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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.

Intrauterine infections during pregnancy by herpes simplex virus (HSV) can cause significant neurodevelopmental deficits in the unborn/newborn, but clinical studies of pathogenesis are challenging, and while animal models can model some aspects of disease, in vitro studies of human neural cells provide a critical platform for more mechanistic studies. We utilized a reductionist approach to model neurodevelopmental outcomes of HSV-1 infection of neural rosettes, which represent the in vitro equivalent of differentiating neural tubes. Specifically, we employed early-stage brain organoids (ES-organoids) composed of human induced pluripotent stem cells (hiPSCs)-derived neural rosettes to investigate aspects of the potential neuropathological effects induced by the HSV-1 infections on neurodevelopment. To allow for the long-term differentiation of ES-organoids, viral infections were performed in the presence of the antiviral drug acyclovir (ACV). Despite the antiviral treatment, HSV-1 infection caused organizational changes in neural rosettes, loss of structural integrity of infected ES-organoids, and neuronal alterations. The inability of ACV to prevent neurodegeneration was associated with the generation of ACV-resistant mutants during the interaction of HSV-1 with differentiating neural precursor cells (NPCs). This study models the effects of HSV-1 infection on the neuronal differentiation of NPCs and suggests that this environment may allow for accelerated development of ACV-resistance.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) occur in children who were exposed to alcohol in utero and are manifested in a wide range of neurocognitive deficits. These deficits could be caused by alterations to the cortical microvasculature that are controlled by post-transcriptional regulators such as microRNAs. METHODS: Using an established mouse model of moderate prenatal alcohol exposure (PAE), we isolated cortices (CTX) and brain microvascular endothelial cells (BMVECs) at embryonic day 18 (E18) and examined the expression of miR-150-5p and potential downstream targets. Cellular transfections and intrauterine injections with LNA™ mimics or inhibitors were used to test miR-150-5p regulation of novel target vascular endothelial zinc finger 1 (Vezf1). Dual-luciferase assays were used to assess the direct binding of miR-150-5p to the Vezf1 3′UTR. The effects of miR-150-5p and Vezf1 on endothelial cell function were determined by in vitro migration and tube formation assays. RESULTS: We found that miR-150-5p was upregulated and Vezf1 was downregulated during PAE in the E18 CTX and BMVECs. Transfection with miR-150-5p mimics resulted in decreased Vezf1 expression in BMVECs, while miR-150-5p inhibition did the opposite. Dual-luciferase assays revealed direct binding of miR-150-5p with the Vezf1 3′UTR. Intrauterine injections showed that miR-150-5p regulates the expression of Vezf1 in vivo during PAE. miR-150-5p overexpression decreased BMVEC migration and tube formation, while miR-150-5p inhibition enhanced migration and tube formation. Vezf1 overexpression rescued the effects of the miR-150-5p mimic. Alcohol treatment of BMVECs increased miR-150-5p expression and inhibited migration and tube formation. Finally, miR-150-5p inhibition and Vezf1 overexpression rescued the negative effects of alcohol on migration and tube formation. CONCLUSIONS: miR-150-5p regulation of Vezf1 results in altered endothelial cell function during alcohol exposure. Further, miR-150-5p inhibition of Vezf1 may adversely alter the development of the cortical microvasculature during PAE and contribute to deficits seen in patients with FASD.

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.

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.

Background: Increased soft-drink consumption has contributed to poor calcium intake with 90% of adolescent girls consuming less than the RDA for calcium.Purpose/objectives: The purpose of this inve...

This review addresses underlying physiological, cellular, and molecular factors that alter the developing fetal brain stress circuits and responses of the hypothalamic-pituitary-adrenal (HPA) axis caused by maternal stress and prenatal alcohol exposure (PAE). An emphasis is placed on the contribution of the placenta following maternal stress separately, and as a co-occurrence with PAE. Altered fetal HPA axis ultimately results in dysregulation of the brain stress-response system long after birth and possibly lifelong. Additional consideration of the role of placentally-derived endocrine and sex hormones, as well as a brief discussion of epigenetic mechanisms of altered placental expression of genes encoding the glucocorticoid receptor and the enzymes 11β-HSD that rapidly convert glucocorticoids into its active or inactive forms are reviewed. Data highlighting the strong, reciprocal interactions between the neuroimmune and neuroendocrine systems during fetal development that are impacted by maternal stress and PAE are considered, emphasizing the role of the placenta as a key contributor to the dysregulation of these systems. In view of the maternal-placental-fetal interface, important physiological, cellular, and molecular factors underlying later life dysregulated stress responses are additionally considered. Literature from animal models of PAE and maternal stress is reviewed that support clinical observations of the effect of maternal stress and alcohol exposure during fetal development on later-life adult stress responses and associated mood dysregulation. An appreciation of dysregulated stress responses in individuals with fetal alcohol spectrum disorders (FASD) are addressed given the greater prevalence of adult dysregulated stress responses and a greater co-occurrence of mood disorders in individuals diagnosed with FASD.

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.

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., Our study is the first to investigate alcohol consumption using Oxtr −/− mice. In females, but not males, we found that genetic disruption of the Oxtr resulted in a greater consumption of alcohol both pre‐ and poststress compared to controls. These data suggest that in females, disruptions in Oxt signaling may contribute to increased vulnerability to alcohol‐associated disorders.

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., Richardson's ground squirrels use alarm calls to warn conspecifics about potential predatory threats. The “chirp” alarm call is thought to indicate high levels of threat, while the “whistle” is thought to indicate low levels of threat. Chirps typically elicit escape behaviors in receivers, which, we show, are supported by call‐specific neural activation patterns.

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.

