Pubertal maternal presence reduces anxiety and increases adult neurogenesis in Kunming mice offspring.

Puberty is a critical period of emotional development and neuroplasticity. However, most studies have focused on early development, with limited research on puberty, particularly the parental presence. In this study, four groups were established, and pubertal maternal presence (PMP) was assessed until postnatal days 21 (PD21), 28 (PD28), 35 (PD35), and 42 (PD42), respectively. The social interaction and anxiety behaviors, as well as the expression of oxytocin (OT) in the paraventricular nucleus (PVN) and supraoptic nucleus (SON), and the number of new generated neurons and the expression of estrogen receptor alpha (ERα) in the dentate gyrus (DG) were assessed. The results suggest that there is a lot of physical contact between the mother and offspring from 21 to 42 days of age, which reduces anxiety in both female and male offspring in adulthood; for example, the PMP increased the amount of time mice spent in the center area in the open field experiment and in the bright area in the light-dark box experiment. PMP increased OT expression in the PVN and SON and the number of newly generated neurons in the DG. However, there was a sexual difference in ERα, with ERα increasing in females but decreasing in males. In conclusion, PMP reduces the anxiety of offspring in adulthood, increases OT in the PVN and SON, and adult neurogenesis; ERα in the DG may be involved in this process.

Effects of bile duct ligation on the inhibitory control of supraoptic vasopressin neurons.

Dilutional hyponatremia due to increased plasma arginine vasopressin (AVP) is associated with liver cirrhosis. However, plasma AVP remains elevated despite progressive hypoosmolality. This study investigated changes to inhibitory control of supraoptic nucleus (SON) AVP neurons during liver cirrhosis. Experiments were conducted with adult male Sprague-Dawley rats. Bile duct ligation was used as a model of chronic liver cirrhosis. An adeno-associated virus containing a construct with an AVP promoter and either green fluorescent protein (GFP) or a ratiometric chloride indicator, ClopHensorN, was bilaterally injected into the SON of rats. After 2 weeks, rats received either BDL or sham surgery, and liver cirrhosis was allowed to develop for 4 weeks. In vitro, loose patch recordings of action potentials were obtained from GFP-labeled and unlabeled SON neurons in response to a brief focal application of the GABAA agonist muscimol (100 µM). Changes to intracellular chloride ([Cl]i) following muscimol application were determined by changes to the fluorescence ratio of ClopHensorN. The contribution of cation chloride cotransporters NKCC1 and KCC2 to changes in intracellular chloride was investigated using their respective antagonists, bumetanide (BU, 10 µM) and VU0240551 (10 µM). Plasma osmolality and hematocrit were measured as a marker of dilutional hyponatremia. The results showed reduced or absent GABAA -mediated inhibition in a greater proportion of AVP neurons from BDL rats as compared to sham rats (100% inhibition in sham vs. 47% in BDL, p = .001). Muscimol application was associated with increased [Cl]i in most cells from BDL as compared to cells from sham rats (χ2 = 30.24, p < .001). NKCC1 contributed to the impaired inhibition observed in BDL rats (p < .001 BDL - BU vs. BDL + BU). The results show that impaired inhibition of SON AVP neurons and increased intracellular chloride contribute to the sustained dilutional hyponatremia in liver cirrhosis.

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.

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.

Nucleus of the lateral olfactory tract: A hub linking the water homeostasis-associated supraoptic nucleus-arginine vasopressin circuit and neocortical regions to promote social behavior under osmotic challenge.

Homeostatic challenges may alter the drive for social interaction. The neural activity that prompts this motivation remains poorly understood. In the present study, we identify direct projections from the hypothalamic supraoptic nucleus to the cortico-amygdalar nucleus of the lateral olfactory tract (NLOT). Dual in situ hybridization with probes for pituitary adenylate cyclase-activating polypeptide (PACAP), as well as vesicular glutamate transporter (VGLUT)1, VGLUT2, V1a and V1b, revealed a population of vasopressin-receptive PACAPergic neurons in NLOT layer 2 (NLOT2). Water deprivation (48 h, WD48) increased sociability compared to euhydrated subjects, as assessed with the three-chamber social interaction test (3CST). Fos expression immunohistochemistry showed NLOT and its main efferent regions had further increases in rats subjected to WD48 + 3CST. These regions strongly expressed PAC1 mRNA. Microinjections of arginine vasopressin (AVP) into the NLOT produced similar changes in sociability to water deprivation, and these were reduced by co-injection of V1a or V1b antagonists along with AVP. We conclude that, during challenge to water homeostasis, there is a recruitment of a glutamatergic-multi-peptidergic cooperative circuit that promotes social behavior.

