In need of a specific antibody against the oxytocin receptor for neuropsychiatric research: A KO validation study.

Antibodies are one of the most utilized tools in biomedical research. However, few of them are rigorously evaluated, as there are no accepted guidelines or standardized methods for determining their validity before commercialization. Often, an antibody is considered validated if it detects a band by Western blot of the expected molecular weight and, in some cases, if blocking peptides result in loss of staining. Neither of these approaches are unquestionable proof of target specificity. Since the oxytocin receptor has recently become a popular target in neuropsychiatric research, the need for specific antibodies to be used in brain has arisen. In this work, we have tested the specificity of six commercially available oxytocin receptor antibodies, indicated by the manufacturers to be suitable for Western blot and with an available image showing the correct size band (45-55 KDa). Antibodies were first tested by Western blot in brain lysates of wild-type and oxytocin receptor knockout mice. Uterus tissue was also tested as control for putative differential tissue specificity. In brain, the six tested antibodies lacked target specificity, as both wild-type and receptor knockout samples resulted in a similar staining pattern, including the expected 45-55 KDa band. Five of the six antibodies detected a selective band in uterus (which disappeared in knockout tissue). These five specific antibodies were also tested for immunohistochemistry in uterus, where only one was specific. However, when the uterine-specific antibody was tested in brain tissue, it lacked specificity. In conclusion, none of the six tested commercial antibodies are suitable to detect oxytocin receptor in brain by either Western blot or immunohistochemistry, although some do specifically detect it in uterus. The present work highlights the need to develop standardized antibody validation methods, including a proper negative control, in order to grant quality and reproducibility of the generated data.

Measuring Skeletal Muscle Thermogenesis in Mice and Rats.

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. Surprisingly little evidence is available to link the neural, myocellular, and molecular mechanisms of thermogenesis directly to measurable changes in muscle temperature. This paper describes a method in which temperature transponders are utilized to retrieve direct measurements of mouse and rat skeletal muscle temperature. Remote transponders are surgically implanted within the muscle of mice and rats, and the animals are given time to recover. Mice and rats must then be repeatedly habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage. Muscle temperature can also be measured during prescribed physical activity (i.e., treadmill walking at a constant speed) to factor out changes in activity as contributors to the changes in muscle temperature induced by these stimuli. This method has been successfully used to elucidate mechanisms underlying muscle thermogenic control at the level of the brain, sympathetic nervous system, and skeletal muscle. Provided are demonstrations of this success using predator odor (PO; ferret odor) as a contextual stimulus and injections of oxytocin (Oxt) as a pharmacological stimulus, where predator odor induces muscle thermogenesis, and Oxt suppresses muscle temperature. Thus, these datasets display the efficacy of this method in detecting rapid changes in muscle temperature.

Paternal Cocaine in Mice Alters Social Behavior and Brain Oxytocin Receptor Density in First Generation Offspring.

It is well established that the damaging effects of drugs of abuse, such as cocaine, can extend beyond the user to their offspring. While most preclinical models of the generational effects of cocaine abuse have focused on maternal effects, we, and others, report distinct effects on offspring sired by fathers treated with cocaine prior to breeding. However, little is known about the effects of paternal cocaine use on first generation (F1) offspring's social behaviors. Here, we expand upon our model of oral self-administered paternal cocaine use to address the idea that paternal cocaine alters first generation offspring social behaviors through modulation of the oxytocin system. F1 cocaine-sired males displayed unaltered social recognition vs. non-cocaine sired controls but showed increased investigation times that were not related to altered olfaction. Paternal cocaine did not alter F1 male-aggression behavior or depression-like behaviors, but cocaine-sired males did display decreased anxiety-like behaviors. Female F1 behavior was similarly examined, but there were no effects of paternal cocaine. Cocaine-sired male mice also exhibited localized oxytocin receptor expression differences vs. controls in several brain regions regulating social behavior. These results provide evidence for effects of paternal cocaine exposure on social behaviors in male offspring with associated alterations in central oxytocin transmission.

Pharmacological manipulation of oxytocin receptor signaling during mouse embryonic development results in sex-specific behavioral effects in adulthood.

