Genetic and Pharmacologic Studies Towards Prevention of opioid use disorder

Endogenous opioids mediate both analgesic and affective responses to stress. While the mu opioid receptor (MOR) produces the reinforcing euphoric effects responsible for the high abuse potential of traditional opioid analgesics, the kappa opioid receptor (KOR) is hypothesized to mediate the dysphoric component of stress. With increasing rates of opioid addiction, extensive research efforts are focused on better understanding each of these systems and how they interact, with the goal of developing less addictive pain management strategies and better approaches to addiction treatment. With this in mind, we conducted both clinical and pre-clinical studies aimed at developing better treatment strategies for opioid use and the management of patients with opioid use disorder (OUD). First, we assessed a local cohort of patients with (OUD) for a collection of single nucleotide polymorphisms (SNPs) related to reward processing, with the goal of furthering efforts to develop a gene-based screening mechanism for OUD risk assessment. Second, we conducted preclinical assessments of the G protein-biased KOR agonist nalfurafine as a potential adjuvant medication for increasing the therapeutic efficacy of opioid-based analgesia. Genetic analysis of OUD patients identified two SNPs within the gene encoding the mu opioid receptor, one SNP within the gene encoding the serotonin 2B (5-HT2B) receptor, and one SNP within the gene encoding Regulator of G protein Signaling 2 (RGS2), with the frequency of each SNP varying significantly from that observed in reference populations of European descent. Preclinical investigations with nalfurafine use in mice demonstrated a greater analgesic synergy when co-administered with morphine than morphine co-administered with the unbiased KOR agonist U50,488. As G protein-biased KOR agonists are hypothesized to produce less dysphoria (a therapeutically limiting side effect of KOR agonists), they may present a viable method for reducing the dose of MOR-targeting analgesic necessary for adequate pain relief, thereby reducing the likelihood of developing OUD. Further research is necessary to identify any anti-therapeutic effects of co-administering these two classes of drugs, as well as the range of pain modalities for which this approach is efficacious

Kappa opioid receptor signaling in the brain: Circuitry and implications for treatment

Kappa opioid receptors (KORs) in the central nervous system have been known to be important regulators of a variety of psychiatry illnesses, including anxiety and addiction, but their precise involvement in these behaviors is complex and has yet to be fully elucidated. Here, we briefly review the pharmacology of KORs in the brain, including KOR's involvement in anxiety, depression, and alcohol addiction. We also review the known neuronal circuitry impacted by KOR signaling, and interactions with corticotrophin-releasing factor (CRF), another key peptide in anxiety-related illnesses, as well as the role of glucocorticoids. We suggest that KORs are a promising therapeutic target for a host of neuropsychiatric conditions

Kappa opioid receptor modulation of neurotransmission in the amygdala

Kappa opioid receptors (KORs) and their endogenous ligand, dynorphin, have been implicated in a variety of neuropsychiatric disorders including anxiety and alcohol addiction. Here, we demonstrate the function and role of KORs in the bed nucleus of the stria terminalis (BNST), a key brain region involved in these diseases. In the first series of experiments, we show that KORs in the BNST inhibit glutamate release via a presynaptic, p38- and calcium- dependent mechanism. This synaptic inhibition is specific to basolateral amygdala (BLA) inputs, a previously identified key pathway in rodent models of anxiety-related behaviors. Additionally, we identified a frequency-dependent, light-evoked, local dynorphin-induced heterosynaptic plasticity of glutamate inputs to the BNST, allowing for optogenetic control of peptidergic transmission. We found differential KOR modulation of the BLA-BNST input based on the postsynaptic neurochemical identity. Collectively, these results demonstrate a local dynorphin- and KOR- dependent mechanism of inhibiting an anxiolytic pathway, providing a discrete therapeutic target for treatment of anxiety disorders. In the second series of experiments, we show that following chronic intermittent iv ethanol exposure (CIE), a model of alcohol exposure, KORs differentially modulate glutamate and GABA in the BNST. KOR inhibition of electrically-evoked glutamate inputs is decreased, while KOR inhibition of electrically-evoked GABA inputs is increased, despite overall properties of glutamatergic and GABAergic transmission remaining intact. This change in synaptic physiology is complementary to a KORdependent behavioral change: mice exposed to ethanol show decreased social preference as compared to air exposed, an effect which is partially rescued by systemic pre-administration of the KOR antagonist JDTic. Taken together, these experiments demonstrate KOR-dependent alterations of synaptic transmission in the BNST following CIE, making the BNST a potential site of action for KOR targeted therapies related to alcohol and anxiety. Jointly, these experiments expand our understanding of how key peptidergic transmission in the extended amygdala can play a role in anxiety and addiction related diseases.Doctor of Philosoph

