Electrophysiological study of the effects of arginine vasopressin in the rat juxtacapsular nucleus of the bed nucleus of the stria terminalis

Arginine vasopressin (AVP), a nine amino acid neuropeptide (CYFQNCPRG- NH2) fulfills a dual function: (i) in the periphery, AVP acts as a peptide hormone and (ii) in the CNS, AVP is a neuromodulatory peptide. AVP produces its effects through 3 AVP receptors (AVPRs). AVPR1a and AVPR1b are expressed in the CNS and periphery, whilst AVPR2 is not found centrally but instead solely expressed in the kidneys. Recent evidence revealed a high density of AVP-binding sites in the juxtacapsular nucleus of the bed nucleus of the stria terminalis (jxBNST). While in other regions of the brain, AVP acts at AVPRs to regulate an array of biological processes, including male-typical social behaviours, social memory, stress adaptation, fear, anxiety, and fluid homeostasis, its role in the jxBNST remains elusive. Furthermore, the neurophysiological properties of AVP in the jxBNST are unknown so this study aimed to examine how AVP modulates synaptic transmission in the rat jxBNST. The BNST being one of the most notable sexually dimorphic brain regions and AVPR expression being influenced by gonadal steroids, we investigated the putative influence of sex on the modulatory effects of AVP in the jxBNST. Finally, due to AVP being released at a substantially higher concentration following periods of water deprivation, we examined changes in AVPs modulatory role following water deprivation. Male and female Long Evans rats were euthanized and brain slice whole-cell voltage-clamp electrophysiology was done in the jxBNST to measure the effects of AVP on synaptic transmission of GABA synapses. Exogenous application of AVP produced three responses; either postsynaptic long-term potentiation (LTP) of GABAA-inhibitory postsynaptic currents (IPSC), postsynaptic long-term depression (LTD) of GABAA-IPSC, or no change in GABAA-IPSC amplitudes. Interestingly, the proportion of neurons responding in each of these ways did not differ between sexes and within females was not estrous cycle-dependent. Finally, although not statistically significant, 24-hour water deprivation abolished GABAA-LTD, an effect that was not a consequence of social isolation. Taken together, our data show that AVP modulates GABAA synaptic transmission in the jxBNST in fluid homeostasis- but not sex-dependent manner.

Glycemic State Regulates Brain-Derived Neurotrophic Factor Responsiveness of Neurons in the Paraventricular Nucleus of the Hypothalamus

Brain derived neurotrophic factor (BDNF) is a member of the family of neurotrophins and binds to the tropomyosin-related kinase B (TrkB) receptor. Like other neurotrophic factors, BDNF is involved in the development and differentiation of neurons. Recently, studies have suggested important roles for BDNF in the regulation of energy homeostasis. The paraventricular nucleus (PVN) is critical for normal energy balance contains high levels of both BDNF and TrkB mRNA. Studies have shown that microinjections of BDNF into the PVN increase energy expenditure, suggesting BDNF plays a role in energy homeostasis through direct actions in this hypothalamic nucleus. We used male Sprague-Dawley rats to perform whole-cell current-clamp experiments from PVN neurons in slice preparation. BDNF was bath applied at a concentration of 2nM and caused depolarizations in 54% of neurons (n = 25; mean change in membrane potential: 8.9 ± 1.2 mV), hyperpolarizations in 23% (n = 11; mean change in membrane potential: -6.7 ± 1.4 mV), while the remaining cells tested were unaffected. Previous studies showing effects of BDNF on γ-aminobutyric acid type A (GABAA) mediated neurotransmission in PVN led us to examine if these BDNF-mediated changes in membrane potential were maintained in the presence of tetrodotoxin (TTX) sodium channel blocker (N = 9; 56% depolarized, 22% hyperpolarized, 22% non-responders) and bicuculline (GABAA antagonist) (N = 12; 42% depolarized, 17% hyperpolarized, 41% non-responders), supporting the conclusion that these effects on membrane potential were postsynaptic. We also evaluated the effects of BDNF on these neurons across varying physiologically relevant extracellular glucose concentrations. At 10 mM 23% (n = 11; mean: -6.7 ± 1.4 mV) of PVN neurons hyperpolarized in response to BDNF treatment, whereas at 0.2 mM glucose, 71% showed hyperpolarizing effects (n = 12; mean: -6.3 ± 2.8 mV). Our findings reveal that BDNF has direct impacts on PVN neurons and that these neurons are capable of integrating multiple sources of metabolically relevant input. Our analysis regarding glucose concentrations and their effects on these neurons’ response to other metabolic signals emphasizes the importance of using physiologically relevant conditions for study of central pathways involved in the regulation of energy homeostasis.

IN SITU AND IN VITRO IMMUNOLOCALIZATION OF OVIDUCTIN BINDING SITES ON HAMSTER UTERINE EPITHELIAL CELLS AND DETECTION OF A HAMSTER OVIDUCTIN HOMOLOGUE IN THE FEMALE RAT REPRODUCTIVE TRACT

Oviductin is an oviduct-specific and high-molecular-weight glycoprotein that has been suggested to play important roles in the early events of reproduction. The present study was undertaken to localize the oviductin binding sites in the uterine epithelial cells of the golden hamster (Mesocricetus auratus) both in situ and in vitro, and to detect a hamster oviductin homologue in the female rat reproductive tract. Immunohistochemical localization of oviductin in the hamster uterus revealed certain uterine epithelial cells reactive to the monoclonal anti-hamster oviductin antibody. In order to study the interaction between hamster oviductin and the endometrium in vitro, a method for culturing primary hamster uterine epithelial cells has been established and optimized. Study with confocal microscopy of the cell culture system showed a labeling pattern similar to what was observed using immunohistochemistry. Pre-embedding immunolabeling of cultured uterine epithelial cells also showed gold particles associated with the plasma membrane and microvilli. These results demonstrated that hamster oviductin can bind to the plasma membrane of certain hamster uterine epithelial cells, suggesting the presence of a putative oviductin receptor on the uterine epithelial cell surface. In the second part of the present study, using the monoclonal anti-hamster oviductin antibody that cross-reacts with the rat tissue, we have been able to detect an oviduct-specific glycoprotein, with a molecular weight of 180~300kDa, in the female rat reproductive tract. Immunohistochemical labeling of the female rat reproductive tract revealed a strong immunolabeling in the non-ciliated oviductal epithelial cells and a faint immunoreaction on the cell surface of some uterine epithelial cells. Ultrastructurally, immunogold labeling was restricted to the secretory granules, Golgi apparatus, and microvilli of the non-ciliated secretory cells of the oviduct. In the uterus, immunogold labeling was observed on the cell surface of some uterine epithelial cells. Furthermore, electron micrographs of ovulated oocytes showed an intense immunolabeling for rat oviductin within the perivitelline space surrounding the ovulated oocytes. The findings of the present study demonstrated that oviductin is present in the rat oviduct and uterus, and it appears that, in the rat, oviductin is secreted by the non-ciliated secretory cells of the oviduct.