The oxytocin (Oxt) system is a known neuromodulator of social behaviors, but also appears to contribute to the development of sex-specific neural circuitry. In this latter role, the Oxt system helps to lay the foundation for sex-specific behaviors across the life span. In mice, the Oxt system emerges in early development, with sex differences in the expression of Oxt and a temporal offset in the expression of the Oxt receptor (Oxtr) relative to Oxt. In females, Oxt mRNA is detectable by embryonic day (E) 16.5, but in males, Oxt mRNA is not measurable until after birth. However, in both sexes, Oxtr mRNA is detectable by E12.5 and binding by E16.5. While the postnatal Oxt system has been studied, little is known about the embryonic Oxt system. Therefore, we hypothesize that it directly contributes to the developmental trajectory of the brain, ultimately affecting adult sex-specific behaviors. To test this hypothesis, Oxtr signaling was transiently disrupted at E16.5 using an Oxtr antagonist (OxtrA) and the effects on adult behavior evaluated. OxtrA-treated adult males displayed increased agonistic behavior, social investigation, and depressive-like behavior compared to vehicle-injected controls, while OxtrA-treated adult females had impaired social recognition memory compared to vehicle-injected controls. These data are the first to identify a functional link between the organizational activity of the embryonic Oxt system and adult behavior. Further, this work suggests that the Oxt system does more than serve as a neuromodulator in adulthood, but rather, may help shape the development of the neural circuitry regulating sex-specific behaviors.

Voluntary alcohol consumption is increased in female, but not male, oxytocin receptor knockout mice.

INTRODUCTION: The oxytocin (Oxt) system, while typically associated with the neural regulation of social behaviors, also plays a role in an individual's vulnerability to develop alcohol use disorders (AUD). In humans, changes to the Oxt system, due to early life experience and/or genetic mutations, are associated with increased vulnerability to AUD. While a considerable amount is known about Oxt's role in AUD in males, less is known or understood, about how Oxt may affect AUD in females, likely due to many clinical and preclinical studies of AUD not directly considering sex as a biological variable. This is unfortunate given that females are more vulnerable to the effects of alcohol and have increased alcohol consumption, as compared to males. Therefore, in the current study we wanted to determine whether genetic disruption of the Oxt receptor (Oxtr), that is, Oxtr knockout (-/-) mice, affected stress-induced alcohol consumption in males and females. We hypothesized that genetic disruption of the Oxtr would result in increased stress-induced alcohol consumption in both males and females compared to wild-type (+/+) controls. Though, we predicted that these disruptions might be greater in female Oxtr -/- mice. METHODS: To test this hypothesis, a two-bottle preference test was utilized along with the forced swim test (FST), and pre- and poststress alcohol consumption and preference measured within each sex (males and females were run separately). As a follow-up experiment, a taste preference test, to control for possible genotypic differences in taste, was also performed. RESULTS: In males, we found no significant genotypic differences in alcohol consumption or preference. However, in females, we found that genetic disruption of the Oxtr resulted in a greater consumption of alcohol both pre- and poststress compared to controls. CONCLUSION: These data suggest that in females, disruptions in Oxt signaling may contribute to increased vulnerability to alcohol-associated addiction.

Increased immediate early gene activation in the basolateral amygdala following persistent peripheral inflammation.

Chronic pain results in a variety of neural adaptations, many of which are maladaptive and result in hypersensitivity to pain. In humans, this hypersensitivity can be debilitating and treatment options are limited. Fortunately, there are numerous animal models that mimic clinical populations and have the potential to aid in the evaluation of underlying mechanisms and ultimately the development of better treatments. One of these is the complete Freund's adjuvant (CFA)-model of chronic inflammatory pain. In rodents, this model requires the injection of CFA into the hindpaw, muscle, or joint, which induces inflammation similar to what might be found in individuals with rheumatoid arthritis or tendonitis. While the mechanistic effects CFA on the spinal cord are well established, less is known about the effects of CFA on the brain. Thus, in this study, neuronal activation, as measured by c-Fos immunocytochemistry, in brain regions important to control of pain was evaluated. Animals that received CFA treatment, and tested 3 days later for mechanical allodynia and edema, had an increase in the number of c-Fos immunopositive cells in the basolateral amygdala, but not in any of the other brain regions that were evaluated. Given that the basolateral amygdala is known to be important for pain-related emotional responses, these data suggest that the CFA-model may provide an opportunity to further explore how pain affects this brain region at a mechanistic level, which in turn may shed light on what may be occurring in clinical populations.

Call-specific patterns of neural activation in auditory processing of Richardson's ground squirrel alarm calls.