The role of environmental stressors, particularly exposure to air pollution, in the development of neurodegenerative disease remains underappreciated. We examined the neurological effects of acute ozone (O(3)) exposure in aged mice, where increased blood–brain barrier (BBB) permeability may confer vulnerability to neuroinflammatory outcomes. C57BL/6 male mice, aged 8–10 weeks or 12–18 months were exposed to either filtered air or 1.0 ppm O(3) for 4 h; animals received a single IP injection of sodium fluorescein (FSCN) 20 h postexposure. One-hour post-FSCN injection, animals were transcardially perfused for immunohistochemical analysis of BBB permeability. β-amyloid protein expression was assessed via ELISA. Flow cytometric characterization of infiltrating immune cells, including neutrophils, macrophages, and microglia populations was performed 20 h post-O(3) exposure. Flow cytometry analysis of brains revealed increased microglia “activation” and presentation of CD11b, F4/80, and MHCII in aged animals relative to younger ones; these age-induced differences were potentiated by acute O(3) exposure. Cortical and limbic regions in aged brains had increased reactive microgliosis and β-amyloid protein expression after O(3) insult. The aged cerebellum was particularly vulnerable to acute O(3) exposure with increased populations of infiltrating neutrophils, peripheral macrophages/monocytes, and Ly6C(+) inflammatory monocytes after insult, which were not significantly increased in the young cerebellum. O(3) exposure increased the penetration of FSCN beyond the BBB, the infiltration of peripheral immune cells, and reactive gliosis of microglia. Thus, the aged BBB is vulnerable to insult and becomes highly penetrable in response to O(3) exposure, leading to greater neuroinflammatory outcomes.

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.

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.

In rodents, the removal of the olfactory bulbs (OBs), i.e. olfactory bulbectomy (OBX), results in numerous alterations in neurotransmitter, endocrine and immune systems, as well as behavioral changes, similar to those observed in depressed patients. Because the behavioral deficits induced in OBX animals are reversed after repeated administration of antidepressants, this is a model often used to test the effectiveness of putative antidepressant agents. Recent evidence suggests that OBX results in the dysfunction of various cellular processes within the hippocampus, including decreases in dentate gyrus neurogenesis, disruption in long-term potentiation in CA1 and CA3 subregions and neuronal atrophy in the CA1 subregion, along with downstream markers, all of which are consistent with abnormal neuronal activity in the hippocampus of clinically depressed populations. Moreover, repeated administration of novel natural and synthetic antidepressant compounds can improve certain aspects of depression-like behavior and hippocampal function. In an effort to bring together the existing literature, this review will focus on the mechanisms by which proposed pharmaceuticals impact hippocampal-dependent processes and behavior.

Background Studies in clinical populations and preclinical models have shown that prenatal alcohol exposure (PAE) is associated with impairments in the acquisition, consolidation and recall of information, with deficits in hippocampal formation-dependent learning and memory being a common finding. The glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and extracellular signal-regulated kinase 2 (ERK2) are key regulators of hippocampal formation development, structure and functioning and, thus, are potential mediators of PAE’s effects on this brain region. In the present studies, we employed a well-characterized mouse model of PAE to identify biochemical mechanisms that may underlie activity-dependent learning and memory deficits associated with PAE. Methods Mouse dams consumed either 10% (w/v) ethanol in 0.066% (w/v) saccharin (SAC) or 0.066% (w/v) SAC alone using a limited (4-h) access, drinking-in-the-dark paradigm. Male and female offspring (∼180-days of age) were trained using a delay conditioning procedure and contextual fear responses (freezing behavior) were measured 24 h later. Hippocampal formation tissue and blood were collected from three behavioral groups of animals: 20 min following conditioning (conditioning only group), 20 min following the re-exposure to the context (conditioning plus re-exposure group), and behaviorally naive (naive group) mice. Plasma corticosterone levels were measured by enzyme immunoassay. Immunoblotting techniques were used to measure protein levels of the GR, MR, ERK1 and ERK2 in nuclear and membrane fractions prepared from the hippocampal formation. Results Adult SAC control male and female mice displayed similar levels of contextual fear. However, significant sex differences were observed in freezing exhibited during the conditioning session. Compared to same-sex SAC controls, male and female PAE mice demonstrated context fear deficits While plasma corticosterone concentrations were elevated in PAE males and females relative to their respective SAC naive controls, plasma corticosterone concentrations in the conditioning only and conditioning plus re-exposure groups were similar in SAC and PAE animals. Relative to the respective naive group, nuclear GR protein levels were increased in SAC, but not PAE, male hippocampal formation in the conditioning only group. In contrast, no difference was observed between nuclear GR levels in the naive and conditioning plus re-exposure groups. In females, nuclear GR levels were significantly reduced by PAE but there was no effect of behavioral group or interaction between prenatal treatment and behavioral group. In males, nuclear MR levels were significantly elevated in the SAC conditioning plus re-exposure group compared to SAC naive mice. In PAE females, nuclear MR levels were elevated in both the conditioning only and conditioning plus re-exposure groups relative to the naive group. Levels of activated ERK2 (phospho-ERK2 expressed relative to total ERK2) protein were elevated in SAC, but not PAE, males following context re-exposure, and a significant interaction between prenatal exposure group and behavioral group was found. No main effects or interactions of behavioral group and prenatal treatment on nuclear ERK2 were found in female mice. These findings suggest a sex difference in which molecular pathways are activated during fear conditioning in mice. Conclusions In PAE males, the deficits in contextual fear were associated with the loss of responsiveness of hippocampal formation nuclear GR, MR and ERK2 to signals generated by fear conditioning and context re-exposure. In contrast, the contextual fear deficit in PAE female mice does not appear to be associated with activity-dependent changes in GR and MR levels or ERK2 activation during training or memory recall, although an overall reduction in nuclear GR levels may play a role. These studies add to a growing body of literature demonstrating that, at least partially, different mechanisms underlie learning, memory formation and memory recall in males and females and that these pathways are differentially affected by PAE.

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

Background The clinical course of individuals exposed to alcohol in utero is influenced by multiple factors, including the social environments of the gravid female and offspring. In the present studies we focused on the effects of prenatal alcohol exposure (PAE) and the prenatal and early-life social environments on the hippocampal formation, as impaired development and functioning of this brain region have been implicated in several of the adverse cognitive outcomes associated with PAE. Methods We combined our PAE mouse model with 2 conditions of housing pregnant dams and their preweanling offspring: the standard nest (SN), in which a dam is individually housed prior to parturition and then remains isolated with her offspring, and the communal nest (CN), in which multiple dams are housed together prior to parturition and then following delivery the moms and their litters share a nest. Mouse dams consumed either 10% (w/v) ethanol in 0.066% (w/v) saccharin (SAC) or 0.066% (w/v) SAC alone using a limited (4-hour) access, drinking-in-the-dark paradigm. Immunoblotting techniques were used to measure levels of the glucocorticoid receptor (GR), 11-β-hydroxysteroid dehydrogenase 1, the FK506-binding proteins 51 and 52, and corticotropin-releasing hormone receptor type 1 in the hippocampal formation isolated from male adolescent offspring. We also determined the effect of PAE and rearing conditions on context discrimination, a hippocampal-dependent learning/memory task. Results SN PAE offspring displayed impaired context discrimination and neurochemical changes in the hippocampal formation consistent with increased GR nuclear localization. These effects of PAE were, in general, ameliorated in mice reared in a CN. The CN also altered neurochemical measures and improved learning/memory in SAC control animals. Conclusions These studies demonstrate a complex interplay between the effects of PAE and social environments. The findings have important translational implications, as well as highlight the importance of considering rearing conditions in the interpretation of research findings on PAE.

