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Estrogen, interneuron maturation and sex differences in depression following alcohol use

MOTIVATION: Depression is a debilitating neuropsychiatric disorder whose constellation of symptoms may include both dampened sensitivity to positive stimuli, and heightened sensitivity to negative emotions. With a lifetime incidence of 20-35%1, depression is one of the leading causes of disability worldwide. Problematic alcohol use and depressive symptoms often co-occur2,3, with data supporting a causal relationship between binge drinking and the expression of depression in social drinkers4. This offers a unique opportunity to unpack the biological mechanisms driving increased vulnerability to a less heterogeneous subtype of depression. Human and rodent studies suggest that biological sex may moderate this relationship5,6, with stronger associations between binge drinking and depression onset shown for females6. Given the growing problem of binge drinking in women and girls, understanding how this pattern of alcohol consumption may enhance vulnerability to depression in females should be a significant women’s health initiative. Dr. Melón began to tackle this question during her graduate training where she showed that the sex-specific development of depressive-like behavior following binge drinking involved long-term upregulation of Gabrd gene expression in the amygdala7. Gabrd encodes a subunit protein, δ, found in a subtype of the chloride permeable GABAA receptors that mediate a significant portion of fast inhibitory signaling in the brain. For her postdoctoral training, Dr. Melón developed and used tools to interrogate GABAergic dysfunction at a cellular level to understand its role in driving stress reactivity and depression vulnerability during the postpartum period8. Dr. Melón also expanded her investigation of alcohol’s effects on this system which revealed a significant role for a specific class of GABA releasing interneurons in the effects of binge drinking9. Dr. Melón's independent lab uses mouse models to determine how binge drinking interacts with factors associated with reproductive transitions to promote sex differences in depression vulnerability across the lifespan.

Fig. 1: The Melón lab will assess and manipulate system wide factors to understand the relationship between gonadal hormones, inhibitory interneuron development and the shaping of windows of vulnerability to depression by environmental factors like binge drinking and peer and infant directed social interaction.

OBJECTIVE: The long-term goal of the Melón Lab is to determine mechanisms that drive sex differences in the development of disorders associated with alcohol use across the lifespan. Our immediate objective is to determine what role fast-spiking parvalbumin interneurons play in the reciprocal regulation of binge drinking and vulnerability to depression across reproductive transitions. By combining innovative technologies in genetics, pharmacology and neurophysiology as well as strong behavioral neuroscience approaches, our short-term efforts will be focused on progressing three fundamental questions:

I. Do developmental shifts in estrogen alter fast-spiking interneuron function to drive changes in depression vulnerability across the reproductive lifecycle in females?

II. How does binge alcohol exposure interact with estrogen to promote vulnerability to depression in females?

III. Are the effects of peripubertal alcohol drinking on infant-directed behaviors (retrieval, nest building, aggression) in adulthood due to perturbations in the estrogen-dependent maturation of GABAergic signaling across the hypothalamo-mesolimbic parental circuit?

Project 1: Reproductive transitions, interneuronopathy and depression vulnerability

SIGNIFICANCE: From the onset of puberty to the end of perimenopause, the lifetime risk for developing depression is 2 fold greater for women than men11. Within a woman’s reproductive lifecycle, unique epochs characterized by significant changes in gonadal hormones- namely, puberty, peripartum and perimenopause- all represent periods of enhanced risk to depression12. My lab will assess how these developmental shifts in estradiol, the main ovarian hormone in women, regulate the dynamic inhibitory connectome and its ability to mediate communication across neural circuits that underlie positive affect.

