Oxytocin promotes prefrontal population activity via the PVN-PFC pathway to regulate pain.

Neurons in the prefrontal cortex (PFC) can provide top-down regulation of sensory-affective experiences such as pain. Bottom-up modulation of sensory coding in the PFC, however, remains poorly understood. Here, we examined how oxytocin (OT) signaling from the hypothalamus regulates nociceptive coding in the PFC. In vivo time-lapse endoscopic calcium imaging in freely behaving rats showed that OT selectively enhanced population activity in the prelimbic PFC in response to nociceptive inputs. This population response resulted from the reduction of evoked GABAergic inhibition and manifested as elevated functional connectivity involving pain-responsive neurons. Direct inputs from OT-releasing neurons in the paraventricular nucleus (PVN) of the hypothalamus are crucial to maintaining this prefrontal nociceptive response. Activation of the prelimbic PFC by OT or direct optogenetic stimulation of oxytocinergic PVN projections reduced acute and chronic pain. These results suggest that oxytocinergic signaling in the PVN-PFC circuit constitutes a key mechanism to regulate cortical sensory processing.

Analysis of the hypothalamic oxytocin system and oxytocin receptor-expressing astrocytes in a mouse model of Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder characterized by hyperphagia, obesity, developmental delay and intellectual disability. Studies suggest dysfunctional signaling of the neuropeptide oxytocin as one of the key mechanisms in PWS, and administration of oxytocin via intranasal or systemic routes yielded promising results in both humans and mouse models. However, a detailed assessment of the oxytocin system in mouse models of PWS such as the Magel2-deficient Magel2tm1.Stw mouse, is lacking. In the present study, we performed an automated counting of oxytocin cells in the entire paraventricular nucleus of the hypothalamus of Magel2tm1.Stw and wild-type control mice and found a significant reduction in the caudal part, which represents the parvocellular subdivision. In addition, based on the recent discovery that some astrocytes express the oxytocin receptor (OTR), we performed detailed analysis of astrocyte numbers and morphology in various brain regions, and assessed expression levels of the astrocyte marker glial fibrillary acidic protein, which was significantly decreased in the hypothalamus, but not other brain regions in Magel2tm1.Stw mice. Finally, we analyzed the number of OTR-expressing astrocytes in various brain regions and found a significant reduction in the nucleus accumbens of Magel2tm1.Stw mice, as well as a sex-specific difference in the lateral septum. This study suggests a role for caudal paraventricular nucleus oxytocin neurons as well as OTR-expressing astrocytes in a mouse model of PWS, provides novel information about sex-specific expression of astrocytic OTRs, and presents several new brain regions containing OTR-expressing astrocytes in the mouse brain.

Efficiency of cell-type specific and generic promoters in transducing oxytocin neurons and monitoring their neural activity during lactation.

Hypothalamic oxytocin (OXT) and arginine-vasopressin (AVP) neurons have been at the center of several physiological and behavioral studies. Advances in viral vector biology and the development of transgenic rodent models have allowed for targeted gene expression to study the functions of specific cell populations and brain circuits. In this study, we compared the efficiency of various adeno-associated viral vectors in these cell populations and demonstrated that none of the widely used promoters were, on their own, effective at driving expression of a down-stream fluorescent protein in OXT or AVP neurons. As anticipated, the OXT promoter could efficiently drive gene expression in OXT neurons and this efficiency is solely attributed to the promoter and not the viral serotype. We also report that a dual virus approach using an OXT promoter driven Cre recombinase significantly improved the efficiency of viral transduction in OXT neurons. Finally, we demonstrate the utility of the OXT promoter for conducting functional studies on OXT neurons by using an OXT specific viral system to record neural activity of OXT neurons in lactating female rats across time. We conclude that extreme caution is needed when employing non-neuron-specific viral approaches/promoters to study neural populations within the paraventricular nucleus of the hypothalamus.

The multiple faces of the oxytocin and vasopressin systems in the brain.

Classically, hypothalamic neuroendocrine cells that synthesise oxytocin and vasopressin were categorised in two major cell types: the magnocellular and parvocellular neurones. It was assumed that magnocellular neurones project exclusively to the pituitary gland where they release oxytocin and vasopressin into the systemic circulation. The parvocellular neurones, on the other hand, project within the brain to regulate discrete brain circuitries and behaviours. Within the last few years, it has become evident that the classical view of these projections is outdated. It is now clear that oxytocin and vasopressin in the brain are released extrasynaptically from dendrites and from varicosities in distant axons. The peptides act principally to modulate information transfer through conventional synapses (such as glutamate synapses) by actions at respective receptors that may be preferentially localised to synaptic regions (on either side of the synapse) to alter the 'gain' of conventional synapses.