INTRODUCTION: Richardson's ground squirrels use alarm calls to warn conspecifics about potential predatory threats. Chirp calls typically indicate high levels of threat from airborne predators, while whistle calls are associated with lower levels of threat from terrestrial predators. These types of calls primarily elicit escape behaviors and increased vigilance in receivers, respectively. While much is known about the neural mechanisms involved in the production of vocalizations, less is known about the mechanisms important for the perception of alarm calls by receivers, and whether changes in perceived risk are associated with unique patterns of neuronal activation. Thus, to determine whether alarm calls associated with different levels of predation risk result in differential neuronal activation, we used immunohistochemistry to identify and quantify c-Fos immunopositive cells in brain regions important in stress, fear, danger, and reward, following alarm call reception. METHODS: We exposed 29 female Richardson's ground squirrels (10 control, 10 whistle receivers, and 9 chirp receivers) to playbacks of whistles, chirps, or a no-vocalization control. We then assessed neuronal activation via c-Fos immunohistochemistry in 12 brain regions. RESULTS: Ground squirrels receiving high-threat "chirp" vocalizations had reduced neuronal activation in the medial amygdala and superior colliculus compared with controls. It is likely that changes in activity in these brain regions serve to alter the balance between approach and avoidance in turn promoting escape behaviors. CONCLUSIONS: Thus, we conclude that in Richardson's ground squirrels, these brain regions are important for the perception of risk resulting from receiving alarm calls and allow for appropriate behavioral responses by receivers.

Subtle sex differences in vasopressin mRNA expression in the embryonic mouse brain.

Arginine vasopressin (AVP) is a neuropeptide which acts centrally to modulate numerous social behaviors. One receptor subtype through which these effects occur is the AVP 1a receptor (AVPR1A). The modulatory effects of Avp via the AVPR1A varies by species as well as sex, since both AVP and the AVPR1A tend to be expressed more prominently in males. Beyond these neuromodulatory effects there are also indications that the AVP system may play a role in early development to, in part, organize sex-specific neural circuitry that is important to sexually dimorphic social behaviors in adulthood. However, to date, AVP's role in early development is poorly understood, particularly with respect to its differential effect on males and females. In order to determine the timing and distribution of the AVP system in early brain development, we examined the brains of male and female C57BL/6J mice between embryonic day (E) 12.5 and postnatal day (P) 2 and quantified Avp and Avpr1a mRNA using qPCR and AVPR1A protein using receptor autoradiography. The mRNA for Avp was measurable in males and females starting at E14.5, with males producing more than females, while Avpr1a mRNA was found as early as E12.5, with no difference in expression between sexes. AVPR1A binding was observed in both sexes starting at E16.5, and while there were no observed sex differences, binding density and the number of neuroanatomical areas did increase over time. These data are significant as they provide the first whole-brain characterization of the vasopressin system in the embryonic mouse. Further, these findings are consistent with data from other species, that have documented a sex difference in the vasopressin system during early brain formation.

Comparison of the distribution of oxytocin and vasopressin 1a receptors in rodents reveals conserved and derived patterns of nonapeptide evolution.

Oxytocin (OT) and vasopressin (VP) are known modulators of social behaviour across rodents. Research has revealed the location of action of these nonapeptides through localization of their associated receptors, which include the oxytocin receptor (OTR) and the vasopressin 1a receptor (V1aR). As research into these complex systems has progressed, studies investigating how these systems modulate behaviour have remained relatively narrow in scope (ie, focused on how a single brain region shapes behaviour in only a handful of species). However, the brain regions that regulate social behaviour are part of interconnected neural networks for which coordinated activity enables behavioural variation. Thus, to better understand how nonapeptide systems have evolved under different selective pressures among rodent species, we conducted a meta-analysis using a multivariate comparative method to examine the patterns of OTR and V1aR density expression in this taxon. Several brain regions were highly correlated based on their OTR and V1aR binding patterns across species, supporting the notion that the distribution of these receptors is highly conserved in rodents. However, our results also revealed that specific patterns of V1aR density differed from OTR density, and within-genus variance for V1aR was low compared to between-genus variance, suggesting that these systems have responded and evolved quite differently to selective pressures over evolutionary time. We propose that, in addition to examining single brain regions of interest, taking a broad comparative approach when studying the OT and VP systems is important for understanding how the systemic action of nonapeptides modulate social behaviour across species.

Central distribution of oxytocin and vasopressin 1a receptors in juvenile Richardson's ground squirrels.

