavior Receptor distribution associated with affiliative behavior May be located in brain as well as pituitary Mutations associated with nephrogenic diabetes insipidus

sion. This suggests that the genes are closely linked and that a single critically placed mutation could influence expression of both oxytocin and vasopressin. Rodents with mutations of oxytocin and vasopressin have been studied for behaviors relevant to autism. We have recently described a mouse with a null mutation of the oxytocin gene (i.e., an oxytocin knockout mouse) (Nishimori et al 1996). Homozygous mice completely lack oxytocin and are unable to lactate. These mice show deficits in social behavior, including decreased social investigation, increased aggression, and decreased vocal response to social separation (author’s unpublished data). As noted above, these mice also show selective deficits in social memory. The consequences of vasopressin deficiency have been studied in the Brattleboro rat, which fails to make vasopressin because of a point mutation leading to a frameshift in the vasopressin gene. In addition to diabetes insipidus, these rats show various cognitive deficits, including impaired extinction of avoidance behavior and decreased social memory (de Wied et al 1988). Brattleboro rats also show a number of neurochemical and neuroanatomical abnormalities, including reduced catecholamine concentrations and smaller brain volume (Boer et al 1982). Brattleboro rats have been reported to show increased oxytocin synthesis, suggesting that some of the observed abnormalities in these animals may not be a direct effect of the deficiency in vasopressin (Boer et al 1988). Neither the oxytocin knockout mouse nor the Brattleboro rat is a convincing animal model of autism, but each exhibits key features of the syndrome. There are no reported cases of human oxytocin gene mutations, although a family with multigenerational problems with lactation has been recently suggested for study (Hans Zingg, personal communication, August, 1997). A

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series of pedigrees with mutations of the human AVP gene have been recently described (Rittig et al 1996). Although the affected individuals have diabetes insipidus, behavioral and cognitive abnormalities have not been assessed.

Receptor Genes There are four known genes in the neurohypophyseal receptor family: oxytocin or OTR (uterus, mammary tissue, and brain), V1a (liver and brain), V1b or V3 (pituitary), and V2 (kidney). All of these have been cloned and sequenced in the past 5 years (reviewed in Barberis and Tribollet 1996; Zingg 1996). These genes are approximately equivalent in size, with 40 – 60% homology in their amino acid sequences. Each codes for a receptor that belongs to the seven transmembrane spanning domain, G-protein coupled receptor family. Large regions, particularly within the transmembrane domains and the second and third extracellular loops, are conserved across the four different OT/AVP receptors. In addition to ligand binding, coupling to G proteins differs across this group. The OTR, V1a, and V1b receptors use Gq/11 to induce phosphoinositol hydrolysis and increase intracellular calcium. The V2 receptor is coupled to Gs to stimulate adenylate cyclase. By far, the greatest attention has been focused on the V2 receptor gene. Over 40 mutations (mostly point mutations) in this gene have been shown to be associated with nephrogenic diabetes insipidus (Merendino et al 1993; Rosenthal et al 1992; Seibold et al 1992). While it seems likely that the other genes in this family might reveal similar functional sequence variability, there has been only limited study of the OTR (Michelini et al 1995) and V1a receptor genes (Thibonnier et al 1994). The human OTR gene is roughly 17 kb, with four exons and three introns (Inoue et al 1994). In the human genome, the OTR gene is localized to chromosome 3p25–3p26 in a region near the loci for Von Hippel–Lindau disease and renal cell carcinoma (Kimura et al 1994; Michelini et al 1995). The promoter sequence for this gene includes several cytokine-responsive elements. A polymorphic tandem repeat sequence in the 39 flanking region of this gene has been described, but not yet characterized functionally (Michelini et al 1995). This area, downstream from the coding sequence, shows sequence variability in an area where the nucleotides cytosine and adenine (CA) are repeated 30 times in most subjects but 28 times in others. As this is not in a regulatory or coding region of the gene, this variability may be entirely insignificant. In other species, this gene has shown remarkable sequence variability, particularly in the promoter region around a tandem repeat sequence 100 – 400 bp upstream from the start site. Although the OTR complementary DNA, which encodes a 388 amino acid polypeptide shows relatively

