Supraoptic Nucleus Neurons Research Articles

Characterisation of a Novel Osmosensitive Pathway from the Supraoptic Nucleus to the Median Preoptic Nucleus

The Supraoptic Nucleus (SON) is well regarded for the neuroendocrine release of vasopressin and oxytocin through the posterior pituitary gland. Evidence also exists for extra-pituitary efferent projections of SON neurons, mainly collaterals to the lateral hypothalamus and central amygdala. The objective of this study was to examine the existence of a central SON projection to the median preoptic nucleus (MnPO) of the hypothalamus. Microinjections of the retrograde tracer Cholera Toxin B Subunit (CTb) into the MnPO of male Wistar rats (n = 3) retrogradely labelled SON neurons. Fluorescence immunohistochemistry revealed equal involvement of vasopressin (AVP) and oxytocin (OXT) — producing SON neurons (n = 107 AVP+/CTb+, n = 110 OXT+/ CTb+, n = 417 total CTb+ neurons). This major collateral projection was also examined in an animal model of Polycystic Kidney Disease, the Lewis Polycystic Kidney (LPK) rat (n = 3 animals; n = 104 AVP+/CTb+, n = 41 OXT+/CTb+, n = 372 total CTb+ neurons) and its healthy Lewis control (n = 3 animals; n = 115 AVP+/CTb+, n = 100 OXT+/CTb+, n = 430 total CTb+ neurons). Although the function of this projection is currently unclear, preliminary data in a subset of LPKs shows these SON neurons with MnPO collateral projections are activated by osmotic stressors (e.g., hypertonic saline). In summary, we report a major collateral projection from the SON to MnPO that is activated by osmotic stress. Given the well-known role of the MnPO in drinking behaviour, we suggest this SON-MnPO circuit may be involved in the coordination of fluid regulation and drinking behaviour. This project was funded by Macquarie University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mitochondria Regulate Spatiotemporal Ca2+ Dynamics Between Cellular Compartments Affecting the Intrinsic Excitability of Vasopressin Neurons

Calcium (Ca2+) is a critical secondary messenger in all neurons that coordinates important mechanisms of cell physiology. In vasopressin (VP) neurons of supraoptic nucleus (SON) it’s required for somatodendritic release of VP and the slow afterhyperpolarization (sAHP), both of which coordinate neuronal output and thus control stimulus-secretion coupling. Recently, our lab demonstrated that VP sAHPs require endoplasmic reticulum (ER) Ca2+ for activation and that mitochondrial Ca2+ buffering shapes the spatiotemporal trajectory. We also have recent data demonstrating electrotonic segregation between cell compartments. Given the Ca2+ dependence of somatodendritic release and sAHPs, delineation of somatodendritic release from axonal release, and the decoupling of somatic and dendritic electrontonic properties, we hypothesized that calcium dynamics also may be compartmentally segregated as well. Utilizing patch clamp electrophysiology, live calcium imaging, and focal UV uncaging, we probed the spatiotemporal trajectory of rapidly evoked Ca2+ responses in VP neuron somas and dendrites with corresponding membrane potential. UV Ca2+ uncaging at the soma evokes a membrane sAHP and robust increase in cytosolic Ca2+ that propagates into the dendrites. Conversely, Ca2+ uncaged in dendrites propagates bidirectionally from the UV flash but does not penetrate the soma; no membrane hyperpolarization was observed under these conditions. Disabling mitochondrial Ca2+ buffering with Ru360 in the pipette amplifies Ca2+ signasl and sAHPs in somas and dendrites; Ca2+ uncaging in dendrites penetrates soma and can evoke sAHPs under these circumstances. Together, these results suggest that under normal conditions, Ca2+ movement between compartments in VP neurons is unidirectional (soma to dendrite), and that sAHP channels require somatic Ca2+ increases. Moreover, Ca2+ uptake by mitochondria is a critical mechanism that tightly controls the magnitude and progagation of dendritic Ca2+ signals between dendritic and somatic compartments, thus playing an important role in regulating intrinsic mechanisms and ultimately, their neuronal output. NIH K99HL168434 (M.K.K.); R01 HL162575-01 (J.E.S.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Brain-Derived Extracellular Vesicles from Dahl Salt-Sensitive Rats with High Salt Diet Increase PVN and SON Vasopressin Levels in Sprague Dawley rats