Excitatory Effects of Astrocytic Hydrogen Sulfide on the Electrical Activity of Oxytocin Neurons in the Supraoptic Nucleus.

INTRODUCTION: In the regulation of oxytocin (OT) neuronal activity, hydrogen sulfide (H2S), a gaseous neurotransmitter, likely exerts an excitatory role. This role is associated with increased expression of astrocytic cystathionine-ß-synthase (CBS), the key enzyme for H2S synthesis. However, it remains unclear whether H2S is mainly produced in astrocytes and contributes to the autoregulation of OT neurons. METHODS: In hypothalamic slices of male rats, OT and H2S-associated drug effects were observed on the firing activity and spontaneous excitatory postsynaptic currents (sEPSCs) of putative OT neurons in the supraoptic nucleus (SON) in whole-cell patch-clamp recording. Expression of glial fibrillary acidic protein (GFAP) in the SON was analyzed in Western blots. In addition, changes in the length of rat pups' hypothalamic astrocytic processes were observed in primary cultures. RESULTS: In brain slices, OT significantly increased the firing rate of OT neurons, which was simulated by CBS allosteric agonist S-adenosyl-L-methionine (SAM) and H2S slow-releasing donor GYY4137 but blocked by CBS inhibitor aminooxyacetic acid (AOAA). L-α-aminoadipic acid (a gliotoxin) blocked SAM-evoked excitation. OT and SAM also increased the frequency and amplitude of sEPSCs; the effect of OT was blocked by AOAA. Both OT and GYY4137 reduced GFAP expression in the SON. Morphologically, OT or GYY4137 time-dependently reduced the length of astrocytic processes in primary cultures. CONCLUSIONS: These findings indicate that the auto-excitatory effect of OT on OT neurons is mediated by H2S from astrocytes at least partially and astrocytic H2S can elicit retraction of astrocytic processes that subsequently increase OT neuronal excitability.

Abdominal surgery increases activity in several phoenixin immunoreactive nuclei.

BACKGROUND: Research on the peptide phoenixin has increased in recent years and greatly widened the known scope of its functions since its discovery in 2013. Involvement of phoenixin has since been shown in anxiety, food intake, reproduction as well as emotional and immunological stress. To further evaluate its involvement in stress reactions, this study aims to investigate the effects of abdominal surgery, a well-established physical stressor, on the activity of phoenixin-immunoreactive brain nuclei. METHODS: Male Sprague-Dawley rats (n = 6/group) were subjected to either an abdominal surgery stress protocol or a sham operation. Animals in the verum group were anesthetized, the abdominal cavity opened and the cecum palpated, followed by closing of the abdomen and recovery. Sham operated animals only received inhalation anesthesia and time for recovery. All animals were subsequently sacrificed and brains processed and evaluated for c-Fos activity as well as phoenixin density. RESULTS: Compared to control, abdominal surgery significantly increased c-Fos activity in the paraventricular nucleus (PVN, 6.4-fold, p < 0.001), the medial part of the nucleus of the solitary tract (mNTS, 3.8-fold, p < 0.001), raphe pallidus (RPa, 3.6-fold, p < 0.001), supraoptic nucleus (SON, 3.2-fold, p < 0.001), ventrolateral medulla (VLM, also called A1C1, 3.0-fold, p < 0.001), dorsal motor nucleus of vagus (DMN, 2.9-fold, p < 0.001), locus coeruleus (LC, 1.8-fold, p < 0.01) and Edinger-Westphal nucleus (EW, 1.6-fold, p < 0.05), while not significantly altering c-Fos activity in the amygdala (CeM, 1.3-fold, p > 0.05). Phoenixin immunoreactivity was not significantly affected by abdominal surgery (p > 0.05). CONCLUSION: The observed abdominal surgery-related increase in activity in phoenixin immunoreactive nuclei compared to sham surgery controls supports the hypothesis of an involvement of phoenixin in stress reactions. Interestingly, various psychological and physical stressors lead to specific changes in activity and immunoreactivity in phoenixin-containing nuclei, giving rise to a stressor-specific involvement of phoenixin.

