Allison M.J. Anacker

The serotonin system in autism spectrum disorder: From biomarker to animal models

Elevated whole blood serotonin, or hyperserotonemia, was the first biomarker identified in autism spectrum disorder (ASD) and is present in more than 25% of affected children. The serotonin system is a logical candidate for involvement in ASD due to its pleiotropic role across multiple brain systems both dynamically and across development. Tantalizing clues connect this peripheral biomarker with changes in brain and behavior in ASD, but the contribution of the serotonin system to ASD pathophysiology remains incompletely understood. Studies of whole blood serotonin levels in ASD and in a large founder population indicate greater heritability than for the disorder itself and suggest an association with recurrence risk. Emerging data from both neuroimaging and postmortem samples also indicate changes in the brain serotonin system in ASD. Genetic linkage and association studies of both whole blood serotonin levels and of ASD risk point to the chromosomal region containing the serotonin transporter (SERT) gene in males but not in females. In ASD families with evidence of linkage to this region, multiple rare SERT amino acid variants lead to a convergent increase in serotonin uptake in cell models. A knock-in mouse model of one of these variants, SERT Gly56Ala, recapitulates the hyperserotonemia biomarker and shows increased brain serotonin clearance, increased serotonin receptor sensitivity, and altered social, communication, and repetitive behaviors. Data from other rodent models also suggest an important role for the serotonin system in social behavior, in cognitive flexibility, and in sensory development. Recent work indicates that reciprocal interactions between serotonin and other systems, such as oxytocin, may be particularly important for social behavior. Collectively, these data point to the serotonin system as a prime candidate for treatment development in a subgroup of children defined by a robust, heritable biomarker.

Life in groups: the roles of oxytocin in mammalian sociality.

In recent decades, scientific understanding of the many roles of oxytocin (OT) in social behavior has advanced tremendously. The focus of this research has been on maternal attachments and reproductive pair-bonds, and much less is known about the substrates of sociality outside of reproductive contexts. It is now apparent that OT influences many aspects of social behavior including recognition, trust, empathy, and other components of the behavioral repertoire of social species. This review provides a comparative perspective on the contributions of OT to life in mammalian social groups. We provide background on the functions of OT in maternal attachments and the early social environment, and give an overview of the role of OT circuitry in support of different mating systems. We then introduce peer relationships in group-living rodents as a means for studying the importance of OT in non-reproductive affiliative behaviors. We review species differences in oxytocin receptor (OTR) distributions in solitary and group-living species of South American tuco-tucos and in African mole-rats, as well as singing mice. We discuss variation in OTR levels with seasonal changes in social behavior in female meadow voles, and the effects of OT manipulations on peer huddling behavior. Finally, we discuss avenues of promise for future investigation, and relate current findings to research in humans and non-human primates. There is growing evidence that OT is involved in social selectivity, including increases in aggression toward social outgroups and decreased huddling with unfamiliar individuals, which may support existing social structures or relationships at the expense of others. OT’s effects reach beyond maternal attachment and pair bonds to play a role in affiliative behavior underlying “friendships”, organization of broad social structures, and maintenance of established social relationships with individuals or groups.

Prairie voles as a novel model of socially facilitated excessive drinking

Social relationships strongly affect alcohol drinking in humans. Traditional laboratory rodents do not exhibit social affiliations with specific peers, and cannot adequately model how such relationships impact drinking. The prairie vole is a socially monogamous rodent used to study social bonds. The present study tested the prairie vole as a potential model for the effects of social affiliations on alcohol drinking. Same‐sex adult sibling prairie voles were paired for five days, and then either separated into individual cages, or housed in pairs. Starting at the time of separation, the voles received unlimited access to alcohol in a two‐bottle choice test versus water. Pair‐housed siblings exhibited higher preference for alcohol, but not saccharin, than singly housed voles. There was a significant correlation between the amount of alcohol consumed by each member of a pair when they were housed together (r = 0.79), but not when housed apart (r = 0.20). Following automated analysis of circadian patterns of fluid consumption indicating peak fluid intake before and after the dark phase, a limited access two‐hour two‐bottle choice procedure was established. Drinking in this procedure resulted in physiologically relevant blood ethanol concentrations and increased Fos immunoreactivity in perioculomotor urocortin containing neurons (but not in nucleus accumbens or central nucleus of the amygdala). The high ethanol preference and sensitivity to social manipulation indicate that prairie voles can serve to model social influences on excessive drinking.

