Increased reactivity of the paraventricular nucleus of the hypothalamus and decreased threat responding in male rats following psilocin administration.

Psychedelics have experienced renewed interest following positive clinical effects, however the neurobiological mechanisms underlying effects remain unclear. The paraventricular nucleus of the hypothalamus (PVN) plays an integral role in stress response, autonomic function, social behavior, and other affective processes. We investigated the effect of psilocin, the psychoactive metabolite of psilocybin, on PVN reactivity in Sprague Dawley rats. Psilocin increased stimulus-independent PVN activity as measured by c-Fos expression in male and female rats. Psilocin increased PVN reactivity to an aversive air-puff stimulus in males but not females. Reactivity was restored at 2- and 7-days post-injection with no group differences. Additionally, prior psilocin injection did not affect PVN reactivity following acute restraint stress. Experimental groups sub-classified by baseline threat responding indicate that increased male PVN reactivity is driven by active threat responders. These findings identify the PVN as a significant site of psychedelic drug action with implications for threat responding behavior.

Oxytocin neurons in the paraventricular nucleus and fear empathy among male mice.

BACKGROUND: Recent studies have identified empathy deficit as a core impairment and diagnostic criterion for people with autism spectrum disorders; however, the improvement of empathy focuses primarily on behavioural interventions without the target regulation. We sought to compare brain regions associated with empathy-like behaviours of fear and pain, and to explore the role of the oxytocin-oxytocin receptor system in fear empathy. METHODS: We used C57BL mice to establish 2 models of fear empathy and pain empathy. We employed immunofluorescence histochemical techniques to observe the expression of c-Fos throughout the entire brain and subsequently quantified the number of c-Fos-positive cells in different brain regions. Furthermore, we employed chemogenetic technology to selectively manipulate these neurons in Oxt-Cre-/+ mice to identify the role of oxytocin in this process. RESULTS: The regions activated by fear empathy were the anterior cingulate cortex, basolateral amygdala, nucleus accumbens, paraventricular nucleus (PVN), lateral habenula, and ventral and dorsal hippocampus. The regions activated by pain empathy were the anterior cingulate cortex, basolateral amygdala, nucleus accumbens, and lateral habenula. We found that increasing the activity of oxytocin neurons in the PVN region enhanced the response to fear empathy. This enhancement may be mediated through oxytocin receptors. LIMITATIONS: This study included only male animals, which restricts the broader interpretation of the findings. Further investigations on circuit function need to be conducted. CONCLUSION: The brain regions implicated in the regulation of fear and pain empathy exhibit distinctions; the activity of PVN neurons was positively correlated with empathic behaviour in mice. These findings highlight the role of the PVN oxytocin pathway in regulating fear empathy and suggest the importance of oxytocin signalling in mediating empathetic responses.

Hypothalamic vasopressin sex differentiation is observed by embryonic day 15 in mice and is disrupted by the xenoestrogen bisphenol A.

Arginine vasopressin (AVP) neurons of the hypothalamic paraventricular region (AVPPVN) mediate sex-biased social behaviors across most species, including mammals. In mice, neural sex differences are thought to be established during a critical window around birth ( embryonic (E) day 18 to postnatal (P) day 2) whereby circulating testosterone from the fetal testis is converted to estrogen in sex-dimorphic brain regions. Here, we found that AVPPVN neurons are sexually dimorphic by E15.5, prior to this critical window, and that gestational bisphenol A (BPA) exposure permanently masculinized female AVPPVN neuronal numbers, projections, and electrophysiological properties, causing them to display male-like phenotypes into adulthood. Moreover, we showed that nearly twice as many neurons that became AVP+ by P0 were born at E11 in males and BPA-exposed females compared to control females, suggesting that AVPPVN neuronal masculinization occurs between E11 and P0. We further narrowed this sensitive period to around the timing of neurogenesis by demonstrating that exogenous estrogen exposure from E14.5 to E15.5 masculinized female AVPPVN neuronal numbers, whereas a pan-estrogen receptor antagonist exposed from E13.5 to E15.5 blocked masculinization of males. Finally, we showed that restricting BPA exposure to E7.5-E15.5 caused adult females to display increased social dominance over control females, consistent with an acquisition of male-like behaviors. Our study reveals an E11.5 to E15.5 window of estrogen sensitivity impacting AVPPVN sex differentiation, which is impacted by prenatal BPA exposure.