Opioids and Their Receptors

Few neurotransmitter systems have fascinated as much as the opioid system (i.e., opioid ligands and their receptors). Over the years, scientific studies of the endogenous opioid system have uncovered a complex and subtle system that exhibits impressive diversity, based on its critical role in modulating a large number of sensory, motivational, emotional and cognitive functions. Additionally, its important therapeutic value for the treatment of many human disorders, including pain, affective and addictive disorders, and gastrointestinal motility disorders, has been of persistent interest. This book specifically covers a broad area of the opioid research, offering up-to-date and new perspectives about opioid drug discovery. The diversity among the discussed topics ranging from medicinal chemistry to opioid pharmacology, from basic science to translational research, is a testimony to the complexity of the opioid system that results from the expression, regulation and functional role of opioid ligands and their receptors. This book will serve as a useful reference to scientists while also stimulating continuous research in the chemistry and pharmacology of the opioid system, with the prospective for finding improved therapies of human diseases where the opioid system plays a central role

Gene-environment interactions in eating disorders and obesity

This thesis investigates gene-environment interplay underlying molecular basis of eating disorders and obesity. We focused the attention on the role of epigenetic mechanisms controlling expression of relevant feeding-modulating homeostatic and hedonic genes, analyzed also in the attempt to suggest nutritional intervention by gut microbiota modulation. The major finding was the selective regulation of genes belonging to the endogenous cannabinoid system in different conditions. We observed epigenetic regulation of cannabinoid receptor type type I gene (Cnr1) in hypothalamus and nucleus accumbens of the activity-based model of anorexia nervosa (ABA), namely a significant reduction of gene expression correlated with higher DNA methylation at specific CpG sites in ABA rats compared to controls. Consistent hypermethylation was observed at Cnr1 in a subset of restricted human subjects where, with different environmental cues (i.e. hyperactivity, stressful events and dieting history) relevant for the epigenetic regulation of the gene. In an animal model of obesity, we reported the exactly opposite direction of changes for Cnr1 in the hypothalamus, that were confirmed in human subjects. We also explored effects of dietary intervention with selected probiotic strain in the anx/anx mice model of anorexia, observing a reverted expression of the central Cnr1 gene, as well as tendency to lower inflammatory markers in the colon. The transcriptional regulation of relevant endogenous system genes, such as those belonging again to the endocannabinoid but also to the opioid system, was also analyzed in an animal model of Diet Induced Obesity. A selective epigenetic regulation was observed for Cnr1 and mu opioid receptor gene just at the beginning of the development of the obese phenotype. Finally, we also investigated the transcriptional regulation of endocannabinoid system components in an animal model of binge eating behavior where the selective altered gene expression of the enzyme responsible for endocannabinoids degradation, fatty acid amide hydrolase, was observed in the hypothalamus of rats displaying binge behavior compared to controls. These changes were correlated with altered histone acetylation at gene promoter. Our findings suggest novel biomarkers for eating disorders and obesity, and due to the reversible nature of the epigenetic hallmark, open avenues for environmental strategies of intervention

