The BDNF Val68Met polymorphism causes a sex specific alcohol preference over social interaction and also acute tolerance to the anxiolytic effects of alcohol, a phenotype driven by malfunction of BDNF in the ventral hippocampus of male mice.

BACKGROUND: The brain-derived neurotrophic factor (BDNF) Valine 66 to Methionine human polymorphism results in impaired activity-dependent BDNF release and has been linked to psychiatric disorders including depression and anxiety. We previously showed that male knock-in mice carrying the mouse Methionine homolog (Met68BDNF) exhibit excessive and compulsive alcohol drinking behaviors as compared to the wild-type Val68BDNF mice. OBJECTIVE: Here, we set out to determine the potential mechanism for the heightened and compulsive alcohol drinking phenotypes detected in Met68BDNF mice. RESULTS: We found that male, but not female Met68BDNF mice exhibit social anxiety-like behaviors. We further show that male Met68BDNF mice exhibit a preference for alcohol over social interaction. In contrast, alcohol place preference without an alternative social reward, is similar in male Met68BDNF and Val68BDNF mice. Since the Met68BDNF mice show social anxiety phenotypes, we tested whether alcohol reliefs anxiety similarly in Met68BDNF and Val68BDNF mice and found that male, but not female Met68BDNF mice are insensitive to the acute anxiolytic action of alcohol. Finally, we show that this acute tolerance to alcohol-dependent anxiolysis can be restored by overexpressing wild-type Val68BDNF in the ventral hippocampus (vHC) of Met68BDNF mice. CONCLUSIONS: Together, our results suggest that excessive alcohol drinking in the Met68BDNF may be attributed, in part, to heighted social anxiety and a lack of alcohol-dependent anxiolysis, a phenotype that is associated with malfunction of BDNF signaling in the vHC of male Met68BDNF mice.

Differential correlation of serum BDNF and microRNA content in rats with rapid or late onset of heavy alcohol use.

Heavy alcohol use reduces the levels of the brain-derived neurotrophic factor (BDNF) in the prefrontal cortex of rodents through the upregulation of microRNAs (miRs) targeting BDNF mRNA. In humans, an inverse correlation exists between circulating blood levels of BDNF and the severity of psychiatric disorders including alcohol abuse. Here, we set out to determine whether a history of heavy alcohol use produces comparable alterations in the blood of rats. We used an intermittent access to 20% alcohol using the two-bottle choice paradigm (IA20%2BC) and measured circulating levels of BDNF protein and miRs targeting BDNF in the serum of Long-Evans rats before and after 8 weeks of excessive alcohol intake. We observed that the drinking profile of heavy alcohol users is not unified, whereas 70% of the rats gradually escalate their alcohol intake (late onset), and 30% of alcohol users exhibit a very rapid onset of drinking (rapid onset). We found that serum BDNF levels are negatively correlated with alcohol intake in both rapid onset and late onset rats. In contrast, increased expression of the miRs targeting BDNF, miR30a-5p, miR-195-5p, miR191-5p and miR206-3p, was detected only in the rapid onset rats. Finally, we report that the alcohol-dependent molecular changes are not due to alterations in platelet number. Together, these data suggest that rats exhibit both late and rapid onset of alcohol intake. We further show that heavy alcohol use produces comparable changes in BDNF protein levels in both groups. However, circulating microRNAs are responsive to alcohol only in the rapid onset rats.

cAMP-Fyn signaling in the dorsomedial striatum direct pathway drives excessive alcohol use.

Fyn kinase in the dorsomedial striatum (DMS) of rodents plays a central role in mechanisms underlying excessive alcohol intake. The DMS is comprised of medium spiny neurons (MSNs) that project directly (dMSNs) or indirectly (iMSNs) to the substantia nigra. Here, we examined the cell-type specificity of Fyn's actions in alcohol use. First, we knocked down Fyn selectively in DMS dMSNs or iMSNs of mice and measured the level of alcohol consumption. We found that downregulation of Fyn in dMSNs, but not in iMSNs, reduces excessive alcohol but not saccharin intake. D1Rs are coupled to Gαs/olf, which activate cAMP signaling. To examine whether Fyn's actions are mediated through cAMP signaling, DMS dMSNs were infected with GαsDREADD, and the activation of Fyn signaling was measured following CNO treatment. We found that remote stimulation of cAMP signaling in DMS dMSNs activates Fyn and promotes the phosphorylation of the Fyn substrate, GluN2B. In contract, remote activation of GαsDREADD in DLS dMSNs did not alter Fyn signaling. We then tested whether activation of GαsDREADD in DMS dMSNs or iMSNs alters alcohol intake and observed that CNO-dependent activation of GαsDREADD in DMS dMSNs but not iMSNs increases alcohol but not saccharin intake. Finally, we examined the contribution of Fyn to GαsDREADD-dependent increase in alcohol intake, and found that systemic administration of the Fyn inhibitor, AZD0503 blocks GαsDREADD-dependent increase in alcohol consumption. Our results suggest that the cAMP-Fyn axis in the DMS dMSNs is a molecular transducer of mechanisms underlying the development of excessive alcohol consumption.

mTORC2 in the dorsomedial striatum of mice contributes to alcohol-dependent F-Actin polymerization, structural modifications, and consumption.