Extrahypothalamic oxytocin neurons drive stress-induced social vigilance and avoidance.

Oxytocin increases the salience of both positive and negative social contexts and it is thought that these diverse actions on behavior are mediated in part through circuit-specific action. This hypothesis is based primarily on manipulations of oxytocin receptor function, leaving open the question of whether different populations of oxytocin neurons mediate different effects on behavior. Here we inhibited oxytocin synthesis in a stress-sensitive population of oxytocin neurons specifically within the medioventral bed nucleus of the stria terminalis (BNSTmv). Oxytocin knockdown prevented social stress-induced increases in social vigilance and decreases in social approach. Viral tracing of BNSTmv oxytocin neurons revealed fibers in regions controlling defensive behaviors, including lateral hypothalamus, anterior hypothalamus, and anteromedial BNST (BNSTam). Oxytocin infusion into BNSTam in stress naïve mice increased social vigilance and reduced social approach. These results show that a population of extrahypothalamic oxytocin neurons plays a key role in controlling stress-induced social anxiety behaviors.

A Fear Memory Engram and Its Plasticity in the Hypothalamic Oxytocin System.

Oxytocin (OT) release by axonal terminals onto the central nucleus of the amygdala exerts anxiolysis. To investigate which subpopulation of OT neurons contributes to this effect, we developed a novel method: virus-delivered genetic activity-induced tagging of cell ensembles (vGATE). With the vGATE method, we identified and permanently tagged a small subpopulation of OT cells, which, by optogenetic stimulation, strongly attenuated contextual fear-induced freezing, and pharmacogenetic silencing of tagged OT neurons impaired context-specific fear extinction, demonstrating that the tagged OT neurons are sufficient and necessary, respectively, to control contextual fear. Intriguingly, OT cell terminals of fear-experienced rats displayed enhanced glutamate release in the amygdala. Furthermore, rats exposed to another round of fear conditioning displayed 5-fold more activated magnocellular OT neurons in a novel environment than a familiar one, possibly for a generalized fear response. Thus, our results provide first evidence that hypothalamic OT neurons represent a fear memory engram.

Chemogenetic activation of oxytocin neurons: Temporal dynamics, hormonal release, and behavioral consequences.

Chemogenetics provides cell type-specific remote control of neuronal activity. Here, we describe the application of chemogenetics used to specifically activate oxytocin (OT) neurons as representatives of a unique class of neuroendocrine cells. We injected recombinant adeno-associated vectors, driving the stimulatory subunit hM3Dq of a modified human muscarinic receptor into the rat hypothalamus to achieve cell type-specific expression in OT neurons. As chemogenetic activation of OT neurons has not been reported, we provide systematic analysis of the temporal dynamics of OT neuronal responses in vivo by monitoring calcium fluctuations in OT neurons, and intracerebral as well as peripheral release of OT. We further provide evidence for the efficiency of chemogenetic manipulation at behavioral levels, demonstrating that evoked activation of OT neurons leads to social motivation and anxiolysis. Altogether, our results will be profitable for researchers working on the physiology of neuroendocrine systems, peptidergic modulation of behaviors and translational psychiatry.

Hypothalamic miR-103 protects from hyperphagic obesity in mice.

The role of neuronal noncoding RNAs in energy control of the body is not fully understood. The arcuate nucleus (ARC) of the hypothalamus comprises neurons regulating food intake and body weight. Here we show that Dicer-dependent loss of microRNAs in these neurons of adult (DicerCKO) mice causes chronic overactivation of the signaling pathways involving phosphatidylinositol-3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) and an imbalance in the levels of neuropeptides, resulting in severe hyperphagic obesity. Similarly, the activation of PI3K-Akt-mTOR pathway due to Pten deletion in the adult forebrain leads to comparable weight increase. Conversely, the mTORC1 inhibitor rapamycin normalizes obesity in mice with an inactivated Dicer1 or Pten gene. Importantly, the continuous delivery of oligonucleotides mimicking microRNAs, which are predicted to target PI3K-Akt-mTOR pathway components, to the hypothalamus attenuates adiposity in DicerCKO mice. Furthermore, loss of miR-103 causes strong upregulation of the PI3K-Akt-mTOR pathway in vitro and its application into the ARC of the Dicer-deficient mice both reverses upregulation of Pik3cg, the mRNA encoding the catalytic subunit p110γ of the PI3K complex, and attenuates the hyperphagic obesity. Our data demonstrate in vivo the crucial role of neuronal microRNAs in the control of energy homeostasis.