Unravelling The Role Of Androgens In Polycystic Ovary Syndrome

Polycystic Ovary Syndrome (PCOS) is a multifaceted hormonal disorder which affects 5-15% of reproductive-aged women worldwide. While classically recognised as an ovarian disorder, PCOS is associated with a variety of reproductive, endocrine and metabolic features including ovulatory dysfunction, infertility, hyperandrogenism, obesity, and an increased risk of type 2 diabetes mellitus and cardiovascular disease. The most consistently present of these is hyperandrogenism – supraphysiological levels of androgens such as testosterone (T) and dihydrotestosterone (DHT). Typically thought of as male steroid hormones, androgens have been shown to play important role in the maintenance of normal female reproductive function. Despite the high prevalence of hyperandrogenism amongst patients, the role of androgens in the etiology and pathogenesis of PCOS has yet to be determined. The aim of this work was to unravel the association between excess androgen exposure and the development and advancement of the PCOS phenotype in mouse model of androgen-induced PCOS. The first study contained within this work (Chapter Three) provides the first comprehensive characterization of a range of reproductive, endocrine and metabolic traits associated with PCOS in four distinct mouse models: a model of prenatal androgenisation utilising the potent non-aromatizable androgen DHT administered during days 16-18 of gestation, and three diverse models of postnatal androgen exposure employing a long-term treatment with either DHT, the proandrogen dehydroepiandrosterone (DHEA), or letrozole (an aromatase inhibitor) for 90 days beginning at 3 weeks of age. Prenatal androgenisation produced some reproductive and endocrine traits, but failed to induce the metabolic abnormalities seen in PCOS. DHEA treatment did not reproduce any features associated with PCOS while treatment with letrozole produced few PCOS-like characteristics and some aberrant changes not typical of the syndrome. Additionally, letrozole treatment did not reproduce any metabolic attributes of PCOS. On the other hand, postnatal exposure to excess androgen, by way of DHT treatment, produced a breadth of reproductive, endocrine and metabolic traits that mimic those seen in human PCOS. This study revealed that a treatment regime of long-term postnatal exposure to DHT reproduced the strongest PCOS-like phenotype in our mice and provides a robust animal model in which to study the pathogenesis of PCOS. The second study (Chapter Four) aimed to explore the involvement of genomic androgen receptor (AR)-mediated actions in the development of these PCOS traits. As a prenatally androgenised mouse model of PCOS had previously been reported to exhibit impaired neuroendocrine hypothalamic feedback of the hypothalamic-pituitary-gonadal (HPG) axis, we took this opportunity to utilise mice from our own prenatal model to investigate the neuroendocrine regulation of the HPG axis in PCOS in addition to the effects of AR inactivation on the PCOS phenotype. PCOS was induced in wild-type (WT) and androgen receptor knockout (ARKO) mice using DHT administered on days 16-18 of gestation. A subset of these mice were also exposed to 17β-estradiol for 7 days prior to collection, via a subdermal implant, to investigate the impaired estradiol negative feedback on the hypothalamus. WT mice with DHT-induced PCOS displayed several reproductive abnormalities including aberrant cycling and ovulatory dysfunction in addition to adipocyte hypertrophy and hepatic steatosis. However, diestrus serum luteinising hormone and follicle stimulating hormone, and estradiol-induced negative feedback as well as hypothalamic expression of several neuropeptides were unaffected by DHT treatment in WT mice. Mice both homozygous and heterozygous for the global inactivation of the AR (ARKO), did not display any PCOS traits when exposed to excess androgens during prenatal life. This study showed the importance of AR signalling in the development of PCOS and revealed that even AR haplosufficiency is adequate to prevent induction of PCOS by prenatal hyperandrogenism. Finally, the third study (Chapter Five) aimed to shed further light on the AR-mediated androgen actions in PCOS with a focus on identifying the tissue-specific targets of these actions. Employing our postnatal model of PCOS induction, this study investigates the effects of: 1) global loss of AR signalling (ARKO), 2) neuronal knockout (NeurARKO) and 3) granulosa cell-specific AR inactivation (GCARKO) on the development of the PCOS phenotype induced by exposure to exogenous DHT. As in our prenatal model, ARKO mice were fully protected from all DHT-induced features of PCOS. Neuron-specific AR signaling was required for the development of a variety of reproductive and metabolic traits including classic polycystic ovaries, dysfunctional ovulation, obesity and dyslipidemia. In contrast, loss of AR signalling in granulosa cells did not impede the pathogenesis of PCOS-like features in GCARKO mice. To further examine the role of extra-ovarian AR signalling in PCOS, reciprocal ovary transplants were carried out in WT and ARKO mice. Results from ARKO hosts with transplanted WT ovaries revealed that excess androgen exposure requires functional extra-ovarian, and not intra-ovarian, AR signalling in order to produce features of PCOS. This study provides strong evidence that neuroendocrine genomic AR signaling is an important mediator in the development of PCOS. The studies contained within this thesis are the first to provide a comprehensive analysis of a mouse model of PCOS encompassing a breadth of reproductive, endocrine and metabolic features. This work has identified the optimal model in which to study this complex, multifactorial condition which affects a significant number of women worldwide. Additionally, our results have shown that the effects of androgens on the pathogenesis of PCOS are mediated via the androgen receptor in a dose-dependent manner such that two functional copies are required for DHT to reproduce features of PCOS in the mouse. Finally, in a crucial study to investigate the locus of androgen actions we have revealed the previously overlooked importance of extra-ovarian neuroendocrine androgen action in the origins and progression of PCOS, despite it being thought of primarily as an ovarian disorder. Overall, these studies have provided valuable insights into both the role of androgens in Polycystic Ovary Syndrome and potential new targets for the development of mechanism-based treatments of this disorder