Our previous studies suggest that prenatal arsenic exposure (50 ppb) modifies epigenetic control of the programming of the glucocorticoid receptor (GR) signaling system in the developing mouse brain. These deficits may lead to long-lasting consequences, including deficits in learning and memory, increased depressive-like behaviors, and an altered set-point of GR feedback throughout life. To understand the arsenic-induced changes within the GR system, we assessed the impact of in utero arsenic exposure on the levels of the GR and growth arrest-specific-5 (Gas5), a noncoding RNA, across a key gestational period for GR programming (gestational days, GD 14–18) in mice. Gas5 contains a glucocorticoid response element (GRE)-like sequence that binds the GR, thereby decreasing GR-GRE-dependent gene transcription and potentially altering GR programming. Prenatal arsenic exposure resulted in sex-dependent and age-dependent shifts in the levels of GR and Gas5 expression in fetal telencephalon. Nuclear GR levels were reduced in males, but unchanged in females, at all gestational time points tested. Total cellular Gas5 levels were lower in arsenic-exposed males with no changes seen in arsenic-exposed females at GD16 and 18. An increase in total cellular Gas-5 along with increased nuclear levels in GD14 arsenic-exposed females, suggests a differential regulation of cellular compartmentalization of Gas5. RIP assays revealed reduced Gas5 associated with the GR on GD14 in the nuclear fraction prepared from arsenic-exposed males and females. This decrease in levels of GR-Gas5 binding continued only in the females at GD18. Thus, nuclear GR signaling potential is decreased in prenatal arsenic-exposed males, while it is increased or maintained at levels approaching normal in prenatal arsenic-exposed females. These findings suggest that females, but not males, exposed to arsenic are able to regulate the levels of nuclear free GR by altering Gas5 levels, thereby keeping GR nuclear signaling closer to control (unexposed) levels.

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.

Background Despite evidence that prenatal alcohol exposure (PAE) can lead to a wide range of impairments in cognitive, social, and emotional behaviors, drinking during pregnancy remains common. Although there is a general understanding that high levels of drinking during pregnancy are unsafe, conflicting evidence regarding the impact of low intake may account for the persistence of this behavior. Methods To investigate the effects of PAE on learning and executive control, we utilized a voluntary paradigm where pregnant mice had access to a saccharin-sweetened 10% alcohol solution for 4 hours, during the dark cycle, throughout gestation. Male and female offspring were tested as adults on a touch-screen discrimination and reversal task mediated by corticostriatal circuits. Results Consistent with previous findings, PAE did not lead to gross morphological, motor, or sensory alterations in offspring. Both PAE and saccharin control female mice were slower to acquire the discrimination than males, but PAE did not impair associative learning in either sex. During reversal, PAE led to a specific and significant impairment in the early phase, where cortical control is most required to flexibly alter choice behavior. PAE mice showed a significant increase in maladaptive perseverative responses but showed intact learning of the new association during late reversal. Conclusions Previously, data from clinical studies have suggested that executive control deficits may underlie cognitive, as well as social, problems seen in adolescents with documented PAE. These data demonstrate that even more moderate alcohol exposure during development can lead to impaired cognitive functioning well into adulthood.

Background Accumulating evidence indicates that several of the long-term consequences of prenatal alcohol exposure (PAE) are the result of changes in the development and function of cortico-limbic structures, including the hippocampal formation. The glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are key regulators of hippocampal formation development, structure, and functioning and, thus, are potential mediators of PAE's effects on this brain region. In the present studies, we assessed the impact of PAE on components of corticosteroid signaling pathways in the mouse hippocampal formation. Methods Throughout pregnancy, mouse dams were offered either 10% (w/v) ethanol sweetened with 0.066% (w/v) saccharin (SAC) or 0.066% (w/v) SAC alone using a limited (4-hour) access, drinking-in-the-dark paradigm. The hippocampal formation was isolated from naive postnatal day 40 to 50 offspring, and subcellular fractions were prepared. Using immunoblotting techniques, we measured the levels of GR, MR, 11-β-hydroxysteroid dehydrogenase 1 (11β-HSD1), and the FK506-binding proteins 51 (FKBP51, FKBP5) and 52 (FKBP52, FKBP4). Finally, we determined the effect of PAE on context discrimination, a hippocampal-dependent learning/memory task. Results PAE was associated with reduced MR and elevated GR nuclear localization in the hippocampal formation, whereas cytosolic levels of both receptors were not significantly altered. FKBP51 levels were reduced, while FKBP52 levels were unaltered, and 11β-HSD1 levels were increased in postnuclear fractions isolated from PAE mouse hippocampal formation. These neurochemical alterations were associated with reduced context discrimination. Conclusions The data support a model in which PAE leads to increased nuclear localization of GRs secondary to reductions in FKBP51 and increases in 11β-HSD1 levels in the adolescent mouse hippocampal formation. Persistent dysregulation of GR subcellular distribution is predicted to damage the hippocampal formation and may underlie many of the effects of PAE on hippocampal-dependent functioning.

Although it is well documented that heavy consumption of alcohol during pregnancy impairs brain development, it remains controversial whether moderate consumption causes significant damage. Using a limited access, voluntary consumption paradigm, we recently demonstrated that moderate prenatal alcohol exposure (MPAE) is associated with dentate gyrus-dependent learning and memory deficits that are manifested in adulthood. Here, we identified a novel mechanism that may underlie this effect of MPAE. We found that MPAE mice exhibit deficits in N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP) in the dentate gyrus. Further, using semi-quantitative immunoblotting techniques, we found that the levels of GluN2B subunits were decreased in the synaptic membrane, while levels of C2’-containing GluN1 and GluN3A subunits were increased, in the dentate gyrus of MPAE mice. These data suggest that MPAE alters the subunit composition of synaptic NMDARs, leading to impaired NMDAR-dependent LTP in the dentate gyrus.