Extracellular vesicles (EVs), naturally secreted by almost all cell types, encapsulate a broad spectrum of biological molecules, and ferry these contents to the local and distal recipient cells. Under disease conditions, EV-to-cell communication can lead to diverse cell responses and changes. Previously we have demonstrated that brain-derived EVs from hypertensive rats increased inflammation and oxidative stress in primary neurons and hypothalamic paraventricular nucleus (PVN) of the brain, however, the long-term effects of EVs are unclear. PVN is a key hub to mediate blood pressure via its endocrine and autonomic control. Increased vasopressin (AVP) levels have been found in Dahl salt sensitive hypertensive rats, which are produced by PVN and supraoptic nucleus (SON). In this study, we hypothesized that brain-derived EVs from hypertensive rats regulate blood pressure via AVP system. To test this hypothesis, we first labeled brain-derived EVs with Rhodamine dye (Rho-EVs) and studied their bio-distribution via intracerebroventricular (i.c.v.) injection into the normal Sprague Dawley (SD) rats. Rats were euthanized 24 hours after injection, and brain sections containing the PVN and SON regions were stained to test the colocalization of Rho-EVs with neurons (NeuN), astrocytes (GFAP) and microglia (Iba1). Then we injected brain-derived EVs obtained from hypertensive Dahl salt sensitive rats (DSS-EVs) and normotensive Sprague Dawley rats (SD-EVs) into the lateral ventricle (8 μg/rat) in the SD male rats, and the injection was repeated 2 weeks later. Blood pressure and heart rates of each rat were monitored each week using telemetry transducers. 4 weeks post first EVs injections, rats were euthanized, their blood were tested for plasma AVP and norepinephrine (NE) levels, and their brains were tested for AVP and Fos-related antigen 1 (Fra1) mRNA levels. Besides, we also performed biweekly PVN (800 ng/rat) and SON (800ng/rat) injections of DSS-EVs and SD-EVs and measured the AVP and/or Fra1 protein levels with immunostaining. Lastly, we determined the colocalization of Rho-EVs with AVP-containing cells in the PVN and SON. Results show that Rho-EVs are dominantly taken up by neurons in both PVN and SON regions. Biweekly i.c.v. injections of DSS-EVs do not increase blood pressure and heart rate. However, DSS-EVs significantly upregulate mRNA levels of AVP (2.4-fold) and Fra1 (2.5-fold) in the PVN compared to SD-EVs. There is an increasing trend of plasma AVP levels in DSS-EV-treated rats compared to control. Biweekly PVN injections of DSS-EVs significantly increase AVP (1.5-fold) protein levels relative to SD-EVs, and biweekly SON injections of DSS-EVs significantly increase AVP (1.5-fold) and Fra1 (2.6-fold) protein levels relative to SD-EVs. Lastly, we found that Rho-EVs are mainly colocalized with vasopressin-containing cells in the SON. These results suggest that brain-derived EVs cause SON and PVN neuron activation, which in turn may regulate AVP system during hypertensive development. R01HL163159 (Shan); R15HL150703 (Shan); AHA 1807047 (Bi). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Potential role of oxytocin in the regulation of memories and treatment of memory disorders

Oxytocin (OXT) is an “affiliative” hormone or neurohormone or neuropeptide consists of nine amino acids, synthesized in magnocellular neurons of paraventricular (PVN) and supraoptic nuclei (SON) of hypothalamus. OXT receptors are widely distributed in various region of brain and OXT has been shown to regulate various social and nonsocial behavior. Hippocampus is the main region which regulates the learning and memory. Hippocampus particularly regulates the acquisition of new memories and retention of acquired memories. OXT has been shown to regulate the synaptic plasticity, neurogenesis, and consolidation of memories. Further, findings from both preclinical and clinical studies have suggested that the OXT treatment improves performance in memory related task. Various trials have suggested the positive impact of intranasal OXT in the dementia patients. However, these studies are limited in number. In the present study authors have highlighted the role of OXT in the formation and retrieval of memories. Further, the study demonstrated the outcome of OXT treatment in various memory and related disorders.