Influences of social stress in the rewarding effects of mdma and alcohol

La adicción a las drogas es un importante problema en nuestra sociedad con graves consecuencias jurídicas, médicas y sociales para los consumidores. Se trata de un trastorno caracterizado por la pérdida de control sobre el uso de la droga, su búsqueda compulsiva y la aparición de un estado emocional negativo en ausencia de la misma. El alcohol es la droga legal más consumida tanto por adultos como por adolescentes. Por otra parte, entre las sustancias ilegales, los datos epidemiológicos apuntan que el éxtasis o MDMA es una sustancia ampliamente consumida entre los adolescentes y adultos jóvenes sobre todo en fiestas “rave” donde los consumidores pasan horas, incluso días, haciendo uso de esta sustancia. La adolescencia constituye un periodo de alta vulnerabilidad debido a la inmadurez cerebral, siendo muy perjudicial en esta fase de la vida el consumo de drogas y la exposición a situaciones ambientales negativas. En concreto, se ha demostrado que el estrés social puede provocar el inicio, escalada y la reinstauración en el consumo de drogas en modelos animales. Por ello, el objetivo del presente trabajo ha sido estudiar los factores implicados en las propiedades reforzantes de MDMA (1.25 y 10 mg/kg) y alcohol (1.25 y 2.5 g/kg), principalmente el efecto del estrés social agudo y repetido en ratones macho adolescentes y adultos jóvenes. Una de las técnicas metodológicas más utilizadas ha sido la inducción de estrés por derrota social mediante encuentros agonísticos (agudos o repetidos). Para evaluar si el estrés social modifica el valor reforzante de la MDMA o el alcohol se ha utilizado principalmente el paradigma de condicionamiento de lugar pero también se ha evaluado el consumo voluntario de alcohol (“two-bottle choice”). Este procedimiento y el de autoadministración (“second order”) se han utilizado para estudiar los efectos de los antagonistas opiáceos mu (naltrexona y GSK 1521498) en el consumo de alcohol en ratas. Asimismo otras técnicas conductuales han sido utilizadas para medir procesos como ansiedad, memoria, aprendizaje, interacción social o actividad motora entre otros tras la administración de derrota, MDMA o su combinación. Los datos obtenidos indican que la exposición al estrés social (tanto agudo como a largo plazo) incrementa los efectos reforzantes de MDMA y alcohol y produce diferentes alteraciones conductuales, por ejemplo una reducción de la conducta social, que pueden estar relacionadas con el incremento en los efectos reforzantes. Asimismo, la combinación de estrés y MDMA induce un importante deterioro de la memoria y el estado emocional. Los antagonistas glutamatérgicos (memantine y CNQX) y la inhibición de la síntesis de óxido nítrico (7-nitroindazole) bloquean los efectos reforzantes de la MDMA y también parecen estar implicados en los efectos del estrés. El antagonismo de los receptores opiáceos mu disminuye la búsqueda y consumo de alcohol. El avance en el conocimiento de los sistemas de neurotransmisión implicados en los efectos reforzantes de la MDMA y el alcohol y en la influencia del estrés sobre estos efectos puede contribuir al desarrollo de estrategias farmacológicas para el tratamiento de la adicción a la estas drogas.Drug addiction is a serious problem in our society and has serious legal, medical and social consequences for consumers. This disorder is characterised by an impaired control over substance use, compulsive drug seeking, and the emergence of a negative emotional state in the absence of the drug. With regard to legality, there are two broad types of substances of abuse: legal and illegal. Alcohol is the most consumed legal substance by adults and adolescents. Among illegal substances, epidemiologic data suggest that ecstasy, or MDMA, is a widely used substance among adolescents and young adults, especially in "raves", where consumers spend hours, even days, using this substance. Adolescence is a period of enhanced vulnerability due to the lack of brain maturation, and consumption of drugs of abuse and exposure to different negative environmental conditions is especially harmful at this stage of life. It has been demonstrated that social stress can trigger onset, escalation and reinstatement of drug use in animal models. Therefore, the objective of this work was to study the effects of acute and repeated social stress on the rewarding properties of alcohol and MDMA using the place preference paradigm (CPP) and the two-bottle choice procedure. In addition, we set out to evaluate the effects of social stress on several behaviours or processes (anxiety, depression, social interaction, learning and memory). We have also studied the neurobiological aspects underlying the rewarding effects of MDMA and their modulation by stress, assessing along the way the role of the glutamatergic system and nitric oxide (NO) pathway. Moreover, since alcohol is the main addictive substance consumed in the world, we have studied the role of the opioid system in the rewarding effects and intake of ethanol. Overall, our results indicate that exposure to social stress (both acute and long-term) increases the rewarding effects of MDMA and ethanol and induces different behavioural alterations, such as a reduction of social interaction, which is related with an increase in drug consumption. Glutamate antagonists and inhibition of NO synthesis block the rewarding effects of MDMA and antagonism of mu opioid receptors decreases alcohol seeking and intake. Advances in knowledge of the neurotransmitter systems implicated in the rewarding effects of MDMA and alcohol are likely to contribute to the development of pharmacological strategies for the treatment of drug addiction

Examination of nucleus accumbens mechanisms underlying the motivation for physical activity