Independent hypothalamic circuits for social and predator fear.

The neural circuits mediating fear to naturalistic threats are poorly understood. We found that functionally independent populations of neurons in the ventromedial hypothalamus (VMH), a region that has been implicated in feeding, sex and aggression, are essential for predator and social fear in mice. Our results establish a critical role for VMH in fear and have implications for selective intervention in pathological fear in humans.

Evoked axonal oxytocin release in the central amygdala attenuates fear response.

The hypothalamic neuropeptide oxytocin (OT), which controls childbirth and lactation, receives increasing attention for its effects on social behaviors, but how it reaches central brain regions is still unclear. Here we gained by recombinant viruses selective genetic access to hypothalamic OT neurons to study their connectivity and control their activity by optogenetic means. We found axons of hypothalamic OT neurons in the majority of forebrain regions, including the central amygdala (CeA), a structure critically involved in OT-mediated fear suppression. In vitro, exposure to blue light of channelrhodopsin-2-expressing OT axons activated a local GABAergic circuit that inhibited neurons in the output region of the CeA. Remarkably, in vivo, local blue-light-induced endogenous OT release robustly decreased freezing responses in fear-conditioned rats. Our results thus show widespread central projections of hypothalamic OT neurons and demonstrate that OT release from local axonal endings can specifically control region-associated behaviors.

Involvement of transducer of regulated cAMP response element-binding protein activity on corticotropin releasing hormone transcription.

We have recently shown that phospho-cAMP response element-binding protein (CREB) is essential but not sufficient for activation of CRH transcription, suggesting the requirement of a coactivator. Here, we test the hypothesis that the CREB coactivator, transducer of regulated CREB activity (TORC), is required for activation of CRH transcription, using the cell line 4B and primary cultures of hypothalamic neurons. Immunohistochemistry and Western blot experiments in 4B cells revealed time-dependent nuclear translocation of TORC1,TORC 2, and TORC3 by forskolin [but not by the phorbol ester, phorbol 12-myristate 13-acetate (PMA)] in a concentration-dependent manner. In reporter gene assays, cotransfection of TORC1 or TORC2 potentiated the stimulatory effect of forskolin on CRH promoter activity but had no effect in cells treated with PMA. Knockout of endogenous TORC using silencing RNA markedly inhibited forskolin-activated CRH promoter activity in 4B cells, as well as the induction of endogenous CRH primary transcript by forskolin in primary neuronal cultures. Coimmunoprecipitation and chromatin immunoprecipitation experiments in 4B cells revealed association of CREB and TORC in the nucleus, and recruitment of TORC2 by the CRH promoter, after 20-min incubation with forskolin. These studies demonstrate a correlation between nuclear translocation of TORC with association to the CRH promoter and activation of CRH transcription. The data suggest that TORC is required for transcriptional activation of the CRH promoter by acting as a CREB coactivator. In addition, cytoplasmic retention of TORC during PMA treatment is likely to explain the failure of phorbolesters to activate CRH transcription in spite of efficiently phosphorylating CREB.

Impaired reproductive behavior by lack of GluR-B containing AMPA receptors but not of NMDA receptors in hypothalamic and septal neurons.

The roles of ionotropic glutamate receptors in mammalian reproduction are unknown. We therefore generated mice lacking a major subtype of (S)-alpha-amino-3-hydroxy-5-methyl-isoxazolepropionic acid (AMPA) receptors or all N-methyl-d-aspartate (NMDA) receptors in GnRH neurons and other mainly limbic system neurons, primarily in hypothalamic and septal areas. Male mice without NMDA receptors in these neurons were not impaired in breeding and exhibited similar GnRH secretion as control littermates. However, male mice lacking GluR-B containing AMPA receptors in these neurons were poor breeders and severely impaired in reproductive behaviors such as aggression and mounting. Testis and sperm morphology, testis weight, and serum testosterone levels, as well as GnRH secretion, were unchanged. Contact with female cage bedding failed to elicit male sexual behavior in these mice, unlike in control male littermates. Their female counterparts had unchanged ovarian morphology, had bred successfully, and had normal litter sizes but exhibited pronounced impairments in maternal behaviors such as pup retrieval and maternal aggression. Our results suggest that NMDA receptors and GluR-B containing AMPA receptors are not essential for fertility, but that GluR-B containing AMPA receptors are essential for male and female reproduction-related behaviors, perhaps by mediating responses to pheromones or odorants.