Sex differences in responses to aggressive encounters among California mice.

Despite how widespread female aggression is across the animal kingdom, there remains much unknown about its neuroendocrine mechanisms, especially in females that engage in aggression outside the peripartum period. Although the impact of aggressive experience on steroid hormone responses have been described, little is known about the impact of these experiences on female behavior or the subsequent neuropeptide responses to performing aggression. In this study, we compared behavioral responses in both male and female adult California mice based on if they had 0, 1, or 3 aggressive encounters using a resident intruder paradigm. We measured how arginine vasopressin and oxytocin cells in the paraventricular nucleus responded to aggression using c-fos immunohistochemistry. We saw that both sexes disengaged from intruders with repeated aggressive encounters, but that on the final day of testing females were more likely to freeze when they encountered intruders compared to no aggression controls - which was not significant in males. Finally, we saw that percent of arginine vasopressin and c-fos co-localizations in the posterior region of the paraventricular nucleus increased in males who fought compared to no aggression controls. No difference was observed in females. Overall, there is evidence that engaging in aggression induces stress responses in both sexes, and that females may be more sensitive to the effects of fighting.

Afferent Renal Denervation Attenuates Sympathetic Overactivation From the Paraventricular Nucleus in Spontaneously Hypertensive Rats.

BACKGROUND: The effectiveness of renal denervation (RDN) in reducing blood pressure and systemic sympathetic activity in hypertensive patients has been established. However, the underlying central mechanism remains unknown. This study aimed to investigate the role of RDN in regulating cardiovascular function via the central renin-angiotensin system (RAS) pathway. METHODS: Ten-week-old spontaneously hypertensive rats (SHR) were subjected to selective afferent renal denervation (ADN) using capsaicin solution. We hypothesized that ADN would effectively reduce blood pressure and rebalance the RAS component of the paraventricular nucleus (PVN) in SHR. RESULTS: The experimental results show that the ADN group exhibited significantly lower blood pressure, reduced systemic sympathetic activity, decreased chronic neuronal activation marker C-FOS expression in the PVN, and improved arterial baroreflex function, compared with the Sham group. Furthermore, ACE and AT1 protein expression was reduced while ACE2 and MAS protein expression was increased in the PVN of SHR after ADN. CONCLUSIONS: These findings suggest that RDN may exert these beneficial effects through modulating the central RAS pathway.

Decreased levels of hydrogen sulfide in the hypothalamic paraventricular nucleus contribute to sympathetic hyperactivity induced by cerebral infarction.

Paroxysmal sympathetic hyperactivity (PSH) is a common clinical feature secondary to ischemic stroke (IS), but its mechanism is poorly understood. We aimed to investigate the role of H2S in the pathogenesis of PSH. IS patients were divided into malignant (MCI) and non-malignant cerebral infarction (NMCI) group. IS in rats was induced by the right middle cerebral artery occlusion (MCAO). H2S donor (NaHS) or inhibitor (aminooxy-acetic acid, AOAA) were microinjected into the hypothalamic paraventricular nucleus (PVN). Compared with the NMCI group, patients in the MCI group showed PSH, including tachycardia, hypertension, and more plasma norepinephrine (NE) that was positively correlated with levels of creatine kinase, glutamate transaminase, and creatinine respectively. The 1-year survival rate of patients with high plasma NE levels was lower. The hypothalamus of rats with MCAO showed increased activity, especially in the PVN region. The levels of H2S in PVN of the rats with MCAO were reduced, while the blood pressure and renal sympathetic discharge were increased, which could be ameliorated by NaHS and exacerbated by AOAA. NaHS completely reduced the disulfide bond of NMDAR1 in PC12 cells. The inhibition of NMDAR by MK-801 microinjected in PVN of rats with MCAO also could lower blood pressure and renal sympathetic discharge. In conclusion, PSH may be associated with disease progression and survival in patients with IS. Decreased levels of H2S in PVN were involved in regulating sympathetic efferent activity after cerebral infarction. Our results might provide a new strategy and target for the prevention and treatment of PSH.