Fast inhibitory GABAergic signaling, mediated by a diverse class of chloride permeable GABAA receptors which open in response to GABA released by an equally diverse class of inhibitory interneurons, make up much of our central nervous system’s inhibitory connectome. Estradiol has been shown to both upregulate the expression of genes that encode proteins for various GABAA receptor subunits and alter phosphorylation and membrane stability of transporter proteins that regulate internal chloride homeostasis. These changes can have substantive impacts on local inhibitory signaling in a cell and circuit restricted manner. In particular, parvalbumin-expressing inhibitory interneurons with fast-spiking characteristics and expansive basket-like structures are physiologically and physically poised to play a significant role in maintaining and regulating high frequency rhythmic activity locally as well as the coherence of these local rhythms with slower rhythms that may mediate long-range communication across nodes of a neuronal circuit. Interestingly, much of the maturation of fast-spiking parvalbumin positive interneurons occurs at puberty, within an environment of rising gonadal hormones. These neuron-associated adaptations, whether driven by the genomic or non-genomic effects of gonadal hormones, offer opportunities for cell and circuit restricted modulations of inhibitory signaling that may be relevant to the onset of depression. Lastly, these hormonal shifts do not occur in isolation. At an environmental level, fundamental changes in our social environment (social stressors –like peer aggression, or social stress buffers-like peer or infant interactions) occur precisely at these transitional phases and may interact with the shifting hormonal milieu, altering interneuron maturation and widening the window of depression vulnerability. Thus, our hypothesis regarding the increased risk in depression for women during these transitional phases of their reproductive life posits a complex interaction of hormones, neural circuits, life stress and biological vulnerability.

AIM: We aim to determine whether developmental shifts in estradiol alter parvalbumin interneuron structure and function and whether these changes underlie vulnerability to depression across the reproductive lifecycle in females. We use immunohistochemistry and qPCR to investigate the relationship between estrogen receptors, parvalbumin and GABAA receptor subunit expression across the positive affect circuit (see figure 1) in pubertal, peripartum and experimentally-induced premenopausal females. Using ELISA, these cellular changes are correlated with serum estradiol levels at these three reproductive phases. To validate the functional relevance of these structural and molecular changes at a whole system level, local field potentials are used to estimate local and circuit-level activity changes associated with these reproductive phases in mice that do or do not express depressive-like behavior. By using mice with a parvalbumin-dependent expression of the Cas9 endonuclease (Rosa26-LSL-Cas9::PV-Cre offspring), engineered single guide RNAs targeting estrogen receptors and chemogenetic tools, we investigate if the estrogen-dependent regulation of interneuron function is involved in susceptibility to depression associated with reproductive transitions.

Project 2: Binge drinking, interneuronopathy

and sex differences in depression vulnerability

SIGNIFICANCE: Binge drinking is loosely defined as the periodic consumption of intoxicating doses of alcohol. The National Institute on Alcohol Abuse and Alcoholism operationally defines the pharmacological threshold for this level of intake as consumption that increases an individual’s blood alcohol content to greater than or equal to 80mg/dL (the 0.08 legal driving limit). The cycling of such psychoactive doses with periods of abstinence is thought to kindle alcohol-use related problems. Recent studies implicate this specific pattern of drinking in the development of negative affect and anxiety during abstinence3,6. Indeed, a general population study of alcohol drinking in men and women found baseline binge drinking to be the best predictor of the expression of depressive symptoms 5 years later1. The relationship between binge drinking and mood disturbances should be of particular concern in view of the increasing rates of binge drinking among young women and girls. Women are already twice as likely to have had a diagnosis of unipolar depression and anxiety related disorders than men and this gender gap is exaggerated among those with alcohol use disorders. Female alcoholics are more likely to drink excessively to alleviate negative mood states10. Non-dependent females who binge drink are also more likely to display changes in mood during abstinence. Unfortunately, there is a dearth of neuroscience research focused on understanding the biological consequences of alcohol use in women or mechanisms that drive their heavy alcohol use. Non-human animal models offer an opportunity to study the role that biological sex and factors associated with it, like ovarian hormones, may play in the association between binge drinking and negative affect (depressive-like behaviors) and identify a neural correlate underlying this association. Using mouse models of reproductive transitions (including pheromone induced precocious puberty) the Melón Lab initially assesses estradiol-dependent regulation of interneurons and its role in the bidirectional relationship between reproductive status and binge drinking.