Oxytocin is released from neurons in the paraventricular hypothalamic nucleus (PVN) in mothers upon suckling and during adult social interactions. However, neuronal pathways that activate oxytocin neurons in social contexts are not yet established. Neurons in the posterior intralaminar complex of the thalamus (PIL), which contain tuberoinfundibular peptide 39 (TIP39) and are activated by pup exposure in lactating mothers, provide a candidate projection. Innervation of oxytocin neurons by TIP39 neurons was examined by double labeling in combination with electron microscopy and retrograde tract-tracing. Potential classic neurotransmitters in TIP39 neurons were investigated by in situ hybridization histochemistry. Neurons activated after encounter with a familiar conspecific female in a familiar environment were mapped with the c-Fos technique. PVN and the supraoptic nucleus oxytocin neurons were closely apposed by an average of 2.0 and 0.4 TIP39 terminals, respectively. Asymmetric (presumed excitatory) synapses were found between TIP39 terminals and cell bodies of oxytocin neurons. In lactating rats, PIL TIP39 neurons were retrogradely labeled from the PVN. TIP39 neurons expressed vesicular glutamate transporter 2 but not glutamic acid decarboxylase 67. PIL contained a markedly increased number of c-Fos–positive neurons in response to social encounter with a familiar conspecific female. Furthermore, the PIL received ascending input from the spinal cord and the inferior colliculus. Thus, TIP39 neurons in the PIL may receive sensory input in response to social interactions and project to the PVN to innervate and excite oxytocin neurons, suggesting that the PIL-PVN projection contributes to the activation of oxytocin neurons in social contexts.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12868-016-0310-7) contains supplementary material, which is available to authorized users.

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.

Recent studies suggest that oxytocin (OXT) may be important for organising the neural circuitry that underlies adult social behaviour. Although most of the work exploring these effects has focused on early postnatal development, there is evidence that OXT may also be important during foetal development. However, without an understanding of how the OXT system develops, the ability to functionally link OXT in foetal life to adult behaviour is limited. To understand where and when OXT could be acting during embryonic development to affect the organisation of neural substrates, we examined the development of the mouse OXT system from embryonic day (E) 12.5 through postnatal day (PND) 2 using OXT receptor (OXTR) binding and a quantitative polymerase chain reaction. In both males and females, OXTR binding was observed by E16.5 in the ventricular and subventricular zones, as well as the developing amygdala. In males, OXT mRNA was not detectable until PND2, whereas it was detectable by E16.5 in females. OXTR mRNA was detected by E12.5 in both sexes, although females appear to have more OXTR mRNA during foetal development than males. The present study is significant because it is the first to reveal an unexpected sex difference in the development of the OXT system and supports the possibility that OXT during foetal development may contribute to sex differences in adult behaviour.

Previous work implicating the neuropeptide oxytocin (Oxt) in the neural regulation of aggression in males has been limited. However, there are reports of heightened aggression in Oxt knockout and Oxt receptor (Oxtr) knockout male mice when they are born to null mutant mothers; suggesting that intrauterine exposure to Oxt may be important to normal aggression in adulthood. To explore this, we examined aggression in two lines of Oxtr mice, a total knockout (Oxtr-/-), in which the Oxtr gene is absent from the time of conception, and a predominantly forebrain specific knockout (Oxtr FB/FB), in which the Oxtr gene is not excised until approximately 21-28days postnatally. Aggression was measured in males from both lines, as well as control littermates, using a resident-intruder behavioral test. Consistent with previous reports, male Oxtr-/- mice had elevated levels of aggression relative to controls. Oxtr FB/FB mice on the other hand displayed levels of aggression similar to control animals. In addition, following a resident-intruder test, Oxtr+/+ mice that displayed aggression had less c-fos immunoreactivity in the ventral portion of the lateral septum than those that did not. Further, Oxtr-/- mice had increased c-fos immunoreactivity in the medial amygdala relative to controls. These data suggest that Oxt may play an important role during development in the organization of the neural circuits that underlie aggressive behavior in adulthood, with its absence resulting in heightened aggression.

To date, much of the work in rodents implicating vasopressin (Avp) in the regulation of social behavior has focused on its action via the Avp 1a receptor (Avpr1a). However, there is mounting evidence that the Avp 1b receptor (Avpr1b) also plays a significant role in Avp's modulation of social behavior. The Avpr1b is heavily expressed on the anterior pituitary cortiocotrophs where it acts as an important modulator of the endocrine stress response. In the brain, the Avpr1b is prominent in the CA2 region of the hippocampus, but can also be found in areas such as the paraventricular nucleus of the hypothalamus and the olfactory bulb. Studies that have employed genetic knockouts or pharmacological manipulation of the Avpr1b point to the importance of central Avpr1b in the modulation of social behavior. However, there continues to be a knowledge gap in our understanding of where in the brain this is occurring, as well as how and if the central actions of Avp acting via the Avpr1b interact with the stress axis. In this review we focus on the genetic and pharmacological studies that have implicated the Avpr1b in the neural regulation of social behaviors, including social forms of aggressive behavior, social memory, and social motivation. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.

We have previously reported that mice with a targeted disruption of their vasopressin 1b receptor gene, Avpr1b, have mild impairments in social recognition and reduced aggression. The reductions in aggression are limited to social forms of aggression, i.e., maternal and inter-male aggression, while predatory aggression remains unaffected. To further clarify the role of the Avpr1b in the regulation of social behavior we first examined anxiety-like and depression-like behaviors in Avpr1b knockout (Avpr1b -/-) mice. We then went on to test the ability of Avpr1b -/- mice to form dominance hierarchies. No major differences were found between Avpr1b -/- and wildtype mice in anxiety-like behaviors, as measured using an elevated plus maze and an open field test, or depression-like behaviors, as measured using a forced swim test. In the social dominance study we found that Avpr1b -/- mice are able to form dominance hierarchies, though in early hierarchy formation dominant Avpr1b -/- mice display significantly more mounting behavior on Day 1 of testing compared to wildtype controls. Further, non-socially dominant Avpr1b -/- mice spend less time engaged in attack behavior than wildtype controls. These findings suggest that while Avpr1b -/- mice may be able to form dominance hierarchies they appear to employ alternate strategies.