Central administration of AICAR attenuates hypertension via AMPK/Nrf2 pathway in the hypothalamic paraventricular nucleus of hypertensive rats.

BACKGROUND: Oxidative stress and inflammatory cytokines in the hypothalamus paraventricular nucleus (PVN) have been implicated in sympathetic nerve activity and the development of hypertension, but the specific mechanisms underlying their production in the PVN remains to be elucidated. Previous studies have demonstrated that activation of nuclear transcription related factor-2 (Nrf2) in the PVN reduced the production of reactive oxygen species (ROS) and inflammatory mediators. Moreover, AMP-activated protein kinase (AMPK), has been observed to decrease ROS and inflammatory cytokine production when activated in the periphery. 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) is an AMPK agonist. However, little research has been conducted on the role of AMPK in the PVN during hypertension. Therefore, we hypothesized that AICAR in the PVN is involved in regulating AMPK/Nrf2 pathway, affecting ROS and inflammatory cytokine expression, influencing sympathetic nerve activity. METHODS: Adult male Sprague-Dawley rats were utilized to induce two-kidney, one-clip (2K1C) hypertension via constriction of the right renal artery. Bilateral PVN was microinjected with either artificial cerebrospinal fluid or AICAR once a day for 4 weeks. RESULTS: Compared to the SHAM group, the PVN of 2K1C hypertensive rats decreased p-AMPK and p-Nrf2 expression, increased Fra-Like, NAD(P)H oxidase (NOX)2, NOX4, tumor necrosis factor-α and interleukin (IL)-1ß expression, elevated ROS levels, decreased superoxide dismutase 1 and IL-10 expression, and elevated plasma norepinephrine levels. Bilateral PVN microinjection of AICAR significantly ameliorated these changes. CONCLUSION: These findings suggest that repeated injection of AICAR in the PVN suppresses ROS and inflammatory cytokine production through the AMPK/Nrf2 pathway, reducing sympathetic nerve activity and improving hypertension.

Interleukin 17A Contributes to Blood-Brain Barrier Disruption of Hypothalamic Paraventricular Nucleus in Rats With Myocardial Infarction.

BACKGROUND: Elevated inflammatory cytokines in the periphery have been identified as active contributors to neuroinflammation and sympathetic overactivity in heart failure (HF). Yet, the exact mechanisms by which these cytokines breach the blood-brain barrier (BBB) to exert their effects on the brain remain elusive. Interleukin 17A has been linked to BBB disruption in various neurologic disorders, and its levels were significantly augmented in circulation and the brain in HF. The present study aimed to determine whether the BBB integrity was compromised within the hypothalamic paraventricular nucleus (PVN), and if so, whether interleukin 17A contributes to BBB disruption in myocardial infarction-induced HF. METHODS AND RESULTS: Male Sprague-Dawley rats underwent coronary artery ligation to induce HF or sham surgery. Some HF rats received bilateral PVN microinjections of an interleukin 17 receptor A small interfering RNA or a scrambled small interfering RNA adeno-associated virus. Four weeks after coronary artery ligation, the permeability of the BBB was evaluated by intracarotid injection of fluorescent dyes (fluorescein isothiocyanate-dextran 10 kDa+rhodamine-dextran 70 kDa). Compared with sham-operated rats, HF rats exhibited an elevated extravasation of fluorescein isothiocyanate-dextran 10 kDa within the PVN but not in the brain cortex. The plasma interleukin 17A levels were positively correlated with fluorescein isothiocyanate 10 kDa extravasation in the PVN. The expression of caveolin-1, a transcytosis marker, was augmented, whereas the expression of tight junction proteins was diminished in HF rats. Interleukin 17 receptor A was identified within the endothelium of PVN microvessels. Treatment with interleukin 17 receptor A small interfering RNA led to a significant attenuation of fluorescein isothiocyanate 10 kDa extravasation in the PVN and reversed expression of caveolin-1 and tight junction-associated proteins in the PVN. CONCLUSIONS: Collectively, these data indicate that BBB permeability within the PVN is enhanced in HF and is likely attributable to increased interleukin 17A/interleukin 17 receptor A signaling in the BBB endothelium, by promoting caveolar transcytosis and degradation of tight junction complexes.