AIM: We are testing the hypothesis that manipulation of inhibitory interneuron activity in relevant nodes of the positive-affect circuit can reverse the enhanced vulnerability to depression that follows a history of binge drinking in females. Relevant nodes of the circuit are determined by assessing changes in capacity for inhibitory signaling (Interneuron markers and GABAA receptor subunit and Chloride co-transporter transcription and protein expression). We determine whether mice displaying depressive-like symptoms during periods of reproductive transition are more likely to prefer alcohol and whether binge drinking during these periods will increase the ratio of mice displaying vulnerability to depression. As we have preliminary data supporting the latter, we will also test the hypothesis that manipulating estrogen receptor (ERα) expression on parvalbumin interneurons in relevant nodes of the positive affect circuit can disrupt the relationship between binge drinking and depression vulnerability in females.

Project 3: Peripubertal alcohol, social circuits

and sex specific effects on parental behavior

SIGNIFICANCE: Early adolescent alcohol use is a strong predictor of the development of alcohol use disorders and associated problems in adulthood13,14. Teenagers report that the social effects associated with alcohol is the single most important motivator of their alcohol use15. Thus, social circuits may be particularly sensitive to alcohol at this period, and may be especially vulnerable to long term perturbations following early adolescent alcohol use. For example, in rodents, early adolescent males exposed to alcohol show both enhanced social interactions after acute alcohol16 and heightened vulnerability to expressing peer directed aggression as adults after withdrawal from chronic alcohol when compared to females exposed at this time or either sex exposed during mid-late adolescence17. As this early adolescent period coincides with pubertal onset, itself a sexually dimorphic process, sex differences in the effects of alcohol on social behaviors may reflect a synergistic effect of gonadal hormones, alcohol and developmental neuroadaptations. My lab will use the onset of parental behavior (including infant directed behaviors, like retrieval and nest building and heightened peer-directed aggression in the presence of infants), as it represents a collection of social behaviors whose expression is restricted to adulthood (i.e. after the exposure to early adolescent alcohol) to assess how sex and age interact to produce sex-specific deficits in social behaviors in response to peripubertal adolescent intake.

AIM: We first characterize sex differences in the effects of early adolescent binge alcohol on parental behaviors in both male mice --who show retrieval of pups after mating in response to 38-kHz vocalizations-- and female mice--who show retrieval and nest-building behaviors as postpartum dams or as virgins trained with dams. In particular, we demonstrate the effects of early adolescent binge drinking and early adolescent isolation on infant directed behaviors (both retrieval and aggression) in adulthood. Alongside these behavioral studies, we evaluate sex differences in the maturation of GABAergic inhibitory neurons within the hypothalamic-mesolimbic parental circuit and changes in the expression of Estrogen receptor alpha (ERα) in the hypothalamic (medial preoptic area) central node of this circuit. Immunohistochemistry and qPCR are used to determine how early adolescent alcohol use changes the mature profile of inhibitory neurons within multiple nodes of this circuit as well as activity of other relevant cell types in the circuit in response to pup exposure. Early manipulation of ERα in inhibitory neurons in the central node of the circuit are used to evaluate a mechanism linking changes in parental behavior and early alcohol use. Lastly, sex differences in local ethanol concentrations of ethanol during early adolescent exposure are confirmed using implanted biosensors to exclude sex differences in pharmacokinetics on any effects noted.

These experiments all involve tools that we have configured and/or validated during projects 1 and 2. During Dr. Melón's postdoctoral career, she validated the use of implanted biosensors to correlate local changes in field potentials with ethanol concentrations in real-time in mice. Further, Dr. Melón has extensive experience with behavioral assessment and the molecular dissection of parental behavior in females and uses this expertise to explore parental behaviors in both sexes.