little difference across species, the sequence variability in the promoter region may account for the profound differences in this receptor’s regional expression in the brains of different mammals (Young et al 1996). For instance, monogamous prairie voles and nonmonogamous montane voles show markedly different patterns of oxytocin receptor distribution in brain (Insel and Shapiro 1992) and as a consequence, respond differently to exogenous peptide (Winslow et al 1993b). A similar variation in the human OTR promoter might lead to changes in the distribution or the developmental regulation of the receptor protein in brain. As a result, a different set of targets would be influenced within the brain, and attachment behavior or cognitive function could be impaired even in the presence of normal oxytocin concentrations. The V1a receptor gene has also been sequenced in several species, including humans (Thibonnier et al 1994). The human V1a receptor gene encodes a 394 amino acid protein and has been studied extensively by transfection into CHO cells (Thibonnier et al 1994). Polymorphisms for the human V1a receptor have been detected in the second intron, and three tandem repeat microsatellites have been found in the 59 flanking region (Thibonnier, personal communication, December, 1996). As with the oxytocin receptor, the V1a receptor also shows marked species variability in the promoter region across species (Young et al unpublished) and even more striking species differences in regional expression (Insel et al 1994b). For instance, the V1a receptor is most heavily expressed in the lateral septum, bed nucleus of the stria terminalis, lateral hypothalamus, and ventral thalamus within the rat brain, yet shows a broad cortical distribution in the primate brain with most intense expression in both cortical and subcortical regions important for memory, such as entorhinal cortex, mammillary bodies, insula, and cingulate cortex (Toloczko et al 1997). Curiously, although there is only a modest homology between neurohypophyseal peptide receptor subtypes, point mutation experiments have shown that changing only a single amino acid residue within the agonist binding domain in the transmembrane part of the V1a receptor results in a shift of binding specificity toward the oxytocin subtype (Chini et al 1995). Therefore, one might expect that a mutation in this region would confer significant functional consequences, just as a change in the promoter sequence appears to be associated with important differences in regional distribution.

Developmental Genes It is also worth considering that developmentally regulated genes involved in cell lineage determination of the PVN or functionally related areas may be altered in autism. A fundamental aspect of neurogenesis is the requirement for

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precise spatial and temporal coordination of sequential events that underlie the appearance of mature cellular phenotypes. Some of the genes active in the formation of the hypothalamus have been characterized. Many of these genes code for transcription factors that are likely to regulate the expression of downstream effector genes. Some of the most intriguing hypothalamic transcription factors that have been identified thus far are in a family of recently discovered proteins called POU proteins. Genes coding for proteins in this family, including Brn-1, Brn-2, and Brn-4, are expressed in magnocellular neurons of the PVN and SON (Rosenfeld et al 1996) and are capable of binding to the distal 59 flanking region of the oxytocin gene, meaning that they may influence transcription. In a remarkable experiment, mice engineered to lack the Brn-2 gene fail to develop a mature PVN and SON (Schonemann et al 1995). Other less well-characterized homeoboxcontaining genes (i.e., genes with homeobox sequences that may be important for determining polarity or orientation in the developing neuraxis) including dbx and otp are also expressed in the anterior hypothalamus, as well as other regions that receive oxytocin and vasopressin projections (Lu et al 1992, 1994; Simeone et al 1994). Alterations in these genes may have the potential to disrupt the normal genetic program, leading to dysfunctional outcomes in anatomically discrete circuits. In summary, if an alteration in oxytocin or vasopressin neurotransmission accounts for the pathophysiology of autism, and autism is a genetic disease, one might expect to find an associated allele in either the oxytocin or vasopressin genes or the genes for their neural receptors (OTR and V1a). Animals with mutations of either the oxytocin or vasopressin genes exhibit some of the features of autism (social deficits in the oxytocin knockout mouse, memory deficits in the vasopressin-deficient rat), but also demonstrate abnormalities not observed in autism (e.g., diabetes insipidus in the Brattleboro rat). Experience with the V2 receptor (localized in kidney) demonstrates that at least one member of this family is subject to functional mutations. What would be the result of a similar alteration in the receptors expressed in brain? We do not yet have animals with null mutations for the OTR or V1a receptors, but comparative studies demonstrate marked species differences in both the promoter regions of these receptor genes and the patterns of expression of these receptors in brain. As species with different patterns of receptor distribution also differ in patterns of social affiliation, we have previously suggested that either the OTR or V1a receptor may represent a molecular basis for the capacity to form selective, enduring social attachments, as seen in monogamous mammals (Insel et al 1996). Given the profound species differences in OTR and V1a receptor distribution, even among primates (Toloczko et al 1997),

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one might expect to find within-species variability in the expression of one or both receptors in the human brain, with associated changes in social behavior.