Oxytocinergic projection from the hypothalamus to supramammillary nucleus drives recognition memory in mice.

Oxytocin (OXT) neurons project to various brain regions and its receptor expression is widely distributed. Although it has been reported that OXT administration affects cognitive function, it is unclear how endogenous OXT plays roles in cognitive function. The present study examined the role of endogenous OXT in mice cognitive function. OXT neurons were specifically activated by OXT neuron-specific excitatory Designer Receptors Exclusively Activated by Designer Drug expression system and following administration of clozapine-N-oxide (CNO). Object recognition memory was assessed with the novel object recognition task (NORT). Moreover, we observed the expression of c-Fos via immunohistochemical staining to confirm neuronal activity. In NORT, the novel object exploration time percentage significantly increased in CNO-treated mice. CNO-treated mice showed a significant increase in the number of c-Fos-positive cells in the supramammillary nucleus (SuM). In addition, we found that the OXT-positive fibers from paraventricular hypothalamic nucleus (PVN) were identified in the SuM. Furthermore, mice injected locally with CNO into the SuM to activate OXTergic axons projecting from the PVN to the SuM showed significantly increased percentage time of novel object exploration. Taken together, we proposed that object recognition memory in mice could be modulated by OXT neurons in the PVN projecting to the SuM.

High-calorie diets uncouple hypothalamic oxytocin neurons from a gut-to-brain satiation pathway via κ-opioid signaling.

Oxytocin-expressing paraventricular hypothalamic neurons (PVNOT neurons) integrate afferent signals from the gut, including cholecystokinin (CCK), to adjust whole-body energy homeostasis. However, the molecular underpinnings by which PVNOT neurons orchestrate gut-to-brain feeding control remain unclear. Here, we show that mice undergoing selective ablation of PVNOT neurons fail to reduce food intake in response to CCK and develop hyperphagic obesity on a chow diet. Notably, exposing wild-type mice to a high-fat/high-sugar (HFHS) diet recapitulates this insensitivity toward CCK, which is linked to diet-induced transcriptional and electrophysiological aberrations specifically in PVNOT neurons. Restoring OT pathways in diet-induced obese (DIO) mice via chemogenetics or polypharmacology sufficiently re-establishes CCK's anorexigenic effects. Last, by single-cell profiling, we identify a specialized PVNOT neuronal subpopulation with increased κ-opioid signaling under an HFHS diet, which restrains their CCK-evoked activation. In sum, we document a (patho)mechanism by which PVNOT signaling uncouples a gut-brain satiation pathway under obesogenic conditions.

Ghrelin Action in the PVH of Male Mice: Accessibility, Neuronal Targets, and CRH Neurons Activation.

The hormone ghrelin displays several well-characterized functions, including some with pharmaceutical interest. The receptor for ghrelin, the growth hormone secretagogue receptor (GHSR), is expressed in the hypothalamic paraventricular nucleus (PVH), a critical hub for the integration of metabolic, neuroendocrine, autonomic, and behavioral functions. Here, we performed a neuroanatomical and functional characterization of the neuronal types mediating ghrelin actions in the PVH of male mice. We found that fluorescent ghrelin mainly labels PVH neurons immunoreactive for nitric oxide synthase 1 (NOS1), which catalyze the production of nitric oxide [NO]). Centrally injected ghrelin increases c-Fos in NOS1 PVH neurons and NOS1 phosphorylation in the PVH. We also found that a high dose of systemically injected ghrelin increases the ghrelin level in the cerebrospinal fluid and in the periventricular PVH, and induces c-Fos in NOS1 PVH neurons. Such a high dose of systemically injected ghrelin activates a subset of NOS1 PVH neurons, which do not express oxytocin, via an arcuate nucleus-independent mechanism. Finally, we found that pharmacological inhibition of NO production fully abrogates ghrelin-induced increase of calcium concentration in corticotropin-releasing hormone neurons of the PVH whereas it partially impairs ghrelin-induced increase of plasma glucocorticoid levels. Thus, plasma ghrelin can directly target a subset of NO-producing neurons of the PVH that is involved in ghrelin-induced activation of the hypothalamic-pituitary-adrenal neuroendocrine axis.

