Characterization of tyrosine hydroxylase- and non-tyrosine hydroxylase-immunoreactive sexually dimorphic oxytocin receptor-expressing neurons in the anteroventral periventricular nucleus of female mice.

New promising work has indicated oxytocin (OXT) receptor activation is blunted in cardiovascular diseases (CVDs) and OXT receptor activation may be a novel target to increase parasympathetic activity to the heart and treat CVDs. This study tests the hypotheses that the release of OXT from hypothalamic paraventricular (PVN) neurons onto cardiac vagal neurons (CVNs) is diminished with chronic intermittent hypoxia‐hypercapnia (CIH/H), an animal model of OSA, and furthermore that selective activation of PVN OXT neurons can reduce adverse cardiovascular consequences that occur with CIH/H. Optogenetic stimulation of channelrhodopsin expressing PVN fibers evoked large transient increases in Ca2+ in CHO cells stably transfected to express the human recombinant OXT receptor and the red fluorescent calcium indicator, R‐GECO1, dispersed adjacent to CVNs. The release of OXT onto CVNs upon photoactivation of PVN fibers was blunted in animals exposed to 21 days of CIH/H. To examine if restoration of OXT neuron activity can prevent the adverse cardiovascular effects of CIH/H in vivo, we selectively expressed excitatory DREADDs in PVN OXT neurons, and implanted animals with telemetry devices to monitor blood pressure (BP) and EKG activity. Selective chronic activation of PVN OXT neurons decreased resting BP and HR, and perhaps more importantly chronic PVN OXT neuron activation prevented the elevations in BP and HR that occur with CIH/H. These results indicate excitation of parvocellular PVN fibers releases OXT at brainstem CVN targets, and selective activation of OXT neurons in the PVN decreases resting BP and HR, and prevents the elevations in BP and HR that occur after 21 days of CIH/H.

Oxytocin (OXT) and OXT receptor (OXTR)-mediated signaling control excitability, firing patterns, and plasticity of hippocampal CA2 pyramidal neurons, which are pivotal in generation of brain oscillations and social memory. Nonetheless, the ionic mechanisms underlying OXTR-induced effects in CA2 neurons are not fully understood. Using slice physiology in a reporter mouse line and interleaved current-clamp and voltage-clamp experiments, we systematically identified the ion channels modulated by OXT signaling in CA2 pyramidal cells (PYRs) in mice of both sexes and explored how changes in channel conductance support altered electrical activity. Activation of OXTRs inhibits an outward potassium current mediated by inward rectifier potassium channels (IKir) and thus favoring membrane depolarization. Concomitantly, OXT signaling also diminishes inward current mediated by hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels (Ih), providing a hyperpolarizing drive. The combined reduction in both IKir and Ih synergistically elevate the membrane resistance and favor dendritic integration while the membrane potential is restrained from quickly depolarizing from rest. As a result, the responsiveness of CA2 PYRs to synaptic inputs is highly sharpened during OXTR activation. Unexpectedly, OXTR signaling also strongly enhances a tetrodotoxin-resistant (TTX-R), voltage-gated sodium current that helps drive the membrane potential to spike threshold and thus promote rhythmic firing. This novel array of OXTR-stimulated ionic mechanisms operates in close coordination and underpins OXT-induced burst firing, a key step in CA2 PYRs' contribution to hippocampal information processing and broader influence on brain circuitry. Our study deepens our understanding of underpinnings of OXT-promoted social memory and general neuropeptidergic control of cognitive states.SIGNIFICANCE STATEMENT Oxytocin (OXT) plays key roles in reproduction, parenting and social and emotional behavior, and deficiency in OXT receptor (OXTR) signaling may contribute to neuropsychiatric disorders. We identified a novel array of OXTR-modulated ion channels that operate in close coordination to retune hippocampal CA2 pyramidal neurons, enhancing responsiveness to synaptic inputs and sculpting output. OXTR signaling inhibits both potassium conductance (IKir) and mixed cation conductance (Ih), engaging opposing influences on membrane potential, stabilizing it while synergistically elevating membrane resistance and electrotonic spread. OXT signaling also facilitates a tetrodotoxin-resistant (TTX-R) Na+ current, not previously described in hippocampus (HP), engaged on further depolarization. This TTX-R current lowers the spike threshold and supports rhythmic depolarization and burst firing, a potent driver of downstream circuitry.