Oxytocin and Vasopressin in Human Psychopathology Several of the results from animal studies suggest that human diseases involving abnormal social attachments could be due to abnormalities of oxytocin or vasopressin pathways. Although there has been remarkably little research on oxytocin or vasopressin in autism (see below), there are several studies that have reported abnormalities of central oxytocin or vasopressin in neuropsychiatric populations (Table 3). The evidence for abnormality in cerebrospinal fluid (CSF) oxytocin or vasopressin in schizophrenia is not compelling (Beckmann et al 1985; Glovinsky et al 1994; Linkowski et al 1984), but a recent postmortem study by Mai et al has described abnormal morphometric profiles of neurophysin-stained cell bodies and terminals in untreated schizophrenics. It is not clear if these abnormalities are specific to schizophrenia or restricted to either oxytocin or vasopressin pathways (Mai et al 1993). Similarly, results from measuring CSF oxytocin or vasopressin concentrations in either untreated (Gjerris et al 1985; Linkowski et al 1984) or treated (Pitts et al 1995) affectively ill patients have not been consistent across studies. More intriguing results emerged in a recent postmortem study that found a 56% increase in vasopressin-positive cells and a 23% increase in oxytocin-positive cells in the hypothalamus of depressed subjects (Purba et al 1996). This study used stereologic techniques to quantify immunoreactive cells in the PVN for 8 depressed patients (all medicated) and 8 age-matched controls (age 23– 88 years). As this same group previously reported no change in the number of vasopressin cells in the PVN or SON with Alzheimer’s disease (Van der Woude et al 1995), a 40% decrease in the number of oxytocin cells but no change in the number of vasopressin cells in AIDS (Purba et al 1993), and a 42% decrease in the number of oxytocin cells in Prader–Willi syndrome (Swaab et al 1995), the findings in depression appear relatively selective. Perhaps the most surprising clinical data suggest that obsessive– compulsive disorder (OCD) patients may have increased oxytocin concentrations in CSF, although this finding appears to be limited to OCD subjects without tics (Leckman et al 1994). One other study including a broad spectrum of OCD subjects found a difference in CSF vasopressin levels relative to normals (Altemus et al 1992), and another found CSF vasopressin to be negatively correlated with OCD symptoms while CSF oxytocin

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Table 3. Oxytocin and Vasopressin in Neuropsychiatric Disorders Disorder Schizophrenia

Affective disorders

OCD

Prader–Willi AIDS Alzheimer’s

Finding

Reference

Decreased NPI (AVP), increased NPII (OT) Increased CSF OT No change in CSF OT Altered neurophysin profiles CSF NPI (AVP) decreased UP, increased BP CSF NPII (OT) no change UP, increased BP Decreased CSF AVP No change in CSF AVP or OT with medication 56% increase of AVP and 23% increase of OT in PVN Increased CSF OT, normal CSF AVP (nontic group) Increased CSF AVP Negative correlation of CSF AVP and severity Positive correlation of CSF OT and depression Decreased OT cells, no change in AVP Decreased OT cells, no change in AVP No change in OT or AVP cells

(Linkowski et al 1984) (Beckmann et al 1985) (Glovinsky et al 1994) (Mai et al 1993) (Linkowski et al 1984) (Linkowski et al 1984) (Gjerris et al 1985) (Pitts et al 1995) (Purba et al 1996) (Leckman et al 1994) (Altemus et al 1992) (Swedo et al 1992) (Swedo et al 1992) (Swaab et al 1995) (Purba et al 1993) (Van der Woude et al 1995)

NP, neurophysin; AVP, vasopressin; OT, oxytocin; CSF, cerebrospinal fluid; UP, unipolar; BP, bipolar; PVN, paraventricular nucleus of hypothalamus.

concentrations were correlated with severity of depressive symptoms in this disorder (Swedo et al 1992). It is not immediately clear how these evolving findings with OCD relate to an abnormality with social attachments, but the evidence that these neuropeptides may stimulate grooming behavior and may influence the extinction of avoidance behaviors could provide a link between oxytocin or vasopressin and OCD. A controlled trial of intranasal oxytocin in 12 OCD patients found no significant effects on obsessions or compulsions (Den Boer and Westenberg 1992).