Energy Expenditure Homeostasis Requires ErbB4, an Obesity Risk Gene, in the Paraventricular Nucleus.

Obesity affects more than a third adult population in the United States; the prevalence is even higher in patients with major depression disorders. GWAS studies identify the receptor tyrosine kinase ErbB4 as a risk gene for obesity and for major depression disorders. We found that ErbB4 was enriched in the paraventricular nucleus of the hypothalamus (PVH). To investigate its role in metabolism, we deleted ErbB4 by injecting a Cre-expressing virus into the PVH of ErbB4-floxed male mice and found that PVH ErbB4 deletion increased weight gain without altering food intake. ErbB4 PVH deletion also reduced nighttime activity and decreased intrascapular brown adipose tissue (iBAT) thermogenesis. Analysis of covariance (ANCOVA) revealed that ErbB4 PVH deletion reduced O2 consumption, CO2 production and heat generation in a manner independent of body weight. Immunostaining experiments show that ErbB4+ neurons in the PVH were positive for oxytocin (OXT); ErbB4 PVH deletion reduces serum levels of OXT. We characterized mice where ErbB4 was specifically mutated in OXT+ neurons and found reduction in energy expenditure, phenotypes similar to PVH ErbB4 deletion. Taken together, our data indicate that ErbB4 in the PVH regulates metabolism likely through regulation of OXT expressing neurons, reveal a novel function of ErbB4 and provide insight into pathophysiological mechanisms of depression-associated obesity.

Paraventricular nucleus projections to the nucleus tractus solitarii are essential for full expression of hypoxia-induced peripheral chemoreflex responses.

The hypothalamic paraventricular nucleus (PVN) is essential to peripheral chemoreflex neurocircuitry, but the specific efferent pathways utilized are not well defined. The PVN sends dense projections to the nucleus tractus solitarii (nTS), which exhibits neuronal activation following a hypoxic challenge. We hypothesized that nTS-projecting PVN (PVN-nTS) neurons contribute to hypoxia-induced nTS neuronal activation and cardiorespiratory responses. To selectively target PVN-nTS neurons, rats underwent bilateral nTS nanoinjection of retrogradely transported adeno-associated virus (AAV) driving Cre recombinase expression. We then nanoinjected into PVN AAVs driving Cre-dependent expression of Gq or Gi designer receptors exclusively activated by designer drugs (DREADDs) to test the degree that selective activation or inhibition, respectively, of the PVN-nTS pathway affects the hypoxic ventilatory response (HVR) of conscious rats. We used immunohistochemistry for Fos and extracellular recordings to examine how DREADD activation influences PVN-nTS neuronal activation by hypoxia. Pathway activation enhanced the HVR at moderate hypoxic intensities and increased PVN and nTS Fos immunoreactivity in normoxia and hypoxia. In contrast, PVN-nTS inhibition reduced both the HVR and PVN and nTS neuronal activation following hypoxia. To further confirm selective pathway effects on central cardiorespiratory output, rats underwent hypoxia before and after bilateral nTS nanoinjections of C21 to activate or inhibit PVN-nTS terminals. PVN terminal activation within the nTS enhanced tachycardic, sympathetic and phrenic (PhrNA) nerve activity responses to hypoxia whereas inhibition attenuated hypoxia-induced increases in nTS neuronal action potential discharge and PhrNA. The results demonstrate the PVN-nTS pathway enhances nTS neuronal activation and is necessary for full cardiorespiratory responses to hypoxia. KEY POINTS: The hypothalamic paraventricular nucleus (PVN) contributes to peripheral chemoreflex cardiorespiratory responses, but specific PVN efferent pathways are not known. The nucleus tractus solitarii (nTS) is the first integration site of the peripheral chemoreflex, and the nTS receives dense projections from the PVN. Selective GqDREADD activation of the PVN-nTS pathway was shown to enhance ventilatory responses to hypoxia and activation (Fos immunoreactivity (IR)) of nTS neurons in conscious rats, augmenting the sympathetic and phrenic nerve activity (SSNA and PhrNA) responses to hypoxia in anaesthetized rats. Selective GiDREADD inhibition of PVN-nTS neurons attenuates ventilatory responses, nTS neuronal Fos-IR, action potential discharge and PhrNA responses to hypoxia. These results demonstrate that a projection from the PVN to the nTS is critical for full chemoreflex responses to hypoxia.