Centrally administered oxytocin elicits exaggerated grooming in oxytocin null mice

The oxytocin (OT) receptor (OTR) mediates a wide spectrum of biological actions and is expressed in a large number of different tissues, including uterine, breast, and lung tumors. To define more completely the intracellular signaling mechanisms linked to OTR activation, we have used a phosphoproteomics approach and have characterized changes in the phosphorylation states of intracellular proteins in response to OTR activation in OTR-expressing cell lines. Using a specific antiphosphothreonine antibody, we observed several distinct changes in the threonine phosphorylation patterns. The most prominent change involved dephosphorylation of a 95-kDa moiety. Purification by ion exchange chromatography combined with one- and two-dimensional polyacrylamide gel electrophoresis followed by N-terminal micro-sequence analysis revealed that the 95-kDa moiety corresponded to eukaryotic elongation factor 2. This protein is a key regulator of cellular protein synthesis and mediates, upon dephosphorylation, the translocation step of peptide chain elongation. Dose-response curves in myometrial cells expressing the endogenous OTR indicated a significant effect of OT on eukaryotic elongation factor 2 dephosphorylation at 1 nM, a concentration close to the dissociation constant (K(d)) of OT. Time course analysis indicates that the effect is rapid with a significant effect occurring at 5 min. To determine directly the effect of OT on protein synthesis, the incorporation of [35S]Met into total protein was assessed. In myometrial cells, OTR activation led to significant 29% increase in total protein synthesis over a 2-h period. These findings establish a novel link between OTR activation and cellular protein synthesis and thus define a mechanism by which OT assumes a so far unrecognized, physiologically relevant trophic function.

1. The effects of Cs+ on the action potential, post-train after-hyperpolarization (AHP), Ca2+-dependent post-spike depolarizing after-potential (DAP) and phasic firing were examined during intracellular recordings from magnocellular neurosecretory cells (MNCs) in superfused rat hypothalamic explants. 2. Extracellular Cs+ reversibly inhibited (IC50, approximately 1 mM) DAPs, and associated after-discharges, that followed brief spike trains in each of sixteen cells tested. Although bath application of Cs+ also provoked a small reversible depolarization, inhibition of the DAP was retained when membrane voltage was kept constant by current injection. 3. Application of Cs+ had no significant effects on spike duration (n = 8), frequency-dependent spike broadening (n = 8), spike hyperpolarizing after-potentials (n = 14), or the amplitude of the isolated AHP (n = 7). Caesium-evoked inhibition of the DAP, therefore, does not result from diminished spike-evoked Ca2+ influx, and may reflect direct blockade of the conductance underlying the DAP. 4. Inhibition of the DAP was associated with an enhancement of the amplitude and duration of the AHP, indicating that the currents underlying the AHP and the DAP overlap in time following a train of action potentials, and that the relative magnitude of these currents is an important factor in determining the shape and time course of post-train after-potentials. 5. Bath application of Cs+ reversibly abolished phasic firing in each of seven cells tested. This effect was reversible and persisted at all subthreshold voltages tested. These results indicate that the current underlying the DAP is necessary for the genesis of plateau potentials and phasic firing in MNCs.

The neurohypophysial hormone oxytocin is involved in the regulation of social behaviors, including social recognition, pair bonding, and sex-specific parental behaviors in a variety of species. Oxytocin triggers these social behaviors by binding to oxytocin receptors (OXTR) in various parts of the brain. Oxytocin-induced sex-typical behavior, therefore, suggests a sexual dimorphic distribution of OXTR in the brain. In recent years, the oxytocin system in the brain received tremendous attention as a potential pharmacological target for treatment of many psychiatric disorders, such as anxiety, autism spectrum disorders, and even sex-specific psychiatric disorder like postpartum depression (PPD). An important problem and a critical barrier to progress in the field is that despite the importance, the cellular characterization and distribution of the OXTR expressing neurons in the brain are still largely unknown. The overall long-term objective of this project is to elucidate the physiological and behavioral significance of the estrogen-dependent, sexually dimorphic OXTR in the AVPV. This study was conducted to map OXTR expressing neurons in the hypothalamic medial preoptic area (MPOA), a region known as sexually dimorphic and hormone-sensitive area. We revealed using OXTR-reporter mice in which a part of the OXTR gene was replaced with fluorescent protein, Venus, that OXTR neurons are predominantly present in the anteroventral periventricular nucleus (AVPV) in the MPOA of females, but not of males. Moreover, the expression of OXTR in neurons of the AVPV in females depends upon estrogen. Ovariectomy (OVX) resulted in total loss of OXTR-Venus neurons in the AVPV. Because the onset of proper maternal behavior at parturition requires activation of OXTR in the MPOA, the female specific expression of OXTR in neurons of the AVPV implies that these neurons are involved in the induction of maternal behavior. The dopaminergic neurons in the AVPV are known to play a role in the regulation of maternal behavior. Our study showed that a fraction of the OXTR neurons are immunoreactive to tyrosine hydroxylase, an enzyme essential for dopamine synthesis, implying that some of the OXTR neurons are dopaminergic as well. Using immunocytochemistry and fluorescence microscopy, the exact distribution of OXTR-expressing dopaminergic neurons was shown and the changes in their expression during pregnancy and postpartum states were quantified. Using a chemogenetic approach to specifically inactivate OXTR neurons, this study shows that OXTR neurons in the AVPV are critical to regulate maternal behavior. Understanding sex differences in the oxytocin system in the brain could ultimately lead to sex-specific