Neural control of fluid homeostasis is engaged below 10°C in hibernation.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) hibernate for several months each winter without access to water,1 but the mechanisms that maintain fluid homeostasis during hibernation are poorly understood. In torpor, when body temperature (TB) reaches 4°C, squirrels decrease metabolism, slow heart rate, and reduce plasma levels of the antidiuretic hormones arginine vasopressin (AVP) and oxytocin (OXT).1 Squirrels spontaneously undergo interbout arousal (IBA) every 2weeks, temporarily recovering an active-like metabolism and a TB of 37°C for up to 48 h.1,2 Despite the low levels of AVP and OXT during torpor, profound increases in blood pressure and heart rate during the torpor-IBA transition are not associated with massive fluid loss, suggesting the existence of a mechanism that protects against diuresis at a low TB. Here, we demonstrate that the antidiuretic hormone release pathway is activated by hypothalamic supraoptic nucleus (SON) neurons early in the torpor-arousal transition. SON neuron activity, dense-core vesicle release from the posterior pituitary, and plasma hormone levels all begin to increase before TB reaches 10°C. Invivo fiber photometry of SON neurons from hibernating squirrels, together with RNA sequencing and c-FOS immunohistochemistry, confirms that SON is electrically, transcriptionally, and translationally active to monitor blood osmolality throughout the dynamic torpor-arousal transition. Our work emphasizes the importance of the antidiuretic pathway during the torpor-arousal transition and reveals that the neurophysiological mechanism that coordinates the hormonal response to retain fluid is active at an extremely low TB, which is prohibitive for these processes in non-hibernators.

A dedicated hypothalamic oxytocin circuit controls aversive social learning.

To survive in a complex social group, one needs to know who to approach and, more importantly, who to avoid. In mice, a single defeat causes the losing mouse to stay away from the winner for weeks1. Here through a series of functional manipulation and recording experiments, we identify oxytocin neurons in the retrochiasmatic supraoptic nucleus (SOROXT) and oxytocin-receptor-expressing cells in the anterior subdivision of the ventromedial hypothalamus, ventrolateral part (aVMHvlOXTR) as a key circuit motif for defeat-induced social avoidance. Before defeat, aVMHvlOXTR cells minimally respond to aggressor cues. During defeat, aVMHvlOXTR cells are highly activated and, with the help of an exclusive oxytocin supply from the SOR, potentiate their responses to aggressor cues. After defeat, strong aggressor-induced aVMHvlOXTR cell activation drives the animal to avoid the aggressor and minimizes future defeat. Our study uncovers a neural process that supports rapid social learning caused by defeat and highlights the importance of the brain oxytocin system in social plasticity.

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.

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.

Glucagon-like Peptide-1 Receptor in the Human Hypothalamus Is Associated with Body Mass Index and Colocalizes with the Anorexigenic Neuropeptide Nucleobindin-2/Nesfatin-1.

Data on animals emphasize the importance of the neuronal glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) for feeding suppression, although it is unclear whether astrocytes participate in the transduction of anorectic GLP-1R-dependent signals. In humans, the brain circuitry underlying these effects remains insufficiently investigated. The present study aimed to explore GLP-1R protein expression in the human hypothalamus and its correlation with body mass index (BMI). Sections of hypothalamus from 28 autopsy cases, 11 with normal weight (BMI < 25 kg/m2) and 17 with non-normal weight (BMI ≥ 25 kg/m2), were examined using immunohistochemistry and double immunofluorescence labeling. Prominent GLP-1R immunoexpression was detected in neurons of several hypothalamic nuclei, including paraventricular, supraoptic, and infundibular nuclei; the lateral hypothalamic area (LH); and basal forebrain nuclei. Interestingly, in the LH, GLP-1R was significantly decreased in individuals with BMI ≥ 25 kg/m2 compared with their normal weight counterparts (p = 0.03). Furthermore, GLP-1R was negatively correlated (τb = −0.347, p = 0.024) with BMI levels only in the LH. GLP-1R extensively colocalized with the anorexigenic and antiobesogenic neuropeptide nucleobindin-2/nesfatin-1 but not with the astrocytic marker glial fibrillary acidic protein. These data suggest a potential role for GLP-1R in the regulation of energy balance in the human hypothalamus. In the LH, an appetite- and reward-related brain region, reduced GLP-1R immunoexpression may contribute to the dysregulation of homeostatic and/or hedonic feeding behavior. Possible effects of NUCB2/nesfatin-1 on central GLP-1R signaling require further investigation.