The dentate gyrus (DG) is the central input region to the hippocampus and is known to play an important role in learning and memory. Previous studies have shown that prenatal alcohol is associated with hippocampal-dependent learning deficits and a decreased ability to elicit long term potentiation (LTP) in the DG in adult animals. Given that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade by N-methyl-D-aspartate (NMDA) receptors is required for various forms of learning and memory, as well as LTP, in hippocampal regions, including the DG, we hypothesized that fetal alcohol-exposed (FAE) adult animals would have deficits in hippocampal NMDA receptor-dependent ERK1/2 activation. We used immunoblotting and immunohistochemistry techniques to detect NMDA-stimulated ERK1/2 activation in acute hippocampal slices prepared from adult FAE mice. We present the first evidence linking prenatal alcohol exposure to deficits in NMDA receptor-dependent ERK1/2 activation specifically in the DG of adult offspring. This deficit may account for the LTP deficits previously observed in the DG, as well as the life-long cognitive deficits, associated with prenatal alcohol exposure.

Microinjection of arginine vasopressin (Avp) into the rostral hypothalamus of Syrian hamsters induces a form of scent marking known as flank marking. The ability of Avp to stimulate flank marking is mediated by the vasopressin 1a receptor (Avpr1a). In hamsters housed in long 'summer-like' photoperiods, the amount of flank marking and the number of Avpr1a receptors in the rostral hypothalamus are regulated by testosterone. However, hamsters housed in short 'winter-like' photoperiods for 6-8 weeks continue to flank mark at high levels despite significant reductions in the circulating levels of testosterone. In the present study, we compared the effects of gonadal steroids on Avp-induced flank marking and Avpr1 a binding and affinity in hamsters housed in short photoperiods and those housed in long photoperiods. In long-photoperiod-housed hamsters, castration significantly reduced the amount of Avp-induced flank marking; however, in short-photoperiod-housed hamsters there were no significant differences between gonadally regressed and castrated hamsters. Surprisingly, Avpr1a receptor binding, but not affinity, in the medial preoptic area and the medial preoptic nucleus was significantly reduced in long-photoperiod-housed castrates as well as short-photoperiod-housed gonadally regressed and castrated hamsters, compared with long-photoperiod-housed gonadally intact hamsters. These data demonstrate that in short photoperiods Avp-induced flank marking is independent of gonadal hormones, despite gonadal steroid-dependent reductions in Avpr1a binding.

Vasopressin (Avp) is mainly synthesized in the magnocellular cells of the hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) whose axons project to the posterior pituitary. Avp is then released into the blood stream upon appropriate stimulation (e.g., hemorrhage or dehydration) to act at the kidneys and blood vessels. The brain also contains several populations of smaller, parvocellular neurons whose projections remain within the brain. These populations are located within the PVN, bed nucleus of the stria terminalis (BNST), medial amygdala (MeA) and suprachiasmatic nucleus (SCN). Since the 1950s, research examining the roles of Avp in the brain and periphery has intensified. The development of specific agonists and antagonists for Avp receptors has allowed for a better elucidation of its contributions to physiology and behavior. Anatomical, pharmacological and transgenic, including "knockout," animal studies have implicated Avp in the regulation of various social behaviors across species. Avp plays a prominent role in the regulation of aggression, generally of facilitating or promoting it. Affiliation and certain aspects of pair-bonding are also influenced by Avp. Memory, one of the first brain functions of Avp that was investigated, has been implicated especially strongly in social recognition. The roles of Avp in stress, anxiety, and depressive states are areas of active exploration. In this review, we concentrate on the scientific progress that has been made in understanding the role of Avp in regulating these and other behaviors across species. We also discuss the implications for human behavior.

The Bruce effect is a pheromonally mediated process whereby exposure to chemosensory cues from an unfamiliar male terminates pregnancy in a recently mated female. Pharmacological and genetic evidence implicates both oxytocin (Oxt) and vasopressin (Avp) in the regulation of social memory in males, but less work has been done in females. We tested the extent to which the Avp receptors (Avprs) 1a and 1b and Oxt are essential for the Bruce effect, a phenomenon that relies on olfactory memory. Adult female mice were paired with stimulus males and monitored for the presence of sperm plugs. Wild-type, heterozygous, and homozygous knockout (KO) females for either the Avpr1a, Avpr1b, or Oxt genes were randomly assigned to one of the following treatment groups: 1) alone (mate removed, no second exposure to another animal); 2) paired continuously (mate kept with female for 10–14 d); 3) familiar male (mate removed, reintroduced 24 h later); or 4) unfamiliar male (mate removed, BalbC male introduced 24 h later). Regardless of genotype, 90–100% of females in the alone or paired continuously groups became pregnant. The Oxt KO females terminated their pregnancies regardless of whether their original mate or an unfamiliar male was reintroduced. The Avpr1b KO mice failed to terminate pregnancy in the presence of an unfamiliar male. The Avpr1a KO mice exhibited a normal Bruce effect. These data demonstrate that both Oxt and the Avpr1b are critical for the normal expression of the Bruce effect but have different effects on the interpretation of social cues.

Vasopressin affects behavior via its two brain receptors, the vasopressin 1a and vasopressin 1b receptors (Avpr1b). Recent work from our laboratory has shown that disruption of the Avpr1b gene reduces intermale aggression and reduces social motivation. Here, we further characterized the aggressive phenotype in Avpr1b -/- (knockout) mice. We tested maternal aggression and predatory behavior. We also analyzed the extent to which food deprivation and competition over food increases intermale aggression. We quantified defensive behavior in Avpr1b -/- mice and later tested offensive aggression in these same mice. Our results show that attack behavior toward a conspecific is consistently reduced in Avpr1b -/- mice. Predatory behavior is normal, suggesting that the deficit is not because of a global inability to detect and attack stimuli. Food deprivation, competition for food and previous experience increase aggression in both Avpr1b +/+ and -/- mice. However, in these circumstances, the level of aggression seen in knockout mice is still less than that observed in wild-type mice. Defensive avoidance behaviors, such as boxing and fleeing, are largely intact in knockout mice. Avpr1b -/- mice do not display as many 'retaliatory' attacks as the Avpr1b +/+ mice. Interestingly, when territorial aggression was measured following the defensive behavior testing, Avpr1b -/- mice typically show less initial aggressive behavior than wild-type mice, but do show a significant increase in aggression with repeated testing. These studies confirm that deficits in aggression in Avpr1b -/- mice are limited to aggressive behavior involving the attack of a conspecific. We hypothesize that Avpr1b plays an important role in the central processing that couples the detection and perception of social cues (which appears normal) with the appropriate behavioral response.