Alcohol Intake in Prairie Voles is Influenced by the Drinking Level of a Peer

BACKGROUND Peer interactions can have important effects on alcohol-drinking levels, in some cases increasing use, and in other cases preventing it. In a previous study, we have established the prairie vole as a model animal for the effects of social relationships on alcohol intake and have observed a correlation of alcohol intake between individual voles housed together as pairs. Here, we investigated this correlated drinking behavior, hypothesizing that 1 animal alters its alcohol intake to match the drinking of its partner. METHODS Adult prairie voles were tested for baseline drinking levels with continuous access to 10% alcohol and water for 4 days. In Experiment 1, high alcohol drinkers (>9 g/kg/d) were paired with low alcohol drinkers (<5 g/kg/d) of the same sex on either side of a mesh divider for 4 days with continuous access to the same 2-bottle choice test. In Experiment 2, high drinkers were paired with high drinkers and low drinkers paired with low drinkers. In both experiments, animals were again separated following pairing, and drinking was retested in isolation. In Experiment 3, alcohol-naïve animals were tested for saccharin consumption (0.05%) first in isolation and then in high saccharin drinkers paired with low saccharin drinkers, and then in another isolation period. RESULTS In Experiment 1, high drinkers paired with low drinkers significantly decreased their alcohol intake and preference from baseline drinking in isolation, and drinking levels remained significantly lower during isolation following pairing. Interestingly, there was variability between pairs in whether the high drinker decreased or the low drinker increased intake. In Experiment 2, high drinkers paired with high drinkers did not significantly change their intake level or preference, nor did low drinkers paired with low drinkers, and no changes occurred during the subsequent isolation. In Experiment 3, there was no change in saccharin intake or preference when high drinkers were paired with high drinkers or low paired with low, or in the subsequent isolation. CONCLUSIONS Alcohol drinking of prairie voles can be altered under social conditions, such that 1 animal changes its alcohol intake to more closely match the intake of the other animal, helping to explain previous findings of correlated alcohol drinking. The effect does not extend to saccharin, a naturally rewarding sweet substance. This behavior can be used to model the peer pressure that can often affect alcohol intake in humans.

Biological Contribution to Social Influences on Alcohol Drinking: Evidence from Animal Models

Social factors have a tremendous influence on instances of heavy drinking and in turn impact public health. However, it is extremely difficult to assess whether this influence is only a cultural phenomenon or has biological underpinnings. Research in non-human primates demonstrates that the way individuals are brought up during early development affects their future predisposition for heavy drinking, and research in rats demonstrates that social isolation, crowding or low social ranking can lead to increased alcohol intake, while social defeat can decrease drinking. Neurotransmitter mechanisms contributing to these effects (i.e., serotonin, GABA, dopamine) have begun to be elucidated. However, these studies do not exclude the possibility that social effects on drinking occur through generalized stress responses to negative social environments. Alcohol intake can also be elevated in positive social situations, for example, in rats following an interaction with an intoxicated peer. Recent studies have also begun to adapt a new rodent species, the prairie vole, to study the role of social environment in alcohol drinking. Prairie voles demonstrate a high degree of social affiliation between individuals, and many of the neurochemical mechanisms involved in regulation of these social behaviors (for example, dopamine, central vasopressin and the corticotropin releasing factor system) are also known to be involved in regulation of alcohol intake. Naltrexone, an opioid receptor antagonist approved as a pharmacotherapy for alcoholic patients, has recently been shown to decrease both partner preference and alcohol preference in voles. These findings strongly suggest that mechanisms by which social factors influence drinking have biological roots, and can be studied using rapidly developing new animal models.