Physical inactivity, a primary contributor to numerous diseases including obesity, type 2 diabetes, depression, and dementia, has reached pandemic levels worldwide. Alarmingly, the percentage of individuals engaging in physical activity is low and decreasing. Accelerometry data shows that > 90% of adults fail to meet the U.S. Physical Activity Guidelines despite the excess of knowledge suggesting exercise improves health. Therefore, beginning to understand the molecular mechanisms which influence physical activity levels is imperative for the development of therapies to reduce sedentary behavior. The work presented in this dissertation made use of three independent experimental paradigms in rats to test the hypothesis that differences in the mesolimbic dopamine system associate with/cause changes in voluntary physical activity. In the first study, rats selectively bred for high (HVR) or low (LVR) voluntary wheel running distance were used to assess inherent differences in opioidergic signaling between HVR and LVR, as well as the influence of dopamine on opioid-induced changes in voluntary wheel running. Mu-opioid receptor expression and function was increased in the nucleus accumbens (NAc) of HVR compared to LVR. Likewise, naltrexone injection decreased dopamine-related mRNA expression in mesolimbic brain regions and reduced wheel running in HVR, but not LVR. Finally, lesion of dopaminergic neurons in the NAc prevented the decrease in running following naltrexone administration in HVR, suggesting opioidergic signaling requires downstream dopaminergic activity to influence voluntary running. In the second study, the transgenerational effect of maternal Western diet (WD) on offspring voluntary wheel running was assessed. Wheel running was increased in female WD offspring from 4-7 weeks of age, but decreased running from 16-19 weeks of age, compared to offspring from chow fed dams. These age-specific changes in wheel running are associated with the up- and down-regulation of dopamine receptor 1 in the NAc at 6 and 18 weeks of age, respectively, in WD female offspring, which in turn was negatively associated with leptin receptor mRNA in the ventral tegmental area. In the third study, age-related influences on wheel running were assessed in 8 and 14 week-old rats. In addition to a [about]60% reduction in running, RNA-sequencing revealed down-regulations in networks related to cAMP-mediated signaling and synaptic plasticity in the NAc from 8 to 14 weeks-old. The down-regulations of these networks was mirrored by reductions in dendritic spine density in the NAc from 8 to 14 weeks-old. Additionally, intra-NAc injection of the Cdk5 inhibitor roscovitine, a known modulator of dendritic density and dopamine signaling, dose-dependently decreased wheel running. Despite the varying experimental models used in this dissertation, these findings collectively suggest that alterations in dopaminergic signaling in the NAc associate with, and influence, voluntary physical activity.Includes biblographical reference