Long-range axonal projections of transplanted mouse embryonic stem cell-derived hypothalamic neurons into adult mouse brain.

The hypothalamus is comprised of heterogenous cell populations and includes highly complex neural circuits that regulate the autonomic nerve system. Its dysfunction therefore results in severe endocrine disorders. Although recent experiments have been conducted for in vitro organogenesis of hypothalamic neurons from embryonic stem (ES) or induced pluripotent stem (iPS) cells, whether these stem cell-derived hypothalamic neurons can be useful for regenerative medicine remains unclear. We therefore performed orthotopic transplantation of mouse ES cell (mESC)-derived hypothalamic neurons into adult mouse brains. We generated electrophysiologically functional hypothalamic neurons from mESCs and transplanted them into the supraoptic nucleus of mice. Grafts extended their axons along hypothalamic nerve bundles in host brain, and some of them even projected into the posterior pituitary (PPit), which consists of distal axons of the magnocellular neurons located in hypothalamic supraoptic and paraventricular nuclei. The axonal projections to the PPit were not observed when the mESC-derived hypothalamic neurons were ectopically transplanted into the substantia nigra reticular part. These findings suggest that our stem cell-based orthotopic transplantation approach might contribute to the establishment of regenerative medicine for hypothalamic and pituitary disorders.

Norepinephrine innervation of the supraoptic nucleus contributes to increased copeptin and dilutional hyponatremia in male rats.

Dilutional hyponatremia associated with liver cirrhosis is due to inappropriate release of arginine vasopressin (AVP). Elevated plasma AVP causes water retention resulting in a decrease in plasma osmolality. Cirrhosis, in this study caused by ligation of the common bile duct (BDL), leads to a decrease in central vascular blood volume and hypotension, stimuli for nonosmotic AVP release. The A1/A2 neurons stimulate the release of AVP from the supraoptic nucleus (SON) in response to nonosmotic stimuli. We hypothesize that the A1/A2 noradrenergic neurons support chronic release of AVP in cirrhosis leading to dilutional hyponatremia. Adult, male rats were anesthetized with 2-3% isoflurane (mixed with 95% O2/5% CO2) and injected in the SON with anti-dopamine ß-hydroxylase (DBH) saporin (DSAP) or vehicle followed by either BDL or sham surgery. Plasma copeptin, osmolality, and hematocrit were measured. Brains were processed for ΔFosB, dopamine ß-hydroxylase (DBH), and AVP immunohistochemistry. DSAP injection: 1) significantly reduced the number of DBH immunoreactive A1/A2 neurons (A1, P < 0.0001; A2, P = 0.0014), 2) significantly reduced the number of A1/A2 neurons immunoreactive to both DBH and ΔFosB positive neurons (A1, P = 0.0015; A2, P < 0.0001), 3) reduced the number of SON neurons immunoreactive to both AVP and ΔFosB (P < 0.0001), 4) prevented the increase in plasma copeptin observed in vehicle-injected BDL rats (P = 0.0011), and 5) normalized plasma osmolality and hematocrit (plasma osmolality, P = 0.0475; hematocrit, P = 0.0051) as compared with vehicle injection. Our data suggest that A1/A2 neurons contribute to increased plasma copeptin and hypoosmolality in male BDL rats.

Rescue of Vasopressin Synthesis in Magnocellular Neurons of the Supraoptic Nucleus Normalises Acute Stress-Induced Adrenocorticotropin Secretion and Unmasks an Effect on Social Behaviour in Male Vasopressin-Deficient Brattleboro Rats.