Social housing and alcohol drinking in male-female pairs of prairie voles (Microtus ochrogaster)

RationaleSocial environment influences alcohol consumption in humans; however, animal models have only begun to address biological underpinnings of these effects.ObjectivesWe investigated whether social influences on alcohol drinking in the prairie vole are specific to the sex of the social partner.MethodsIn Experiment 1, control, sham, and gonadectomized voles were placed either in mesh-divided housing with a same-sex sibling or isolation with access to ethanol. In Experiment 2, animals were given an elevated plus maze test (EPM) and then females were paired with a castrated male followed by isolation or mesh-divided housing with access to ethanol. In Experiment 3, subjects categorized as low or high drinkers based on initial ethanol intake were placed in mesh-divided housing with an opposite-sex partner of the same or opposite drinking group and ethanol access. Subjects were then moved back to isolation for a final ethanol access period.ResultsSame-sex pairs showed social facilitation of drinking similar to previous reports. Gonadectomy did not affect alcohol drinking. Opposite-sex paired animals in Experiment 2 did not differ in alcohol drinking based on social housing. EPM measures suggested a relationship between anxiety-like behaviors and drinking that depended on social environment. Experiment 3 identified moderate changes in alcohol preference based on social housing, but these effects were influenced by the animal’s own drinking behavior and were independent of their partner’s drinking.ConclusionsSocial influences on alcohol self-administration in prairie voles differ based on the sex of a social partner, consistent with human drinking behavior.

Differential sensitivity of the perioculomotor urocortin-containing neurons to ethanol, psychostimulants and stress in mice and rats

The perioculomotor urocortin-containing population of neurons (pIIIu: otherwise known as the non-preganglionic Edinger-Westphal nucleus) is sensitive to alcohol and is involved in the regulation of alcohol intake. A recent study indicated that this brain region is also sensitive to psychostimulants. Since pIIIu has been shown to respond to stress, we investigated how psychostimulant-induced pIIIu activation compares to stress- and ethanol-induced activation, and whether it is independent from a generalized stress response. Several experiments were performed to test how the pIIIu responds to psychostimulants by quantifying the number of Fos immunoreactive nuclei after acute i.p. injections of saline, 10-30 mg/kg cocaine, 5 mg/kg methamphetamine, 5 mg/kg amphetamine, 2.5 g/kg ethanol, 2 h of restraint stress, 10 min of swim stress, or six applications of mild foot shock in male C57BL/6 J mice. We also compared Fos immunoreactivity in pIIIu after acute (20 mg/kg cocaine) and repeated cocaine exposure (7 days of 20 mg/kg cocaine) injections in male C57BL/6 J mice in order to investigate the potential habituation of this response. Finally, we quantified the number of Fos immunoreactive nuclei in pIIIu after administration of saline, 2.5 g/kg ethanol, 20 mg/kg cocaine, or 2 h of restraint stress in male Sprague-Dawley rats. We found that exposure to psychostimulants and ethanol induced significantly higher Fos levels in pIIIu compared to stress in mice. Furthermore, repeated cocaine injections did not decrease Fos immunoreactivity as would be expected if this response were due to stress. In rats, exposure to ethanol, psychostimulant and restraint stress all induced pIIIu Fos immunoreactivity compared to saline-injected controls. In both mice and rats, ethanol- and cocaine-induced Fos immunoreactivity occurred exclusively in urocortin 1-positive, but not in tyrosine hydroxylase-positive, cells. These results provide evidence that the pIIIu Fos-response to psychostimulants is independent of a generalized stress in mice, but not rats. They additionally show that the pIIIu response to stress differs significantly between species.

Dissection of corticotropin-releasing factor system involvement in locomotor sensitivity to methamphetamine.