Anthocyanin attenuates high salt-induced hypertension via inhibiting the hyperactivity of the sympathetic nervous system.

BACKGROUND: Anthocyanin plays a protective role in cardiovascular disease through antioxidant effect. Whether anthocyanin can reduce salt-induced hypertension and the related mechanisms remain unclear. METHODS: Chronic infusion of vehicle (artificial cerebrospinal fluid, aCSF, 0.4 µL/h) or anthocyanin (10 mg/kg, 0.4 µL/h) into bilateral paraventricular nucleus (PVN) of Sprague-Dawley rats was performed. Then, the rats were fed a high salt diet (8% NaCl, HS) or normal salt diet (0.9%, NaCl, NS) for 4 weeks. RESULTS: High salt diet induced an increase in blood pressure and peripheral sympathetic nerve activity (increased LF/HF and decreased SDNN and RMSSD), which was accompanied by increased reactive oxygen species (ROS) production and angiotensin II type-1 receptor (AT1R) expression and function in the PVN. Moreover, the NOD-like receptor protein 3 (NLRP3) and related inflammatory proteins (caspase-1) expression, the pro-inflammatory cytokine levels including IL-1ß and TNF-α were higher in PVN of rats with a high salt diet. Bilateral PVN infusion of anthocyanin attenuated NLRP3-dependent inflammation (NLRP3, caspase-1, IL-1ß and TNF-α) and ROS production, reduced AT1R expression and function in PVN and lowered peripheral sympathetic nerve activity and blood pressure in rats with salt-induced hypertension. CONCLUSIONS: Excessive salt intake activates NLRP3-dependent inflammation and oxidative stress and increased AT1R expression and function in the PVN. Bilateral PVN infusion of anthocyanin lowers peripheral sympathetic nerve activity and blood pressure in rats with salt-induced hypertension by improvement of expression and function of AT1R in the PVN through inhibiting NLRP3 related inflammatory and oxidative stress.

Kisspeptin neuron projections to oxytocin neurons are not necessary for parturition in the mouse.

Oxytocin is synthesized by hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) neurons and is released from the posterior pituitary gland to trigger uterine contractions during parturition. In rats, oxytocin neuron innervation by periventricular nucleus (PeN) kisspeptin neurons increases over pregnancy and intra-SON kisspeptin administration excites oxytocin neurons only in late pregnancy. To test the hypothesis that kisspeptin neurons excite oxytocin neurons to trigger uterine contractions during birth in C57/B6J mice, double-label immunohistochemistry for kisspeptin and oxytocin first confirmed that kisspeptin neurons project to the SON and PVN. Furthermore, kisspeptin fibers expressed synaptophysin and formed close appositions with oxytocin neurons in the mouse SON and PVN before and during pregnancy. Stereotaxic viral delivery of caspase-3 into the AVPV/PeN of Kiss-Cre mice before mating reduced kisspeptin expression in the AVPV, PeN, SON and PVN by > 90% but did not affect the duration of pregnancy or the timing of delivery of each pup during parturition. Therefore, it appears that AVPV/PeN kisspeptin neuron projections to oxytocin neurons are not necessary for parturition in the mouse.

Isoflurane Rapidly Modifies Synaptic and Cytoskeletal Phosphoproteomes of the Supraoptic Nucleus of the Hypothalamus and the Cortex.