The relevance of vasopressin (AVP) of magnocellular origin to the regulation of the endocrine stress axis and related behaviour is still under discussion. We aimed to obtain deeper insight into this process. To rescue magnocellular AVP synthesis, a vasopressin-containing adeno-associated virus vector (AVP-AAV) was injected into the supraoptic nucleus (SON) of AVP-deficient Brattleboro rats (di/di). We compared +/+, di/di, and AVP-AAV treated di/di male rats. The AVP-AAV treatment rescued the AVP synthesis in the SON both morphologically and functionally. It also rescued the peak of adrenocorticotropin release triggered by immune and metabolic challenges without affecting corticosterone levels. The elevated corticotropin-releasing hormone receptor 1 mRNA levels in the anterior pituitary of di/di-rats were diminished by the AVP-AAV-treatment. The altered c-Fos synthesis in di/di-rats in response to a metabolic stressor was normalised by AVP-AAV in both the SON and medial amygdala (MeA), but not in the central and basolateral amygdala or lateral hypothalamus. In vitro electrophysiological recordings showed an AVP-induced inhibition of MeA neurons that was prevented by picrotoxin administration, supporting the possible regulatory role of AVP originating in the SON. A memory deficit in the novel object recognition test seen in di/di animals remained unaffected by AVP-AAV treatment. Interestingly, although di/di rats show intact social investigation and aggression, the SON AVP-AAV treatment resulted in an alteration of these social behaviours. AVP released from the magnocellular SON neurons may stimulate adrenocorticotropin secretion in response to defined stressors and might participate in the fine-tuning of social behaviour with a possible contribution from the MeA.

AVP-eGFP was significantly upregulated by hypovolemia in the parvocellular division of the paraventricular nucleus in the transgenic rats.

Arginine vasopressin (AVP) is produced in the paraventricular (PVN) and supraoptic nuclei (SON). Peripheral AVP, which is secreted from the posterior pituitary, is produced in the magnocellular division of the PVN (mPVN) and SON. In addition, AVP is produced in the parvocellular division of the PVN (pPVN), where corticotrophin-releasing factor (CRF) is synthesized. These peptides synergistically modulate the hypothalamic-pituitary-adrenal (HPA) axis. Previous studies have revealed that the HPA axis was activated by hypovolemia. However, the detailed dynamics of AVP in the pPVN under hypovolemic state has not been elucidated. Here, we evaluated the effects of hypovolemia and hyperosmolality on the hypothalamus, using AVP-enhanced green fluorescent protein (eGFP) transgenic rats. Polyethylene glycol (PEG) or 3% hypertonic saline (HTN) was intraperitoneally administered to develop hypovolemia or hyperosmolality. AVP-eGFP intensity was robustly upregulated at 3 and 6 h after intraperitoneal administration of PEG or HTN in the mPVN. While in the pPVN, eGFP intensity was significantly increased at 6 h after intraperitoneal administration of PEG with significant induction of Fos-immunoreactive (-ir) neurons. Consistently, eGFP mRNA, AVP hnRNA, and CRF mRNA in the pPVN and plasma AVP and corticosterone were significantly increased at 6 h after intraperitoneal administration of PEG. The results suggest that AVP and CRF syntheses in the pPVN were activated by hypovolemia, resulting in the activation of the HPA axis.

Local kisspeptin excitation of rat oxytocin neurones in late pregnancy.

The hormone, oxytocin, is synthesised by magnocellular neurones of the supraoptic and paraventricular nuclei and is released from the posterior pituitary gland into the circulation to trigger uterine contractions during parturition. Kisspeptin fibre density increases around the supraoptic nucleus over pregnancy and intracerebroventricular kisspeptin excites oxytocin neurones only in late pregnancy. However, the mechanism of this excitation is unknown. Here, we found that microdialysis administration of kisspeptin into the supraoptic nucleus consistently increased the action potential (spike) firing rate of oxytocin neurones in urethane-anaesthetised late-pregnant rats (gestation day 18-21) but not in non-pregnant rats. Hazard analysis of action potential firing showed that kisspeptin specifically increased the probability of another action potential firing immediately after each action potential (post-spike excitability) in late-pregnant rats. Patch-clamp electrophysiology in hypothalamic slices showed that bath application of kisspeptin did not affect action potential frequency or baseline membrane potential in supraoptic nucleus neurones. Moreover, kisspeptin superfusion did not affect the frequency or amplitude of excitatory postsynaptic currents or inhibitory postsynaptic currents in supraoptic nucleus neurones. Taken together, these studies suggest that kisspeptin directly activates oxytocin neurones in late pregnancy, at least in part, via increased post-spike excitability. KEY POINTS: Oxytocin secretion is triggered by action potential firing in magnocellular neurones of the hypothalamic supraoptic and paraventricular nuclei to induce uterine contractions during birth. In late pregnancy, kisspeptin expression increases in rat periventricular nucleus neurones that project to the oxytocin system. Here, we show that intra-supraoptic nucleus administration of kisspeptin increases the action potential firing rate of oxytocin neurones in anaesthetised late-pregnant rats, and that the increased firing rate is associated with increased oxytocin neurone excitability immediately after each action potential. By contrast, kisspeptin superfusion of hypothalamic slices did not affect the activity of supraoptic nucleus neurones or the strength of local synaptic inputs to supraoptic nucleus neurones. Hence, kisspeptin might activate oxytocin neurons in late pregnancy by transiently increasing oxytocin neuron excitability after each action potential.