Actin is highly enriched at dendritic spines, and actin remodeling plays an essential role in structural plasticity. The mammalian target of rapamycin complex 2 (mTORC2) is a regulator of actin polymerization. Here, we report that alcohol consumption increases F-actin content in the dorsomedial striatum (DMS) of mice, thereby altering dendritic spine morphology in a mechanism that requires mTORC2. Specifically, we found that excessive alcohol consumption increases mTORC2 activity in the DMS, and that knockdown of Rictor, an essential component of mTORC2 signaling, reduces actin polymerization, and attenuates the alcohol-dependent alterations in spine head size and the number of mushroom spines. Finally, we show that knockdown of Rictor in the DMS reduces alcohol consumption, whereas intra-DMS infusion of the mTORC2 activator, A-443654, increases alcohol intake. Together, these results suggest that mTORC2 in the DMS facilitates the formation of F-actin, which in turn induces changes in spine structure to promote and/or maintain excessive alcohol intake.

Prosapip1-Dependent Synaptic Adaptations in the Nucleus Accumbens Drive Alcohol Intake, Seeking, and Reward.

The mammalian target of rapamycin complex 1 (mTORC1), a transducer of local dendritic translation, participates in learning and memory processes as well as in mechanisms underlying alcohol-drinking behaviors. Using an unbiased RNA-seq approach, we identified Prosapip1 as a novel downstream target of mTORC1 whose translation and consequent synaptic protein expression are increased in the nucleus accumbens (NAc) of mice excessively consuming alcohol. We demonstrate that alcohol-dependent increases in Prosapip1 levels promote the formation of actin filaments, leading to changes in dendritic spine morphology of NAc medium spiny neurons (MSNs). We further demonstrate that Prosapip1 is required for alcohol-dependent synaptic localization of GluA2 lacking AMPA receptors in NAc shell MSNs. Finally, we present data implicating Prosapip1 in mechanisms underlying alcohol self-administration and reward. Together, these data suggest that Prosapip1 in the NAc is a molecular transducer of structural and synaptic alterations that drive and/or maintain excessive alcohol use.

Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum.

The tyrosine kinase Fyn plays an important role in synaptic plasticity, learning, and memory. Here we report that Fyn is activated in response to 15 min D1 receptor (D1R) but not D2 receptor (D2R) stimulation specifically in the dorsomedial striatum (DMS) of mice but not in the other substriatal regions, the dorsolateral striatum (DLS), and the nucleus accumbens (NAc). Once activated Fyn phosphorylates its substrate GluN2B, and we show that GluN2B is phosphorylated only in the DMS but not in the other striatal regions. Striatal neurons are divided into D1R expressing medium spiny neurons (MSNs) and D2R expressing MSNs. Thus, to explore the cell-type specificity of this signaling pathway in the DMS, we developed a Cre-dependent Flip Excision (FLEX) approach to knockdown Fyn in D1R MSNs or D2R MSNs, and proved that the D1R-dependent Fyn activation is localized to DMS D1R MSNs. Importantly, we provide evidence to suggest that the differential association of Fyn and GluN2B with the scaffolding RACK1 is due to the differential localization of Fyn in lipid rafts.Our data further suggest that the differential cholesterol content in the three striatal regions may determine the region specificity of this signaling pathway. Together, our data show that the D1R-dependent Fyn/GluN2B pathway is selectively activated in D1R expressing MSNs in the DMS, and that the brain region specificity of pathway depends on the molecular and cellular compartmentalization of Fyn and GluN2B.

The influence of early maternal care on perceptual attentional set shifting and stress reactivity in adult rats.

Stress influences a wide variety of outcomes including cognitive processing. In the rat, early life maternal care can influence developing offspring to affect both stress reactivity and cognitive processes in adulthood. The current study assessed if variations in early life maternal care can influence cognitive performance on a task, the ability to switch cognitive sets, dependent on the medial prefrontal cortex. Early in life, offspring was reared under High or Low maternal Licking conditions. As adults, they were trained daily and then tested on an attentional set-shifting task (ASST), which targets cognitive flexibility in rodents. Stress-sensitive behavioral and neural markers were assayed before and after the ASST. High and Low Licking offspring performed equally well on the ASST despite initial, but not later, differences in stress axis functioning. These results suggest that early life maternal care does not impact the accuracy of attentional set-shifting in rats. These findings may be of particular importance for those interested in the relationship between early life experience and adult cognitive function.