INTRODUCTION: Despite the widespread use of general anaesthetics, the mechanisms mediating their effects are still not understood. Although suppressed in most parts of the brain, neuronal activity, as measured by FOS activation, is increased in the hypothalamic supraoptic nucleus (SON) by numerous general anaesthetics, and evidence points to this brain region being involved in the induction of general anaesthesia (GA) and natural sleep. Posttranslational modifications of proteins, including changes in phosphorylation, enable fast modulation of protein function which could be underlying the rapid effects of GA. In order to identify potential phosphorylation events in the brain-mediating GA effects, we have explored the phosphoproteome responses in the rat SON and compared these to cingulate cortex (CC) which displays no FOS activation in response to general anaesthetics. METHODS: Adult Sprague-Dawley rats were treated with isoflurane for 15 min. Proteins from the CC and SON were extracted and processed for nano-LC mass spectrometry (LC-MS/MS). Phosphoproteomic determinations were performed by LC-MS/MS. RESULTS: We found many changes in the phosphoproteomes of both the CC and SON in response to 15 min of isoflurane exposure. Pathway analysis indicated that proteins undergoing phosphorylation adaptations are involved in cytoskeleton remodelling and synaptic signalling events. Importantly, changes in protein phosphorylation appeared to be brain region specific suggesting that differential phosphorylation adaptations might underlie the different neuronal activity responses to GA between the CC and SON. CONCLUSION: In summary, these data suggest that rapid posttranslational modifications in proteins involved in cytoskeleton remodelling and synaptic signalling events might mediate the central mechanisms mediating GA.

The antidepressant-like effects of escitalopram in mice require salt-inducible kinase 1 and CREB-regulated transcription co-activator 1 in the paraventricular nucleus of the hypothalamus.

BACKGROUND: The salt-inducible kinase 1 (SIK1)-CREB-regulated transcription co-activator 1 (CRTC1) system in the paraventricular nucleus (PVN) of the hypothalamus has been demonstrated to participate in not only depression neurobiology but also the antidepressant mechanisms of fluoxetine, paroxetine, venlafaxine, and duloxetine. Like fluoxetine and paroxetine, escitalopram is also a well-known selective serotonin (5-HT) reuptake inhibitor (SSRI). However, recently it has been found that escitalopram can modulate a lot of targets other than the 5-HT system. Here, we speculate that escitalopram produces effects on the SIK1-CRTC1 system in the PVN. METHODS: Two mice models of depression (chronic social defeat stress (CSDS) and chronic unpredictable mild stress (CUMS)), various behavioral tests, enzyme linked immunosorbent assay (ELISA), western blotting, co-immunoprecipitation (Co-IP), quantitative real-time reverse transcription PCR (qRT-PCR), immunofluorescence, and adeno-associated virus (AAV)-mediated gene transfer were used together in the present study. RESULTS: It was found that escitalopram administration not only significantly prevented the hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis induced by CSDS and CUMS, but also notably reversed the effects of CSDS and CUMS on SIK1, CRTC1, and CRTC1-CREB binding in the PVN of mice. AAV-based genetic knock-down of SIK1 in PVN neurons evidently abolished the antidepressant-like effects of escitalopram in mice. LIMITATION: A shortage of this study is that only rodent models of depression were used, while human samples were not included. CONCLUSIONS: In summary, regulating the SIK1-CRTC1 system in the PVN participates in the antidepressant mechanism of escitalopram, which extends the knowledge of the pharmacological actions of escitalopram.

Hypothalamic PVN CRH Neurons Signal Through PVN GABA Neurons to Suppress GnRH Pulse Generator Frequency in Female Mice.

Corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN) are central to the stress response. Chemogenetic activation of PVN CRH neurons decreases LH pulse frequency but the mechanism is unknown. In the present study, optogenetic stimulation of PVN CRH neurons suppressed LH pulse frequency in estradiol-replaced ovariectomized CRH-cre mice, and this effect was augmented or attenuated by intra-PVN GABAA or GABAB receptor antagonism, respectively. PVN CRH neurons signal to local GABA neurons, which may provide a possible indirect mechanism by which PVN CRH neurons suppress LH pulse frequency. Optogenetic stimulation of potential PVN GABAergic projection terminals in the hypothalamic arcuate nucleus in ovariectomized estradiol-replaced Vgat-cre-tdTomato mice via an optic fiber implanted in the arcuate nucleus suppressed LH pulse frequency. To further determine whether PVN CRH neurons signal through PVN GABA neurons to suppress LH pulsatility, we combined recombinase mice with intersectional vectors to selectively target these neurons. CRH-cre::Vgat-FlpO mice expressing the stimulatory opsin ChRmine in non-GABAergic CRH neurons alone or in combination with the inhibitory opsin NpHR3.3 in non-CRH-expressing GABA neurons in the PVN were used. Optogenetic stimulation of non-GABAergic CRH neurons suppressed pulsatile LH secretion; however, LH pulse frequency was not affected when CRH neurons were stimulated and PVN GABA neurons were simultaneously inhibited. Together, these studies demonstrate that suppression of LH pulse frequency in response to PVN CRH neuronal activation is mediated by GABAergic signalling intrinsic to the PVN and may incorporate PVN GABAergic projection to the hypothalamic GnRH pulse generator.