Oxytocin and vasopressin are well-conserved peptides important to the regulation of numerous aspects of social behavior, including sociality. Research exploring the distribution of the receptors for oxytocin (Oxtr) and for vasopressin (Avpr1a) in mammals has revealed associations between receptor distribution, sociality, and species' mating systems. Given that sociality and gregariousness can be tightly linked to reproduction, these nonapeptides unsurprisingly support affiliative behaviors that are important for mating and offspring care. We localized these receptors in juvenile Richardson's ground squirrel brains to determine whether distribution patterns of Oxtr and Avpr1a that are associated with promiscuous mating systems differ in rodents that also exhibit non-reproductive affiliation. These squirrels are social, colonial, and engage in nepotistic alarm calling behavior and affiliation outside of a reproductive context. Juveniles are the most affiliative age-class and are non-reproductive; making them ideal for examining these associations. We found that juveniles had dense Oxtr binding in the dentate gyrus of the hippocampus, amygdala, lateral septum, bed nucleus of the stria terminalis and medial geniculate nucleus. Juveniles had low to modest levels of Avpr1a binding in the medial preoptic area, olfactory bulbs, nucleus accumbens, superior colliculus, and inferior colliculus. We noted Oxtr and Avpr1a binding in the social behavior neural network (SBNN), further supporting a role of these nonapeptides in modulating social behavior across taxa. Oxtr and Avpr1a binding was also present in brain regions important to auditory processing that have known projections to the SBNN. We speculate that these neural substrates may be where these nonapeptides regulate communication.

Effects of arginine vasopressin on Richardson's ground squirrel social and vocal behavior.

In nearly every vertebrate species examined thus far arginine vasopressin (AVP) and its homologues modulate behavior; thus, providing rich systems for comparative research. In rodents, AVP is best known for its modulation of social behavior; however, to date, research on AVPs effects on behavior have been limited to laboratory models and a few experiments using large outdoor enclosures. To extend our understanding of AVPs role in modulating social behavior and communication in an ecologically relevant context, we examined the effects of AVP on behavior of free-living Richardson's ground squirrels (Urocitellus richardsonii). To test the hypothesis that AVP influences social behavior and communication, we implanted osmotic minipumps into Richardson's ground squirrels and centrally administered AVP or saline as a control. Three different behavioral experiments quantifying behavior before and after AVP or saline administration were performed: a general behavior survey, a predator model presentation, and a social challenge test. AVP administration increased male vocalization rate when approached by a conspecific, but not when presented with a predator model. In males, social aggression decreased, but antipredator vigilance increased with AVP administration. Finally, AVP-treated females had increased "anxiety-like" behaviors during the social challenge test. Our data reveal that AVP has sex-specific effects on vocalizations and antipredator vigilance, as well as other social behaviors. Further, our data illustrate the importance of social context to AVPs modulation of behavior. (PsycINFO Database Record

Social Context, Stress, Neuropsychiatric Disorders, and the Vasopressin 1b Receptor.

The arginine vasopressin 1b receptor (Avpr1b) is involved in the modulation of a variety of behaviors and is an important part of the mammalian hormonal stress axis. The Avpr1b is prominent in hippocampal CA2 pyramidal cells and in the anterior pituitary corticotrophs. Decades of research on this receptor has demonstrated its importance to the modulation of social recognition memory, social forms of aggression, and modulation of the hypothalamic-pituitary-adrenal axis, particularly under conditions of acute stress. Further, work in humans suggests that the Avpr1b may play a role in human neuropsychiatric disorders and its modulation may have therapeutic potential. This paper reviews what is known about the role of the Avpr1b in the context of social behaviors, the stress axis, and human neuropsychiatric disorders. Further, possible mechanisms for how Avpr1b activation within the hippocampus vs. Avpr1b activation within anterior pituitary may interact with one another to affect behavioral output are proposed.

Oxytocin and Vasopressin: Powerful Regulators of Social Behavior.

For many, the terms oxytocin and vasopressin immediately evoke images of animals interacting with one another, as both of these neuropeptides have been implicated as being part of the neurochemical "glue" that socially binds animals. However, social environments and social interactions are complex and include behaviors that bring animals together as well as behaviors that keep animals apart. It is at the intersection of social context, social experience, and an individual's sex that oxytocin and vasopressin act to modulate social behavior and social cognition. In this review, this complexity will be explored across mammalian species, with a focus on social memory, cooperative behaviors, and competitive behaviors. Implications for humans as well as future directions will also be considered.

Oxytocin and behavior: Lessons from knockout mice.

It is well established that the nonapeptide oxytocin (Oxt) is important for the neural modulation of behaviors in many mammalian species. Since its discovery in 1906 and synthesis in the early 1950s, elegant pharmacological work has helped identify specific neural substrates on which Oxt exerts its effects. More recently, mice with targeted genetic disruptions of the Oxt system-i.e., both the peptide and its receptor (the Oxtr)-have further defined Oxt's actions and laid some important scientific groundwork for studies in other species. In this article, we highlight the scientific contributions that various mouse knockouts of the Oxt system have made to our understanding of Oxt's modulation of behavior. We specifically focus on how the use of these mice has shed light on our understanding of social recognition memory, maternal behavior, aggression, and several nonsocial behaviors. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 190-201, 2017.