Maternal care is crucial for the survival and development of offspring. Oxytocin modulates maternal behavior by binding to oxytocin receptors (OXTRs) in various parts of the brain. Previously, we showed that OXTRs are expressed in the anteroventral periventricular nucleus (AVPV) of female, but not male mice. Because the AVPV is involved in the regulation of maternal behavior and oxytocin enhances its induction, this finding leads to the hypothesis that the female specific population of OXTR neurons in the AVPV regulates maternal behavior. To address this hypothesis, OXTR-Venus reporter mice were used to assess if expression levels of OXTR in the AVPV are changed during the postpartum period. The total number of OXTR-Venus neurons was significantly greater in postpartum dams compared to virgin females. To assess efferent projections of the AVPV-OXTR neurons, a Cre-dependent fluorescent protein (tdTomato) expressing a viral vector was injected into one side of the AVPV of female OXTR-Cre mice. Fibers expressing tdTomato were found in hypothalamic areas containing oxytocin neurons (the supraoptic and paraventricular nuclei) and the midbrain areas (the ventral tegmental area and periaqueductal gray) that are involved in the regulation of maternal motivation. To assess if activity of the AVPV-OXTR neurons is involved in the regulation of maternal behaviors, a chemogenetic approach was employed. Specific inhibition of activity of AVPV-OXTR neurons completely abolished pup retrieval and nest building behaviors. Collectively, these findings demonstrate that AVPV-OXTR neurons in postpartum female mice constitute an important node in the neural circuitry that regulates maternal behavior.

Background: Although it is known that moderate-to-high doses of the neurohypophysial hormones oxytocin and vasopressin are natriuretic, doubts remain over the identity of the receptors responsible. To address this issue, we have used highly selective antagonists of oxytocin and vasopressin receptors in animals with elevated endogenous circulating levels of the 2 hormones. Methods: Rats were anaesthetised and prepared surgically for clearance studies, thereby raising plasma oxytocin and vasopressin concentrations. Sodium excretion, glomerular filtration rate and lithium clearance (an index of end-proximal fluid delivery) were measured: first during a control period, then after administration of the selective oxytocin receptor antagonist desGly-NH₂,d(CH₂)₅[D-Trp²,Thr⁴,Dap⁵]OVT, the selective vasopressin V_1a receptor antagonist d(CH₂)₅[Tyr(Me)²,Dab⁵]AVP, or vehicle alone. Results: Absolute and fractional sodium excretion fell in rats given the oxytocin antagonist (by 32 and 27%, respectively, compared with corresponding values in vehicle-infused rats), but not in those given the V_1a antagonist or vehicle. Antinatriuresis was associated with a small reduction in the ratio of sodium clearance to lithium clearance (an index of the fraction of distally delivered sodium that escapes reabsorption in the distal nephron). Conclusions: These results corroborate previous studies showing that activation of oxytocin receptors increases sodium excretion and imply that the natriuretic effect of elevated plasma vasopressin concentrations results from stimulation of oxytocin receptors.

Oxytocin (OT) and arginine vasopressin (AVP), in their capacities as neuromodulators, are believed to play an important role in mood control, including regulation of the anxiety response. In the present study, the contributions of oxytocin and vasopressin receptor modulation to anxiety-like behaviors were examined in male Sprague-Dawley rats. The behavioral effects of the OT receptor agonist, carbetocin (intracerebroventricular, intravenous and intraperitoneal routes), the AVP receptor agonist desmopressin (intravenous route), and the OT/AVP(1A) receptor antagonist atosiban (intravenous route) were evaluated in the elevated plus maze. The benzodiazepine diazepam was included as a positive control. Central but not systemic administration of carbetocin produced pronounced anxiolytic-like behavioral changes comparable to those measured following systemic diazepam treatment. The anxiolytic efficacy of carbetocin was maintained following 10 days of once-daily treatment, contrasting with the effects of diazepam which were no longer distinguishable from saline treatment. Systemic administration of desmopressin produced anxiogenic-like effects whereas systemic atosiban produced anxiolytic-like effects. Co-administration of desmopressin with atosiban resulted in saline-like behavioral responses, implicating an AVP(1A) receptor mechanism in the anxiolytic and anxiogenic effects of these neuropeptides following systemic administration. A peripherally-mediated antidiuretic effect of desmopressin on water consumption was also demonstrated. These results highlight the potential therapeutic utility of AVP(1A) receptor blockade in the modulation of anxiety-related behaviors; AVP(1A) receptor blockade appears to be a more promising pharmacological target than does OT receptor activation following systemic drug administration.