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 β-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 β-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.

Osmoregulatory neurons clockwork is altered during metabolic disorder induced by high energy diet in the Sand rat Psammomys obesus

ABSTRACT Increasing evidences highlight the role of vasopressin (AVP) in the regulation of metabolic functions and the association between hydration state and metabolic disorders. Supraoptic nuclei (SON) neurons are particularly sensitive to metabolic changes, as shown by the loss of plasma AVP rhythmicity during diabetes. We aimed in this work to determine the effect of high energy (HE) feeding on SON clock functioning in Psammomys obesus, a desert herbivore rodent in which metabolic syndrome is nutritionally induced. Animals were subjected to either normal or HE feeding for 3 months. Immunohistochemical study indicates that AVP and the circadian transcription factor CLOCK profiles follow daily oscillations with higher content in the beginning of the rest period in control group fed with low energy (LE) diet. Our data show that HE feeding induces critical changes through impairment of daily AVP and CLOCK cycling in the SON. These findings show clearly that SON clockwork is altered by HE diet-induced metabolic changes. Further studies are needed to better understand SON neurons responses to metabolic derangement.

Central Kisspeptin Does Not Affect ERK1/2 or p38 Phosphorylation in Oxytocin Neurons of Late-Pregnant Rats.

Oxytocin is secreted by hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) oxytocin neurons to induce uterine contractions during parturition. Increased activation of oxytocin neurons at parturition involves a network of afferent inputs that increase oxytocin neuron excitability. Kisspeptin fibre density increases around oxytocin neurons during pregnancy, and central kisspeptin administration excites oxytocin neurons only in late pregnancy. Kisspeptin signals via extracellular regulated kinase 1/2 (ERK1/2) and p38. Therefore, to determine whether kisspeptin excites oxytocin neurons via ERK1/2-p38 signalling in late-pregnant rats, we performed immunohistochemistry for phosphorylated ERK1/2 (pERK1/2) and phosphorylated p38 (p-p38) in oxytocin neurons of non-pregnant and late-pregnant rats. Intracerebroventricular (ICV) kisspeptin administration (2 µg) did not affect pERK1/2 or p-p38 expression in SON and PVN oxytocin neurons of non-pregnant or late-pregnant rats. Furthermore, ICV kisspeptin did not affect pERK1/2 or p-p38 expression in brain areas with major projections to the SON and PVN: the nucleus tractus solitarius, rostral ventrolateral medulla, locus coeruleus, dorsal raphe nucleus, organum vasculosum of the lamina terminalis, median preoptic nucleus, subfornical organ, anteroventral periventricular nucleus, periventricular nucleus and arcuate nucleus. Hence, kisspeptin-induced excitation of oxytocin neurons in late pregnancy does not appear to involve ERK1/2 or p38 activation in oxytocin neurons or their afferent inputs.

Bile Duct Ligation Changes the Inhibitory Control of Vasopressin Neurons in Male Rats