Sensitivity to the euphoric and locomotor‐activating effects of drugs of abuse may contribute to risk for excessive use and addiction. Repeated administration of psychostimulants such as methamphetamine (MA) can result in neuroadaptive consequences that manifest behaviorally as a progressive escalation of locomotor activation, termed psychomotor sensitization. The present studies addressed the involvement of specific components of the corticotropin‐releasing factor (CRF) system in locomotor activation and psychomotor sensitization induced by MA (1, 2 mg/kg) by utilizing pharmacological approaches, as well as a series of genetic knockout (KO) mice, each deficient for a single component of the CRF system: CRF‐R1, CRF‐R2, CRF, or the CRF‐related peptide Urocortin 1 (Ucn1). CRF‐R1 KO mice did not differ from wild‐type mice in sensitization to MA, and pharmacological blockade of CRF‐R1 with CP‐154,526 (15, 30 mg/kg) in DBA/2J mice did not selectively attenuate either the acquisition or expression of MA‐induced sensitization. Deletion of either of the endogenous ligands of CRF‐R1 (CRF, Ucn1) either enhanced or had no effect on MA‐induced sensitization, providing further evidence against a role for CRF‐R1 signaling. Interestingly, deletion of CRF‐R2 attenuated MA‐induced locomotor activation, elucidating a novel contribution of the CRF system to MA sensitivity, and suggesting the participation of the endogenous urocortin peptides Ucn2 and Ucn3. Immunohistochemistry for Fos was used to visualize neural activation underlying CRF‐R2‐dependent sensitivity to MA, identifying the basolateral and central nuclei of the amygdala as neural substrates involved in this response. Our results support further examination of CRF‐R2 involvement in neural processes associated with MA addiction.

Drinking alcohol has sex-dependent effects on pair bond formation in prairie voles

Significance This study provides the first evidence to our knowledge that the effects of alcohol on social bonding can be mediated by biological mechanisms. The observed effects differed between males and females, such that alcohol inhibited social bonding in males and facilitated the partner preference in females. In addition to affecting behavior, alcohol affected neuropeptide systems known to be involved in social and stress/anxiety-like behaviors. These findings allow us to understand the factors involved in regulation of social behaviors, and effects of alcohol on them, better. Identification of these factors can help develop ways to prevent or treat the devastating effects of alcohol abuse on social relationships. Alcohol use and abuse profoundly influences a variety of behaviors, including social interactions. In some cases, it erodes social relationships; in others, it facilitates sociality. Here, we show that voluntary alcohol consumption can inhibit male partner preference (PP) formation (a laboratory proxy for pair bonding) in socially monogamous prairie voles (Microtus ochrogaster). Conversely, female PP is not inhibited, and may be facilitated by alcohol. Behavior and neurochemical analysis suggests that the effects of alcohol on social bonding are mediated by neural mechanisms regulating pair bond formation and not alcohol’s effects on mating, locomotor, or aggressive behaviors. Several neuropeptide systems involved in the regulation of social behavior (especially neuropeptide Y and corticotropin-releasing factor) are modulated by alcohol drinking during cohabitation. These findings provide the first evidence to our knowledge that alcohol has a direct impact on the neural systems involved in social bonding in a sex-specific manner, providing an opportunity to explore the mechanisms by which alcohol affects social relationships.

Identification of subpopulations of prairie voles differentially susceptible to peer influence to decrease high alcohol intake

Peer influences are critical in the decrease of alcohol (ethanol) abuse and maintenance of abstinence. We previously developed an animal model of inhibitory peer influences on ethanol drinking using prairie voles and here sought to understand whether this influential behavior was due to specific changes in drinking patterns and to variation in a microsatellite sequence in the regulatory region of the vasopressin receptor 1a gene (avpr1a). Adult prairie voles’ drinking patterns were monitored in a lickometer apparatus that recorded each lick a subject exhibited during continuous access to water and 10% ethanol during periods of isolation, pair housing of high and low drinkers, and subsequent isolation. Analysis of fluid consumption confirmed previous results that high drinkers typically decrease ethanol intake when paired with low drinkers, but that a subset of voles do not decrease. Analysis of bout structure revealed differences in the number of ethanol drinking bouts in the subpopulations of high drinkers when paired with low drinkers. Lickometer drinking patterns analyzed by visual and by cross-correlation analyses demonstrated that pair housing did not increase the rate of subjects drinking in bouts occurring at the same time. The length of the avpr1a microsatellite did not predict susceptibility to peer influence or any other drinking behaviors. In summary, subpopulations of high drinkers were identified, by fluid intake and number of drinking bouts, which did or did not lower their ethanol intake when paired with a low drinking peer, and these subpopulations should be explored for testing the efficacy of treatments to decrease ethanol use in groups that are likely to be responsive to different types of therapy.