Dissecting alterations to arcuate nucleus neuropeptide Y neural circuitry in a model of polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is the most common form of infertility among women of reproductive age. Despite the prevalence of PCOS, the underlying mechanisms behind how PCOS arises and causes infertility remain poorly understood. While primarily considered a disorder of the ovary, a growing body of evidence suggests that PCOS is associated with dysfunctional neuroendocrine circuitry in the hypothalamus that orchestrates gonadotrophin hormone secretion. Women with PCOS frequently exhibit rapid pulsatile luteinizing hormone (LH) secretion. While LH secretion is tightly regulated by ovarian steroid hormonal feedback in fertile women, women with PCOS are unresponsive to these hormones. Elevated LH secretion likely reflects dysfunctions to the gonadotropin-releasing hormone (GnRH) neuronal network in the hypothalamus that gates LH secretion and integrates steroid hormone feedback. As GnRH neurons themselves do not express the receptors necessary for feedback to occur, research has sought to identify alterations to GnRH neural afferents that may arise in PCOS. Prior work within our lab has utilized a prenatal androgen (PNA) treated mouse model of PCOS to identify enhancement of excitatory GABAergic inputs to GnRH neurons. In particular, GABA neurons in the hypothalamic arcuate nucleus (ARN) have been identified with enhanced input to GnRH neurons, and reduced sensitivity to an ovarian steroid hormone, progesterone. Neuropeptide Y (NPY) neurons compose a large proportion of GABA neurons within the ARN, and are known to play an important role in restraint of LH secretion via inhibition of GnRH neurons. Interestingly, plasma NPY is often reduced in women with PCOS, indicating impairment of its neural secretion. The overarching aim of work presented in this thesis was therefore to determine whether NPY neurons within the ARN also exhibit PNA-induced alterations, which may advance our understanding of how unrestrained LH secretion occurs in PCOS. Initially, the proportion of NPY neurons that are GABAergic within the ARN was assessed by pairing immunohistochemical (IHC) labelling of NPY with transgenic reporter identification of GABA neurons, in both PNA-treated mice and fertile controls. Additionally, the molecular identity of ARN GABA neurons was further dissected to assess whether they express other peptides known to regulate GnRH neurons. This work established that ARN NPY neurons compose approximately one-third of ARN GABAergic neurons, a far larger proportion than any other neural phenotype examined, and demonstrated that the vast majority (> 93%) of ARN NPY neurons are GABAergic. While fertile male mice were observed to have a larger population of ARN NPY neurons compared with both fertile (p = 0.0006) and PNA-treated females (p = 0.0192), no differences were found between female groups, indicating the size of this population is not masculinized by PNA-treatment. To investigate remodelling of NPY neurons in the ARN specifically, the validity of an agouti-related peptide (AgRP)-Cre mouse line was characterised as AgRP is highly co-expressed with NPY, and exclusively expressed in the ARN. After crossing AgRP-Cre mice onto a Cre-dependant GFP reporter it was demonstrated that within the ARN, GFP reporter was highly specific and effective at identifying NPY neurons. Additionally, GFP expression was overwhelmingly confined to the ARN only. Whole brain mapping of axon-associated GFP expression revealed that this reporter was highly effective for visualising distal axons. In order to assess whether ARN NPY neurons projecting to