Oxytocin mediates social affiliation behaviors and social memory in rodents. It has been suggested that disruptions in oxytocin contribute to the deficits in reciprocal social interactions that characterize autism. The present experiments employed a new social approach task for mice which is designed to detect low levels of sociability, representing the first diagnostic criterion for autism. Two lines of oxytocin knockout mice were tested, the National Institute of Mental Health line in Bethesda, and the Baylor/Emory line at the University of North Carolina in Chapel Hill. Similar methods were used for each line to evaluate tendencies to spend time with a stranger mouse versus with an inanimate novel object with no social valence. Adult C57BL/6J males were tested identically, as controls to confirm the robustness of the methods used in the social task. Comprehensive phenotyping of general health, neurological reflexes, olfactory and other sensory abilities, and motor functions was employed to assess both lines. No genotype differences were detected in any of the control measures for either line. Normal sociability, measured as time spent with a novel stranger mouse as compared to time spent with a novel object, was seen in both the NIMH and the Baylor/Emory lines of oxytocin null mutants, heterozygotes, and wild-type littermate controls. Normal preference for social novelty, measured as time spent with a second novel stranger as compared to time spent with a more familiar mouse, was seen in both the NIMH and the Baylor/Emory lines of oxytocin null mutants, heterozygotes, and wild-type littermate controls, with minor exceptions. Similar behavioral results from two independent targeted gene mutations, generated with different targeting vectors, bred on different genetic backgrounds, and tested in different laboratory environments, corroborates the negative findings on sociability in oxytocin mutant mice. Intact tendencies to spend time with another mouse versus with a novel object, in both lines of oxytocin knockouts, supports an interpretation that oxytocin plays a highly specific role in social memory, but is not essential for general spontaneous social approach in mice.

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.

Studies of the role of vasopressin (Avp) in mediating the effects of ethanol have focused on Avp’s role in altering kidney function via its action through the vasopressin 2 receptor. However, alcohol consumption also has central effects that are poorly understood. There is evidence that Avp may mediate ethanol consumption as well as some of ethanol’s behavioral effects. Centrally only two Avp receptor subtypes are expressed: the 1a receptor (Avpr1a) and the 1b receptor (Avpr1b). To determine the extent to which these receptors mediate the behavioral effects of alcohol, we used mice with targeted disruptions of either their Avpr1a or Avpr1b gene. We examined the effects of genotype on the acute intoxicating effects of ethanol as well as on voluntary ethanol consumption. Surprisingly, our findings indicate that there is no interaction between either the Avpr1a or Avpr1b and ethanol on motor coordination, hypothermia, mood, or voluntary ethanol consumption.

The extracellular signal-regulated protein kinases (ERKs) are proline-directed, serine/threonine kinases that regulate a variety of cellular functions, including proliferation, differentiation, and plasticity. In the present report, we provide evidence that ERK2 and phosphatidylinositol-specific phospholipase C (PLC)-beta and -gamma isozymes interact in the rat hippocampal formation. We found that anti-PLC-beta1a, -beta2, -beta4, -gamma1 and -gamma2, but not -beta3, immune complexes isolated from rat hippocampal formation postnuclear fractions contain anti-ERK2 immunoreactivity. Further, we show that PLC catalytic activity is associated with anti-ERK2 immunoprecipitates isolated from the hippocampal formation, and that the amount of enzyme activity is significantly increased following fear-conditioned learning. The observed interactions may be mediated by consensus sequences conforming to an ERK2 docking site, termed a D-domain, that we identified in PLC-beta1a, -beta2, -beta4 -gamma1 and -gamma2. Based on these results, we propose that PLC-beta and PLC-gamma isozymes form signaling complexes with ERK2 in rat brain, and these complexes play critical roles in learning and memory, as well as a variety of other neuronal functions.

Syrian hamsters are photoperiodic and become sexually quiescent when exposed to short "winter-like" photoperiods. In short photoperiods, male hamsters display significantly higher levels of aggression than males housed in long photoperiods. Arginine-vasopressin (AVP) within the anterior hypothalamus (AH) has been reported to modulate aggression in hamsters housed in long photoperiods. Previous studies have shown that AVP can facilitate aggression and its effects appear to be mediated by AVP V(1a) receptors (V(1a)R). In the present study, we investigated whether the increased levels of aggression observed after exposure to short photoperiod were the result of an increased responsiveness to AVP within the AH. Injections of AVP into the AH significantly increased aggression in hamsters housed in a long photoperiod, but had no effect in hamsters housed in a short photoperiod. In addition, injection of a V(1a)R antagonist into the AH significantly inhibited aggression in hamsters housed in long photoperiod, but had no effect in hamsters housed in a short photoperiod. These findings indicate that AVP within the AH increases aggression in hamsters housed in long photoperiods, but not in hamsters housed in short photoperiods.

Background: Previous studies in our laboratory indicated that metabotropic glutamate receptor (mGluR)-stimulated phosphoinositide hydrolysis is markedly reduced in the hippocampal formation of adult rat offspring whose mothers drank moderate amounts of ethanol during pregnancy. In the present study, we extended these observations by measuring the impact of prenatal ethanol exposure on proteins associated with the mGluR 5 receptor-effector system along with two mGluR 5 agonist-mediated responses in dentate gyrus of adult offspring. Methods: Sprague-Dawley rat dams consumed one of three diets throughout gestation: (1) a BioServ liquid diet that contained 5% ethanol (v/v), (2) pair-fed an isocalorically equivalent amount of 0% ethanol liquid diet, or (3) lab chow ad libitum. Microdissected slices of dentate gyrus were prepared from adult female offspring from each diet group and used for (1) Western blot analyses of mGluR 5 , the G-proteins Gα q and Gα 1 1 , and phospholipase C-pl; (2) 2-chloro-5-hydroxyphenylglycine (CHPG)-stimulated growth associated protein 43 (GAP-43) phosphorylation; or (3) CHPG potentiation of electrically evoked [ 3 H]-D-aspartate (D-ASP) release from dentate gyrus slices. Results: In tissue prepared from untreated control rats, CHPG produced a dose-dependent increase in 3 2 phosphate incorporation into GAP-43, with maximal agonist stimulation occurring at 20 μM of CHPG. CHPG produced a quantitatively similar dose-dependent increase in the potentiation of electrically evoked D-ASP release from dentate gyrus slices from untreated controls. Fetal ethanol exposure reduced the amount of dentate gyrus mGluR 5 receptor protein by 36% compared with the diet control groups. There were no significant differences between diet groups in the two G-proteins or phospholipase C-β1 protein. Fetal ethanol exposure reduced CHPG-stimulated GAP-43 phosphorylation to approximately one half the amount of CHPG stimulation observed in the control diet groups. Prenatal ethanol exposure also reduced CHPG potentiation of D-ASP release to a similar degree compared with control. Conclusions: These results indicate that prenatal exposure to moderate quantities of ethanol reduces mGluR 5 expression in the dentate gyrus of adult offspring. Although the subcellular site(s) for reduced mGluR 5 expression cannot be discerned from Western blot data, the quantitatively similar effects of prenatal ethanol exposure on mGluR 5 agonist stimulation of presynaptically localized GAP-43 phosphorylation and CHPG potentiation of evoked D-ASP release suggest that the presynaptic nerve terminal is one site where prenatal ethanol exposure has reduced mGluR 5 receptor number and function. Furthermore, these data implicate these neurochemical alterations as one factor contributing to the hippocampal synaptic plasticity and behavioral deficits that we have observed previously in prenatal ethanol-exposed offspring.