Effects of electroacupuncture at KI3 and ST36 on the hypothalamic paraventricular nucleus in a rat model of chronic glomerulonephritis.

OBJECTIVE: The hypothalamic paraventricular nucleus (PVN) acts as a critical integrating center of endocrine/autonomic responses and regulates visceral functional activities. However, its involvement in electroacupuncture (EA) treatment of chronic glomerulonephritis (CGN) remains unclear. METHODS: Over four experiments, we randomized 111 rats into: control, untreated model (CGN) or EA-treated model (CGN + EA) groups, a model group receiving EA after PVN damage (CGN + EA + Lesion) or untreated model groups injected with adeno-associated viral vectors encoding human M4 muscarinic receptor (CGN + hM4D) or enhanced green fluorescent protein (CGN + EGFP). CGN was modeled by intraperitoneal injection of bovine serum albumin for 2 weeks. Rats in the CGN + EA and CGN + EA + Lesion groups received EA at bilateral ST36 and KI3 for 14 days. Urine/serum samples were collected to evaluate inflammatory factors and changes in renal function. RESULTS: EA inhibited the release of interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-1ß, and decreased urine protein (PRO), creatinine (Cre) and blood urea nitrogen (BUN) levels. PVN damage influenced the effect of EA on the levels of these parameters. EA appeared to inhibit the firing frequency and spectral energy of PVN neurons. In the viral vector experiment, levels of PRO, Cre, IL-6, IL-1ß and TNF-α in the CGN group were increased in CGN versus control groups (p < 0.0001), decreased in CGN + hM4D versus CGN groups (p < 0.05) and did not differ between CGN + EGFP and control groups (p > 0.05). CONCLUSION: Our findings indicate that EA at ST36 and KI3 improves CGN in this rat model by weakening the activity of PVN neurons, alleviating impairment of renal function impairment and restricting the release of inflammatory factors.

Dynamic modulation of pulsatile activities of oxytocin neurons in lactating wild-type mice.

Breastfeeding, which is essential for the survival of mammalian infants, is critically mediated by pulsatile secretion of the pituitary hormone oxytocin from the central oxytocin neurons located in the paraventricular and supraoptic hypothalamic nuclei of mothers. Despite its importance, the molecular and neural circuit mechanisms of the milk ejection reflex remain poorly understood, in part because a mouse model to study lactation was only recently established. In our previous study, we successfully introduced fiber photometry-based chronic imaging of the pulsatile activities of oxytocin neurons during lactation. However, the necessity of Cre recombinase-based double knock-in mice substantially compromised the use of various Cre-dependent neuroscience toolkits. To overcome this obstacle, we developed a simple Cre-free method for monitoring oxytocin neurons by an adeno-associated virus vector driving GCaMP6s under a 2.6 kb mouse oxytocin mini-promoter. Using this method, we monitored calcium ion transients of oxytocin neurons in the paraventricular nucleus in wild-type C57BL/6N and ICR mothers without genetic crossing. By combining this method with video recordings of mothers and pups, we found that the pulsatile activities of oxytocin neurons require physical mother-pup contact for the milk ejection reflex. Notably, the frequencies of photometric signals were dynamically modulated by mother-pup reunions after isolation and during natural weaning stages. Collectively, the present study illuminates the temporal dynamics of pulsatile activities of oxytocin neurons in wild-type mice and provides a tool to characterize maternal oxytocin functions.