Genotypic differences in intruder-evoked immediate early gene activation in male, but not female, vasopressin 1b receptor knockout mice.

BACKGROUND: The neuropeptide arginine vasopressin (Avp) modulates social behaviors via its two centrally expressed receptors, the Avp 1a receptor and the Avp 1b receptor (Avpr1b). Recent work suggests that, at least in mice, Avp signaling through Avpr1b within the CA2 region of the hippocampus is critical for normal aggressive behaviors and social recognition memory. However, this brain area is just one part of a larger neural circuit that is likely to be impacted in Avpr1b knockout (-/-) mice. To identify other brain areas that are affected by altered Avpr1b signaling, genotypic differences in immediate early gene activation, i.e. c-FOS and early growth response factor 1 (EGR-1), were quantified using immunocytochemistry following a single exposure to an intruder. RESULTS: In females, no genotypic differences in intruder-evoked c-FOS or EGR-1 immunoreactivity were observed in any of the brain areas measured. In males, while there were no intruder-evoked genotypic differences in c-FOS immunoreactivity, genotypic differences were observed in EGR-1 immunoreactivity within the ventral bed nucleus of the stria terminalis and the anterior hypothalamus; with Avpr1b -/- males having less EGR-1 immunoreactivity in these regions than controls. CONCLUSIONS: These data are the first to identify specific brain areas that may be a part of a neural circuit that includes Avpr1b-expressing cells in the CA2 region of the hippocampus. It is thought that this circuit, when working properly, plays a role in how an animal evaluates its social context.

Oxytocin, Vasopressin, and the Motivational Forces that Drive Social Behaviors.

The motivation to engage in social behaviors is influenced by past experience and internal state, but also depends on the behavior of other animals. Across species, the oxytocin (Oxt) and vasopressin (Avp) systems have consistently been linked to the modulation of motivated social behaviors. However, how they interact with other systems, such as the mesolimbic dopamine system, remains understudied. Further, while the neurobiological mechanisms that regulate prosocial/cooperative behaviors have been extensively examined, far less is understood about competitive behaviors, particularly in females. In this chapter, we highlight the specific contributions of Oxt and Avp to several cooperative and competitive behaviors and discuss their relevance to the concept of social motivation across species, including humans. Further, we discuss the implications for neuropsychiatric diseases and suggest future areas of investigation.

Oxytocin during Development: Possible Organizational Effects on Behavior.

Oxytocin (Oxt) is a neurohormone known for its physiological roles associated with lactation and parturition in mammals. Oxt can also profoundly influence mammalian social behaviors such as affiliative, parental, and aggressive behaviors. While the acute effects of Oxt signaling on adult behavior have been heavily researched in many species, including humans, the developmental effects of Oxt on the brain and behavior are just beginning to be explored. There is evidence that Oxt in early postnatal and peripubertal development, and perhaps during prenatal life, affects adult behavior by altering neural structure and function. However, the specific mechanisms by which this occurs remain unknown. Thus, this review will detail what is known about how developmental Oxt impacts behavior as well as explore the specific neurochemicals and neural substrates that are important to these behaviors.

Lesions to the CA2 region of the hippocampus impair social memory in mice.

The function of the CA2 region of the hippocampus is poorly understood. Although the CA1 and CA3 regions have been extensively studied, for years the CA2 region has primarily been viewed as a linking area between the two. However, the CA2 region is known to have distinct neurochemical and structural features that are different from the other parts of the hippocampus and in recent years it has been suggested that the CA2 region may play a role in the formation and/or recall of olfactory-based memories needed for normal social behavior. Although this hypothesis has been supported by hippocampal lesion studies that have included the CA2 region, no studies have attempted to specifically lesion the CA2 region of the hippocampus in mice to determine the effects on social recognition memory and olfaction. To fill this knowledge gap, we sought to perform excitotoxic N-methyl-D-aspartate lesions of the CA2 region and determine the effects on social recognition memory. We predicted that lesions of the CA2 region would impair social recognition memory. We then went on to test olfaction in CA2-lesioned mice, as social memory requires a functional olfactory system. Consistent with our prediction, we found that CA2-lesioned animals had impaired social recognition. These findings are significant because they confirmed that the CA2 region of the hippocampus is a part of the neural circuitry that regulates social recognition memory, which may have implications for our understanding of the neural regulation of social behavior across species.