Astrocytic Hydrogen Sulfide Regulates Supraoptic Cellular Activity in the Adaptive Response of Lactating Rats to Chronic Social Stress.

Maternal social stress among breastfeeding women can be adapted in chronic process. However, neuroendocrine mechanisms underlying such adaptation remain to be identified. Here, we report the effects of 2 hr/day unfamiliar male rat invasion (UMI) stress on maternal behaviors in lactating rats during postpartum day 8 (UMI8) to postpartum day 12 (UMI12). Rat dams at UMI8 presented signs of maternal anxiety, depression, and attacks toward male intruder. These changes partially reversed at UMI12 except the sign of anxiety. In the supraoptic nucleus (SON), UMI12 but not UMI8 significantly increased the expression of c-Fos and phosphorylated extracellular signal-regulated protein kinase 1/2. At UMI8 but not UMI12, length of glial fibrillary acidic protein (GFAP, astrocytic cytoskeletal element) filaments around oxytocin (OT) neurons was significantly longer than that of their controls; the amount of GFAP fragments at UMI12 was significantly less than that at UMI8. Expression of cystathionine ß-synthase (CBS, enzyme for H2S synthesis) at UMI12 was significantly higher than that at UMI8. CBS expression did not change significantly in the somatic zone of the SON but decreased significantly at the ventral glia lamina at UMI8. In brain slices of the SON, aminooxyacetate (a CBS blocker) significantly increased the expression of GFAP proteins that were molecularly associated with CBS. Aminooxyacetate also reduced the firing rate of OT neurons whereas Na2S, a donor of H2S, increased it. The adaptation during chronic social stress is possibly attributable to the increased production of H2S by astrocytes and the subsequent retraction of astrocytic processes around OT neurons.

Sex Differences in the Regulation of Vasopressin and Oxytocin Secretion in Bile Duct-Ligated Rats.

INTRODUCTION: Hyponatremia due to elevated arginine vasopressin (AVP) secretion increases mortality in liver failure patients. No previous studies have addressed sex differences in hyponatremia in liver failure animal models. OBJECTIVE: This study addressed this gap in our understanding of the potential sex differences in hyponatremia associated with increased AVP secretion. METHODS: This study tested the role of sex in the development of hyponatremia using adult male, female, and ovariectomized (OVX) female bile duct-ligated (BDL) rats. RESULTS: All BDL rats had significantly increased liver to body weight ratios compared to sham controls. Male BDL rats had hyponatremia with significant increases in plasma copeptin and FosB expression in supraoptic AVP neurons compared to male shams (all p < 0.05; 5-7). Female BDL rats did not become hyponatremic or demonstrate increased supraoptic AVP neuron activation and copeptin secretion compared to female shams. Plasma oxytocin was significantly higher in female BDL rats compared to female sham (p < 0.05; 6-10). This increase was not observed in male BDL rats. Ovariectomy significantly decreased plasma estradiol in sham rats compared to intact female sham (p < 0.05; 6-10). However, circulating estradiol was significantly elevated in OVX BDL rats compared to the OVX and female shams (p < 0.05; 6-10). Adrenal estradiol, testosterone, and dehydroepiandrosterone (DHEA) were measured to identify a possible source of circulating estradiol in OVX BDL rats. The OVX BDL rats had significantly increased adrenal estradiol along with significantly decreased adrenal testosterone and DHEA compared to OVX shams (all p < 0.05; 6-7). Plasma osmolality, hematocrit, copeptin, and AVP neuron activation were not significantly different between OVX BDL and OVX shams. Plasma oxytocin was significantly higher in OVX BDL rats compared to OVX sham. CONCLUSIONS: Our results show that unlike male BDL rats, female and OVX BDL rats did not develop hyponatremia, supraoptic AVP neuron activation, or increased copeptin secretion compared to female shams. Adrenal estradiol might have compensated for the lack of ovarian estrogens in OVX BDL rats.