Oxytocin and Vasopressin in Autism Analogous data are not yet available for autism. A recent study reported that the plasma concentration of oxytocin in autistic children is about half that observed in healthy age-matched control subjects, and that autistic children failed to show the normal developmental increase of plasma oxytocin with age or interpersonal skills (Modahl et al 1998), consistent with a genetic deficiency of oxytocin. Although there is little relationship of blood oxytocin to CSF concentrations, a genetic defect in oxytocin synthesis might be expected to lower both pools. To date, CSF data on oxytocin and vasopressin in autism have not been reported. Systemic administration of synthetic oxytocin to autistic children reportedly increases social interaction (Hollander, oral communication, December, 1996), which could also support the notion of an oxytocin deficit in autism. It is not clear, however, that systemic peptide crosses into the brain. In short, there is no current compelling clinical evidence that central pathways for either oxytocin or vasopressin are important for this disorder.

Nevertheless, the available results from preclinical research suggest that these pathways are critical for normal social behavior and memory, and that these pathways are a reasonable place to look for abnormalities in autism. Specifically, we hypothesize that abnormalities in either the OTR or V1a receptor genes, or in developmental genes active in the specification of oxytocin or vasopressin pathways, are involved in the etiology of some forms of autism. This hypothesis is supported by the results of comparative research, the discovery of polymorphisms in both receptor genes, and the anatomical localization of these receptors in the primate brain to sites important for social behavior and cognition. Although the animal studies may prove important in guiding the search for what to look for and where to look in humans with impaired attachment behavior, the tests of this hypothesis remain to be done in people with autism. Traditional studies of plasma or CSF concentrations will be important if there is a mutation in either the peptide genes or developmental genes critical for hypothalamic differentiation. Normal concentrations of oxytocin or vasopressin will not preclude a mutation of the receptor genes. As the changes in very discrete clusters of receptors appear important for regulating social behaviors in animal studies, it seems likely that measures of CSF or plasma hormone concentrations will not be definitive in human neuropsychiatric illness. Three techniques will be important. • First, the study of candidate genes should allow the detection of sequence variations in the OTR and V1a receptor genes in DNA from autistic patients. As both OTR and V1a receptors can be expressed in cell

Oxytocin, Vasopressin, and Autism

lines, the functional significance of any variation in sequence can be determined. Association studies will be necessary to determine if functional alleles are relevant to some subset of patients with autism (Risch and Merikangas 1996). If sequence variations in the OTR and V1a receptor genes are not associated with autism, then the candidate gene search should be extended to the peptide genes and the family of developmental genes that specify for differentiation of the endocrine hypothalamus. • Second, pharmacologic techniques can be used to assess the responsiveness of OTR and V1a receptors in brain. Although systemically and intranasally administered peptides are unlikely to cross the blood– brain barrier, the recent development of nonpeptide ligands for both receptors provides an opportunity for assessing central receptors after peripheral administration (Evans et al 1993; Imaizumi et al 1992). • Finally, neuroanatomical studies should prove informative. We know that both receptors are highly variable in their regional expression. Localization of these receptors, either in vivo with positron-emission tomography imaging or in vitro with receptor autoradiography, will be an important test of the hypothesis. It may also be useful to complete postmortem studies of the PVN in autism and compare both messengerRNA and protein levels to previous reports in depression, AIDS, and Prader–Willi syndrome. Although the discovery of an etiology for autism would be an important advance, the development of a treatment of a genetic defect of oxytocin or vasopressin neurotransmission may prove an even greater challenge. Thus far, we have been successful in developing nonpeptide analogues to antagonists but not to agonists (Manning et al 1995), so replacement therapy remains a problem. If the receptor is either not responsive or expressed in an abnormal pattern, replacement therapy will not suffice. We have recently described a transgenic mouse with an altered promoter sequence capable of guiding OTR expression to specific brain sites (Young et al 1997). Using a similar gene therapy strategy, it may soon be possible to induce receptor expression in a predictable pattern based on the construction of a normal receptor gene. In summary, molecular, cellular, and behavioral studies demonstrate a role for oxytocin and vasopressin neural pathways in species-typical social behavior, cognition, communication, and motor stereotypies. We hypothesize that these neural systems are involved in autism. Experience with the structurally related V2 receptor that is found in the kidney demonstrates that this family of receptor genes is prone to functional mutations. We predict that similar mutations in the OTR and V1a receptors, or in relevant developmental genes expressed in the brain, will

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be found in autism. We now have the tools with which to test this prediction. If this prediction is borne out, even in a subset of patients with autism, a strategy for treatment can be devised to increase either oxytocin or vasopressin neurotransmission.

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