Background: The incidence of fetal alcohol spectrum disorders is estimated to be as high as 1 in 100 births. Efforts to better understand the basis of prenatal ethanol-induced impairments in brain functioning, and the mechanisms by which ethanol produces these defects, will rely on the use of animal models of fetal alcohol exposure (FAE). Methods: Using a saccharin-sweetened alcohol solution, we developed a free-choice, moderate alcohol access model of prenatal alcohol exposure. Stable drinking of a saccharin solution (0.066%) was established in female mice. Ethanol then was added to the saccharin in increasing concentrations (2%, 5%, 10% w/v) every 2 days. Water was always available, and mice consumed standard pellet chow. Control mice drank saccharin solution without ethanol. After a stable baseline of ethanol consumption (14 g/kg/day) was obtained, females were impregnated. Ethanol consumption continued throughout pregnancy and then was decreased to 0% in a step-wise fashion over a period of 6 days after pups were delivered. Characterization of the model included measurements of maternal drinking patterns, blood alcohol levels, food consumption, litter size, pup weight, pup retrieval times for the dams, and effects of FAE on performance in fear-conditioned learning and novelty exploration. Results: Maternal food consumption, maternal care, and litter size and number were all found to be similar for the alcohol-exposed and saccharin control animals. FAE did not alter locomotor activity in an open field but did increase the time spent inspecting a novel object introduced into the open field. FAE mice displayed reduced contextual fear when trained using a delay fear conditioning procedure. Conclusions: The mouse model should be a useful tool in testing hypotheses about the neural mechanisms underlying the learning deficits present in fetal alcohol spectrum disorders. Moreover, a mouse prenatal ethanol model should increase the opportunity to use the power of genetically defined and genetically altered mouse populations.

In vitro quantification of the catalytic activity of an enzyme isoform requires the availability of selective agents that allow for either measurements in the presence of the other enzyme isoforms or purification of the isoform and subsequent performance of these measurements on the purified enzyme. Isozyme-specific antibodies are useful tools for these types of analyses. In the present report, we detail a method for the measurement of phospholipase C-gamma1 enzyme activity employing native enzyme that is immobilized on microtiter plates. The method uses biotinylated antiglobulin bound to streptavidin-coated microtiter plates to immobilize antiphospholipase C-gamma1 antibody and subsequently capture phospholipase C-gamma1 from brain tissue lysates. This method avoids biotinylation of the primary (antiphospholipase C-gamma1) antibody, making it less labor intensive than previously described methods for using streptavidin-coated plates; in addition, it is highly reproducible and sensitive and allows for quantification of enzyme activity. We employ the technique to show that one or more tyrosine kinases copurify with rat brain phospholipase C-gamma1. The method is applicable to the study of any enzyme isoform for which antibodies that capture the native form of the enzyme are available and could easily be employed in high-throughput procedures.

We investigated a novel strategy for measuring the synthesis rate of proteins in skeletal and cardiac muscle. Mass isotopomer distribution analysis allows measurement of the isotopic enrichment of the true biosynthetic precursor for proteins (tRNA-amino acids), but cannot easily be applied to slow turnover muscle proteins due to insufficient isotope incorporation into multiply labeled species. Using a rapid turnover protein from the same tissue, however, might reveal tRNA-amino acid enrichment. We tested this strategy in rats on muscle creatine kinase (CK). A trypsinization peptide (3647 u) containing 5 leucine repeats was identified by computer-simulated digestion of CK and then isolated from trypsin hydrolysates. Mass isotopomer abundances were determined by electrospray ionization-magnetic sector-mass spectrometry after in vivo administration of [ 2 H 3 ]leucine. Myosin heavy chain was also isolated and hydrolyzed to free amino acids. Muscle tRNA-amino acids were well labeled, by direct measurement. Enrichments of M+1 and M+2 mass isotopomers in the CK-peptide were measurable but low (consistent with a CK half-life of 3–10 days). Incorporation into skeletal muscle myosin indicated a half-life of 54 days. In conclusion, the general strategy of measuring protein kinetics by quantifying mass isotopomer abundances of mid-sized peptides from protein hydrolysates is effective, but CK does not turn over rapidly in muscle, contrary to previous reports. Identification of a rapid turnover muscle protein would be useful for this purpose.

One brain region that has been implicated in the regulation of lordosis is the medial preoptic-anterior hypothalamic continuum (MPOA-AH). Previous studies have suggested that this zone may be part of the circuit mediating the effects of serotonin (5-HT) on sexual receptivity. In the present experiments, we investigated the role of 5-HT 1a/7 and 5-HT 2 receptor subtypes in the MPOA-AH in the control of lordosis. In two experiments, either 5-HT 1a/7 or 5-HT 2 agonists were injected unilaterally into the MPOA-AH of ovariectomized, hormonally primed female hamsters. In the first experiment, microinjections of the 5-HT 1a/7 agonist 8-hydroxy-2,9-(di- n -propylamino)tetralin resulted in an attenuation of the lordosis posture by causing a decrease in the mean lordosis duration and an increase in the number of lordosis episodes over the entire testing period. In the second experiment, microinjections of the 5-HT 2b/2c agonist m -chlorophenylpiperazine did not result in any changes in sexual receptivity. However, microinjections of the 5-HT 2 agonist (2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl facilitated lordosis by increasing the mean lordosis duration and decreasing the number of lordosis episodes in the first 5 min of the testing period. These data indicate that serotonin may act in the MPOA-AH via 5-HT 1a/7 receptors to attenuate and 5-HT 2 receptors to facilitate sexual receptivity.