Spexin-expressing neurons in the magnocellular nuclei of the human hypothalamus.

Neuropeptides are involved in numerous brain activities being responsible for a wide spectrum of higher mental functions. The purpose of this concise, structural and qualitative investigation was to map the possible immunoreactivity of the novel neuropeptide spexin (SPX) within the human magnocellular hypothalamus. SPX is a newly identified peptide, a natural ligand for the galanin receptors (GALR) 2/3, with no molecular structure similarities to currently known regulatory factors. SPX seems to have multiple physiological functions, with an involvement in reproduction and food-intake regulation recently revealed in animal studies. For the first time we describe SPX expressing neurons in the supraoptic (SON) and paraventricular (PVN) nuclei of the human hypothalamus using immunohistochemical and fluorescent methods, key regions involved in the mechanisms of osmotic homeostasis, energy expenditure, consummatory behaviour, reproductive processes, social recognition and stress responses. The vast majority of neurons located in both examined neurosecretory nuclei show abundant SPX expression and this may indirectly implicate a potential contribution of SPX signalling to the hypothalamic physiology in the human brain.

Effect of amisulpride, olanzapine, quetiapine, and aripiprazole single administration on c-Fos expression in vasopressinergic and oxytocinergic neurons of the rat hypothalamic supraoptic nucleus.

OBJECTIVE: The goal of this study was to reveal the impact of four types of atypical antipsychotics including amisulpride (AMI), olanzapine (OLA), quetiapine (QUE), and aripiprazole (ARI), with different receptor-affinity profile and dissociation constant, on the activity of hypothalamic supraoptic nucleus (SON) vasopressinergic and oxytocinergic neurons. METHODS: Male Sprague Dawley rats received a single injection of vehicle (VEH) (0.1 ml/100g), AMI (20 mg/kg), OLA (5 mg/kg), QUE (15 mg/kg/) or ARI (10 mg/kg). Ninety min after treatment, the animals were fixed by transcardial perfusion, the brains removed, and cryocut into serial coronal sections of 35 µm thickness. The sections were processed for c-Fos staining using an avidin-biotin-peroxidase complex and visualized by nickel intensified diaminobenzidine to reach black end product. Afterwards, the sections were exposed to vasopressin (AVP) and oxytocin (OXY) antibodies and the reaction product visualized by biotin-labeled fluorescent Alexa Fluor 568 dye. The data were evaluated from c-Fos and AVP or OXY merged sections. RESULTS: The present study shows that all four antipsychotics applied induced c-Fos expression in the SON. With respect to the stimulation efficacy of the individual antipsychotics, estimated based on the quantity of c-Fos-labeled AVP and OXY neurons, could be a preferential action assigned to QUE over moderate effect of ARI and lower effect to OLA and reduced effect of AMI (VEH < AMI < OLA < ARI < QUE). CONCLUSION: The present data for the first time provide an insight into the quantitative pattern of brain activity within the clusters of SON AVP and OXY cells in response to different atypical antipsychotics single treatment.

Mapping of c-Fos expression in male tree shrew forebrain.

The tree shrew is susceptible to stimuli. However, mapping of c-Fos expression in male tree shrew forebrain has not been explored. The present results provided the first detailed mapping of c-Fos expression in the forebrain of the tree shrew (Tupaia belangeri chinensis). Acute restraint stress rapidly increased the density of c-Fos-immunoreactive (-ir) neurons in the medial orbital cortex (MO), infralimbic cortex, intermediate part of the lateral septal nucleus (LSi), ventral part of the lateral septal nucleus (LSv), anterior part of the bed nucleus of the stria terminalis, posterior part of the bed nucleus of the stria terminalis (STP), paraventricular nucleus of the hypothalamus, supraoptic nucleus, lateral hypothalamic area, ventromedial hypothalamic nucleus (VMH), and medial amygdaloid nucleus (MeA). Furthermore, a significant increase in c-Fos expression was observed in the MO, LSi, LSv, STP, VMH, arcuate hypothalamic nucleus, anterior amygdaloid area, MeA, and cortical amygdaloid nucleus immediately after acute footshock stress. In addition, the distinct patterns of c-Fos expression in the forebrain were shown in context-, restraint-, or footshock-treated tree shrews. In general, the present study provides the first detailed maps of c-Fos expression in male tree shrew forebrain immediately after various stimuli.