Glucocorticoids and the Brain: Neural Mechanisms Regulating the Stress Response.

In this chapter, we describe the central role of the brain in the glucocorticoid mediated stress response. We describe the mechanisms by which the brain gauges the severity of stress, mechanisms of hypothalamic-pituitary-adrenal axis (HPA) regulation, and how various sub-systems of the brain respond to glucocorticoid (GC) signaling to regulate stress behavior. In particular, we focus on the hippocampus, pre-frontal cortex, and amygdala, where GCs can induce a series of changes. Finally, we briefly discuss an apparent paradox in GC signaling: while exposure to glucocorticoids promotes the survival of an organism during acute stress, these same hormones in chronic excess can also cause damage and promote illness.

Influence of housing variables on the development of stress-sensitive behaviors in the rat.

Diverse environments early in mammalian life can have profound influences on the physiology and behavior of developing offspring. Environmental factors can influence offspring development directly or through perturbations in parental care. In the current study, we wished to determine if the influence of a single environmental variable, type of bedding material used in laboratory cages, is capable of altering physiological and behavioral outcomes in offspring. Female rats were housed in cages containing wood pulp or corncob bedding and allowed to mature. These rats, while housed on assigned bedding material, were bred and allowed to give birth. At weaning, male offspring were housed on one of the two bedding conditions and tested later in adulthood on stress-sensitive behavioral measures. Postmortem analysis of glucocorticoid receptor expression and CRH mRNA levels were also measured. Maternal care directed at the pups reared in the two different bedding conditions was also recorded. Rats reared from birth on corncob bedding exhibited decreased anxiety-like behavior, as adults, in both open field and light-dark box tasks compared to wood pulp reared animals. Animals that received similar overall levels of maternal care, regardless of bedding condition, also differed in anxiety-like behaviors as adults, indicating that the bedding condition is capable of altering phenotype independent of maternal care. Despite observed behavioral differences in adult offspring reared in different bedding conditions, no changes in glucocorticoid receptor expression at the level of the hippocampus, frontal cortex, or corticotrophin releasing hormone (CRH) mRNA expression in the hypothalamus were observed between groups. These results highlight the importance of early life housing variables in programming stress-sensitive behaviors in adult offspring.

Maternal programming of sexual attractivity in female Long Evans rats.

In mammals, maternal care influences the developing offspring across multiple domains. In Long Evans rats, for example, the quality of maternal care received as a pup influences later cognitive function, neuroendocrine responses to stress and behavioral measures of emotionality. Data from humans, non-human primates, and rodents also suggest that early life events may similarly perturb measures of sexual reproduction, with possible consequences for reproductive fitness. The current study examined whether or not male conspecifics differentially prefer females, as adult mating partners, that were reared under varying maternal conditions (assessed via the quantity of licking and grooming received; LG). Additionally, the impact of maternal care on adult female sexual motivation and behavior were quantified to determine if these behavioral characteristics are associated with any preference observed. In a mate preference task, male rats chose, almost exclusively, to mount, copulate and ejaculate with female rats reared under Low LG conditions. Under non-paced mating conditions, female Low LG rats display significantly more paracopulatory and copulatory behaviors compared to High LG rats. Due to its critical role in female paracopulatory behavior, progesterone receptor immunoreactivity (PR-ir) in the ventromedial nucleus of the hypothalamus (VMH) was also assessed in both groups of female rats. Estradiol induced PR-ir in the VMH was significantly higher in Low LG relative to High LG rats. Together, these data suggests that early life parental care may developmentally program aspects of behavior and physiology that subsequently influence sexual attractivity and behavior in adult females.

Toward an integrative understanding of social behavior: new models and new opportunities.

Social interactions among conspecifics are a fundamental and adaptively significant component of the biology of numerous species. Such interactions give rise to group living as well as many of the complex forms of cooperation and conflict that occur within animal groups. Although previous conceptual models have focused on the ecological causes and fitness consequences of variation in social interactions, recent developments in endocrinology, neuroscience, and molecular genetics offer exciting opportunities to develop more integrated research programs that will facilitate new insights into the physiological causes and consequences of social variation. Here, we propose an integrative framework of social behavior that emphasizes relationships between ultimate-level function and proximate-level mechanism, thereby providing a foundation for exploring the full diversity of factors that underlie variation in social interactions, and ultimately sociality. In addition to identifying new model systems for the study of human psychopathologies, this framework provides a mechanistic basis for predicting how social behavior will change in response to environmental variation. We argue that the study of non-model organisms is essential for implementing this integrative model of social behavior because such species can be studied simultaneously in the lab and field, thereby allowing integration of rigorously controlled experimental manipulations with detailed observations of the ecological contexts in which interactions among conspecifics occur.