The preputial glands of house mice express the gene for the fifth melanocortin receptor (MC5-R) and are a primary source of urinary pheromones involved in inter-male aggression. A 'resident-intruder' behavioral model was used to compare the responses of resident males to urine from mice with an engineered disruption of the fifth melanocortin receptor (MC5-RKO) with residents' responses to urine from wild-type mice (WT). Each type of urine was presented in combination with a castrated intruder male to provide the appropriate biological context. Resident males responded with a longer latency to bite when the urine was from gonadally intact WT males compared with urine from MC5-RKO mice. These results are consistent with the hypothesis that activation of the fifth melanocortin receptor in the preputial glands of male house mice causes excretion of urinary pheromones that delay aggressive responses by other males.

The function of the CA2 region of the hippocampus is poorly understood. While 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 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. While 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 (NMDA) lesions of the CA2 region in mice 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 since social memory requires a functional olfactory system. Consistent with our prediction, we found that CA2 lesioned animals have impaired social recognition. These findings are significant because they confirm 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.

The D3-phosphoinositides act as second messengers by recruiting, and thereby activating, diverse signaling proteins. We have previously described the purification of a rat phosphatidylinositol 3-phosphate [PtdIns(3)P] 3-phosphatase, comprising a heterodimer of a 78-kDa adapter subunit in complex with a 65-kDa catalytic subunit. Here, we have cloned and characterized the cDNA encoding the human 3-phosphatase adapter subunit (3-PAP). Sequence alignment showed that 3-PAP shares significant sequence similarity with the protein and lipid 3-phosphatase myotubularin, and with several other members of the myotubularin gene family including SET-binding factor 1. However, unlike myotubularin, 3-PAP does not contain a consensus HCX 5 R catalytic motif. The 3-PAP sequence contains several motifs that predict interaction with proteins containing Src homology-2 (SH2) domains, phosphotyrosine-binding (PTB) domains, members of the 14-3-3 family, as well as proteins with SET domains. Northern blot analysis identified two transcripts (5.5 kb and 2.5 kb) with highest abundance in human liver, kidney, lung, and placenta. 3-PAP immunoprecipitates isolated from platelet cytosol hydrolyzed the D3-phosphate from PtdIns(3)P and PtdIns 3,4-bisphosphate [PtdIns(3,4)P 2 ]. However, insect cell-expressed 3-PAP recombinant protein was catalytically inactive, confirming our prior prediction that this polypeptide represents an adapter subunit.

In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE receptor (FcepsilonRI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P3 production. Using immune complex phospholipase assays, we show that FcepsilonRI cross-linking activates both PLCgamma1 and PLCgamma2. Activation is accompanied by the increased phosphorylation of both PLCgamma isoforms on serine and tyrosine in antigen-treated cells. We also show that the two PLCgamma isoforms have distinct subcellular localizations. PLCgamma1 is primarily cytosolic in resting RBL-2H3 cells, with low levels of plasma membrane association. After antigen stimulation, PLCgamma1 translocates to the plasma membrane where it associates preferentially with membrane ruffles. In contrast, PLCgamma2 is concentrated in a perinuclear region near the Golgi and adjacent to the plasma membrane in resting cells and does not redistribute appreciably after FcepsilonRI cross-linking. The activation of PLCgamma1, but not of PLCgamma2, is blocked by wortmannin, a PI 3-kinase inhibitor previously shown to block antigen-stimulated ruffling and to inhibit Ins(1,4,5)P3 synthesis. In addition, wortmannin strongly inhibits the antigen-stimulated phosphorylation of both serine and tyrosine residues on PLCgamma1 with little inhibition of PLCgamma2 phosphorylation. Wortmannin also blocks the antigen-stimulated translocation of PLCgamma1 to the plasma membrane. Our results implicate PI 3-kinase in the phosphorylation, translocation, and activation of PLCgamma1. Although less abundant than PLCgamma2, activated PLCgamma1 may be responsible for the bulk of antigen-stimulated Ins(1,4,5)P3 production in RBL-2H3 cells.

The metabolic peptide hormone nesfatin-1 has been linked to the reproductive axis in fishes. The purpose of this study was to determine how energy availability after spawning affects plasma levels of nesfatin-1, the metabolic peptide hormone ghrelin, and sex steroid hormones in rematuring female rainbow trout (Oncorhynchus mykiss). To limit reproductive maturation, a group of female trout was food-restricted after spawning and compared with a control group that was fed a standard broodstock ration. The experiment was conducted twice, once using two-year-old trout (second-time spawners) and once using three-year-old trout (third-time spawners). During monthly sampling, blood was collected from all fish, and a subset of fish from each treatment was sacrificed for pituitaries. Pituitary follicle-stimulating hormone-beta (fsh-β) mRNA expression was analyzed with q-RT-PCR; plasma hormone levels were quantified by radioimmunoassay (17β-estradiol and ghrelin) and enzyme-linked immunosorbent assay (11-keto-testosterone and nesfatin-1). Although plasma nesfatin-1 levels increased significantly in the months immediately after spawning within both feeding treatments, plasma nesfatin-1 did not differ significantly between the two treatments at any point. Similarly, plasma ghrelin levels did not differ significantly between the two treatments at any point. Food restriction arrested ovarian development by 15–20 weeks after spawning, shown by significantly lower plasma E2 levels among restricted-ration fish. Pituitary fsh-β mRNA levels were higher among control-ration fish than restricted-ration fish starting at 20 weeks, but did not differ significantly between treatment groups until 30 weeks after spawning. Within both treatment groups, plasma 11-KT was elevated immediately after spawning and rapidly decreased to and persisted at low levels; starting between 20 and 25 weeks after spawning, plasma 11-KT was higher among control-ration fish than restricted-ration fish. The results from these experiments do not provide support for plasma nesfatin-1 as a signal for the initiation of reproductive development in rematuring female rainbow trout.