Depolarizing afterpotentials (DAPs) that follow action potentials in magnocellular neurosecretory cells (MNCs) are thought to underlie the generation of phasic firing, a pattern that optimizes vasopressin release from the neurohypophysis. Previous work has suggested that the DAP may result from the Ca(2+)-dependent reduction of a resting K(+) conductance. Here we examined the effects of flufenamic acid (FFA), a blocker of Ca(2+)-dependent non-selective cation (CAN) channels, on DAPs and phasic firing using intracellular recordings from supraoptic MNCs in superfused explants of rat hypothalamus. Application of FFA, but not solvent (0.1 % DMSO), reversibly inhibited (IC(50) = 13.8 microM; R = 0.97) DAPs and phasic firing with a similar time course, but had no significant effects (P > 0.05) on membrane potential, spike threshold and input resistance, nor on the frequency and amplitude of spontaneous synaptic potentials. Moreover, FFA did not affect (P > 0.05) the amplitude, duration, undershoot, or frequency-dependent broadening of action potentials elicited during the spike trains used to evoke DAPs. These findings suggest that FFA inhibits the DAP by directly blocking the channels responsible for its production, rather than by interfering with Ca(2+) influx. They also support a role for DAPs in the generation of phasic firing in MNCs. Finally, the absence of a depolarization and increased membrane resistance upon application of FFA suggests that the DAP in MNCs may not be due to the inhibition of resting K(+) current, but to the activation of CAN channels.

Oxytocin receptors modulate a social salience neural network in male prairie voles

The oxytocinergic system is critically involved in the regulation of maternal behavior, which includes maternal aggression. Because aggression has been linked to anxiety, we investigated the maternal aggression and the role of brain oxytocin in lactating Wistar rats selectively bred for high anxiety-related behavior (HAB) or low anxiety-related behavior (LAB) during the 10 min maternal defense test. HAB dams displayed more maternal aggression against a virgin intruder compared with LAB dams, resulting in more defensive behavior and higher anxiety of HAB-defeated virgins. The different levels of aggression were accompanied by opposite oxytocin release patterns within the paraventricular nucleus (PVN; HAB, increase; LAB, decrease). Furthermore, oxytocin release was higher within the central nucleus of the amygdala (CeA) of HAB dams compared with LABs. A direct correlation between the offensive behavior displayed during the maternal defense test and local oxytocin release was found in both the PVN and CeA. Using retrodialysis, blockade of endogenous oxytocin action by infusion of an oxytocin receptor antagonist (des-Gly-NH2,d(CH2)5[Tyr(Me)2,Thr4]OVT) into the PVN or CeA reduced maternal aggression of HAB dams, whereas infusion of synthetic oxytocin into the PVN tended to increase aggression toward the intruder in LAB dams. There were no significant differences in oxytocin receptor mRNA expression or oxytocin receptor binding between lactating HAB and LAB dams. Therefore, differences in intracerebral release patterns of oxytocin, rather than differences at the level of oxytocin receptors, are critical for the regulation of maternal aggressive behavior.

Recent work has shown that oxytocin is involved in more than lactation and uterine contraction. The paraventricular nucleus of the hypothalamus (PVN) contains neuroendocrine neurons that control the release of hormones, including vasopressin and oxytocin. Other populations of PVN neurons do not release hormones, but rather project to and release neurotransmitters onto other neurons in the CNS involved in fluid retention, thermoregulation, sexual behavior and responses to stress. Activation of oxytocin receptors can be cardioprotective and reduces the adverse cardiovascular consequences of anxiety and stress, yet how oxytocin can affect heart rate and cardiac function is unknown. While anatomical work has shown the presence of peptides, including oxytocin, in the projections from the PVN to parasympathetic nuclei, electrophysiological studies to date have only demonstrated release of glutamate and activation of fast ligand gated receptors in these pathways. In this study, using rats, we directly show, using sniffer CHO cells that express oxytocin receptors and the Ca2+ indicator R-GECO, that optogenetic activation of channelrhodopsin-2 (ChR2) expressing PVN fibers in the brainstem activates oxytocin receptors in the dorsomotor nucleus of the vagus (DMNV). We also demonstrate that while a single photoactivation of PVN terminals only activates glutamatergic receptors in brainstem cardiac vagal neurons (CVNs), neurons that dominate the neural control of heart rate, both the paired pulse facilitation, and sustained enhancement of glutamate release in this pathway is mediated by activation of oxytocin receptors. Our results provide direct evidence that a pathway from the PVN likely releases oxytocin and enhances short-term plasticity of this critical autonomic connection.

A self‐activating intrinsic brake on bursting in CA3 neurons

Afterpotential characteristics and firing patterns in maturing rat hippocampal CA1 neurones in in vitro slices