Acute myocardial infarction activates magnocellular vasopressin and oxytocin neurones.

Myocardial infarction (MI) is a leading cause of death worldwide. For those who survive the acute insult, the progressive dilation of the ventricle associated with chronic heart failure is driven by an adverse increase in circulating levels of the antidiuretic hormone, vasopressin, which is secreted from hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) nerve terminals. Although increased vasopressin neuronal activity has been demonstrated in the latter stages of chronic heart failure, we hypothesised that vasopressin neurones become activated immediately following an acute MI. Male Sprague-Dawley rats were anaesthetised and an acute MI was induced by ligation of the left anterior descending coronary artery. After 90 minutes of myocardial ischaemia, brains were collected. Dual-label immunohistochemistry was used to quantify the expression of Fos protein, a marker of neuronal activation, within vasopressin- or oxytocin-labelled neurones of the hypothalamic PVN and SON. Fos protein and tyrosine hydroxylase within the brainstem were also quantified. The results obtained show that the expression of Fos in both vasopressin and oxytocin neurones of the PVN and SON was significantly elevated as soon as 90 minutes post-MI compared to sham rats. Moreover, Fos protein was also elevated in tyrosine hydroxylase neurones in the nucleus tractus solitarius and rostral ventrolateral medulla of MI rats than sham rats. We conclude that magnocellular vasopressin and oxytocin neuronal activation occurs immediately following acute MI, rather than in the later stages of chronic heart failure. Therefore, prompt vasopressin antagonist therapy as an adjunct treatment for acute MI may impede the progression of ventricular dilatation, which remains a key adverse hallmark of chronic heart failure.

Effects of salt-loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging.

Salt-loading (SL) impairs GABAA inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down-regulating the activity of K+ /Cl- co-transporter2 (KCC2) and up-regulating Na+ /K+ /Cl- co-transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl- ]i in SON AVP neurones. The study combined virally-mediated chloride imaging with ClopHensorN with a single-cell western blot analysis. An adeno-associated virus with ClopHensorN and a vasopressin promoter (AAV2-0VP1-ClopHensorN) was bilaterally injected in the SON of adult male Sprague-Dawley rats that were either euhydrated (Eu) or salt-loaded (SL) for 7 days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl- ]i in response to focal application of the GABAA agonist muscimol (100 µmol L-1 ). SON AVP neurones from Eu rats showed muscimol-induced chloride influx (P < 0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P < 0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and ß-actin. Neurones that did not respond during chloride imaging did not show KCC2 and ß-actin protein expression. The KCC2 antagonist (VU0240551,10 µmol L-1 ) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10 µmol L-1 ) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol-induced Cl- influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA-12) (50 µmol L-1 ) and protein kinase inhibitor (K252a) (100 nmol L-1 ) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt-loading increases [Cl- ]i in SON AVP neurones via a TrKB-KCC2-NKCC1-dependent mechanism in rats.

A Common Neuroendocrine Substrate for Diverse General Anesthetics and Sleep.

How general anesthesia (GA) induces loss of consciousness remains unclear, and whether diverse anesthetic drugs and sleep share a common neural pathway is unknown. Previous studies have revealed that many GA drugs inhibit neural activity through targeting GABA receptors. Here, using Fos staining, ex vivo brain slice recording, and in vivo multi-channel electrophysiology, we discovered a core ensemble of hypothalamic neurons in and near the supraoptic nucleus, consisting primarily of neuroendocrine cells, which are persistently and commonly activated by multiple classes of GA drugs. Remarkably, chemogenetic or brief optogenetic activations of these anesthesia-activated neurons (AANs) strongly promote slow-wave sleep and potentiates GA, whereas conditional ablation or inhibition of AANs led to diminished slow-wave oscillation, significant loss of sleep, and shortened durations of GA. These findings identify a common neural substrate underlying diverse GA drugs and natural sleep and reveal a crucial role of the neuroendocrine system in regulating global brain states. VIDEO ABSTRACT.