GnRH neurons could be targeted more specifically, a fluorescent retrograde tracer was injected into the preoptic area where the majority of GnRH neurons reside. This revealed that approximately 10% of ARN NPY neurons project to the area where GnRH neurons are located, and that these neurons are scattered throughout the ARN rather than composing an anatomically defined population. These experiments demonstrated that AgRP-GFP mice are an appropriate model for further dissection of ARN NPY neurons. Using this model, the hypothesis that PNA exposure leads to remodelling of ARN NPY sensitivity to steroid hormones, and their projections to GnRH neurons was addressed. Interestingly, ARN NPY neurons did not express the progesterone receptor or estrogen receptor . However, a greater proportion of ARN NPY neurons expressed androgen receptor (AR) in PNA mice compared with fertile controls (p = 0.045). When close appositions between ARN NPY axons and GnRH neurons was examined, indicating putative innervation, it was revealed that there were no differences in the close appositions onto GnRH neurons in the PNA-treated mouse. Overall, these results suggest that ARN NPY neurons are not remodelled in the same way as ARN GABA neurons, but are subject to their own distinct alterations in PNA-treated mice in the form of elevated androgen sensitivity. The role of leptin, a major regulator of ARN NPY neurons and permissive cue in fertility, was then examined in PNA-treated mice compared to fertile controls. Plasma leptin was measured in fed and fasted states, and there were no differences in leptin levels in PNA-treated mice. Interestingly, fasting reduced plasma leptin in PNA-treated mice similar to controls, contrasting previous findings indicating derangements to fasted leptin levels. Furthermore, leptin responsiveness indicated by induction of pSTAT3 was not different in PNA-treated mice. These results indicate that leptin signalling in the ARN is normal in PNA-treated mice and leptin acting within this region is not likely to contribute to altered GnRH neuron activity. While ARN NPY neurons and circuitry were found to be largely unaffected by PNA-treatment, a sensitive NanoString assay for gene expression revealed evidence for potential reductions in downstream signalling within hypothalamic regions important for GnRH neural regulation. Expression of the NPY Y1 receptor was downregulated in the rostral periventricular region of the third ventricle, a region important for generating the preovulatory LH surge. Additionally, a sizable reduction in leptin receptor gene expression was observed in the preoptic area in the vicinity of GnRH neurons, an area in which leptin signalling plays a role in regulating LH secretion. Taken together these results do not support the hypothesis that alterations within ARN NPY-to-GnRH neuron circuitry plays a major role underpinning neuroendocrine disruption in PNA-treated mice and PCOS. While ARN NPY neurons do not appear to compose a subset of ARN GABA neurons that are remodelled following PNA-treatment, elevated androgen sensitivity was identified. As androgens disrupt negative feedback in PCOS, this opens up the possibility that androgens act via ARN NPY neurons to disrupt appropriate response by other steroid hormone sensitive afferent populations. Differences in NPY receptor expression were discovered in hypothalamic regions important for regulation of GnRH neurons, and potentially indicates altered NPY signalling within the GnRH afferent network. These findings establish a platform upon which future research will elucidate the importance of these circuit alterations in generating neuroendocrine disruptions in PCOS