Dilutional hyponatremia typically is caused by increased Arginine Vasopressin (AVP) secretion. This increases the risk of complications and mortality in patients with liver cirrhosis. Liver cirrhosis in male rats increases AVP release but the underlying mechanisms are still under investigation. In other models of chronic AVP release, changes in chloride homeostasis alters the inhibitory control of AVP neurons in the supraoptic nucleus (SON) causing GABA mediated excitation instead of inhibition. In the bile duct ligation (BDL) model of cirrhosis, changes in either intracellular chloride concentration or cell firing activity have not been directly tested. We hypothesize that liver cirrhosis increases intracellular chloride concentration leading to a decrease in GABA‐mediated inhibition of SON neurons from male rats.To test this hypothesis, we injected an adeno‐associated virus with a vasopressin promoter and a ratiometric chloride indicator, AAV2‐0VP1‐ClopHensorN into the SON of adult male Sprague‐Dawley rats. The rats either received BDL surgery, which was used to model liver cirrhosis, or a sham ligation surgery. All BDL rats had significantly higher liver weight to body weight ratio (n = 10‐20, p &lt; 0.05), lower plasma osmolality (n = 10‐20, p &lt; 0.001) and lower hematocrit (n = 10‐20, p &lt; 0.05) as compared to sham rats. All rats were anesthetized with isoflurane (2‐3%), their brains were collected, and slices containing the SON were prepared. Loose‐cell patch recordings were made at 31±1oC using standard artificial cerebrospinal fluid. Labeled AVP cells and unlabeled SON cells were tested with focal application of muscimol (100µM), a GABAA receptor agonist.Cells from BDL rats showed impaired GABAA‐mediated inhibition in response to muscimol, as compared to those from sham rats. All putative AVP SON neurons from sham rats (4/4) showed decreased activity following muscimol while half of the putative AVP SON neurons (7/14) from BDL rats were excited (p &lt; 0.001). Similar results were observed in experiments were the injections missed the SON. In these experiments, all SON neurons from sham rats were inhibited by muscimol (26/26) while thirty‐five percent of SON neurons from BDL rats (16/45) were excited by muscimol (p = 0.001). ClopHensorN based chloride imaging showed that muscimol application was associated with decreased intracellular chloride concentrations in putative AVP cells from BDL rats as compared to sham rats (n = 31‐32, p = 0.001). The results show that liver cirrhosis impairs the normal inhibitory response of SON neurons to GABAA receptor activation which could contribute to increased cell activity and dilutional hyponatremia in male rats.

α-Melanocyte-stimulating hormone inhibition of oxytocin neurons switches to excitation in late pregnancy and lactation.

Oxytocin is secreted into the periphery by magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei (SON and PVN) to trigger uterine contraction during birth and milk ejection during suckling. Peripheral oxytocin secretion is triggered by action potential firing, which is regulated by afferent input activity and by feedback from oxytocin secreted into the extracellular space from magnocellular neuron somata and dendrites. A prominent input to oxytocin neurons arises from proopiomelanocortin neurons of the hypothalamic arcuate nucleus that secrete an alpha‐melanocyte‐stimulating hormone (α‐MSH), which inhibits oxytocin neuron firing in non‐pregnant rats by increasing somato‐dendritic oxytocin secretion. However, α‐MSH inhibition of oxytocin neuron firing is attenuated in mid‐pregnancy and somato‐dendritic oxytocin becomes auto‐excitatory in late‐pregnancy and lactation. Therefore, we hypothesized that attenuated α‐MSH inhibition of oxytocin neuron firing marks the beginning of a transition from inhibition to excitation to facilitate peripheral oxytocin secretion for parturition and lactation. Intra‐SON microdialysis administration of α‐MSH inhibited oxytocin neuron firing rate by 33 ± 9% in non‐pregnant rats but increased oxytocin neuron firing rate by 37 ± 12% in late‐pregnant rats and by 28 ± 10% in lactating rats. α‐MSH‐induced somato‐dendritic oxytocin secretion measured ex vivo with oxytocin receptor‐expressing “sniffer” cells, was of similar amplitude in PVN slices from non‐pregnant and lactating rats but longer‐lasting in slices from lactating rats. Hence, α‐MSH inhibition of oxytocin neuron activity switches to excitation over pregnancy while somato‐dendritic oxytocin secretion is maintained, which might enhance oxytocin neuron excitability to facilitate the increased peripheral secretion that is required for normal parturition and milk ejection.

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.

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.

The influence of melatonin on stress-induced ultramicroscopic changes of the neurons of the supraoptic nuclei of the rat hypothalamus

The aim: to study the effect of melatonin on the ultrastructural state of the supraoptic nuclei of the hypothalamus of rats under immobilization stress.Materials and methods. The experiments were performed on non-linear male white rats weighing 200-220 g. The animals were divided into 3 study series, in each of which the biomaterial was collected at 2 p.m. and at 2 a.m. using electron microscopic method. Long immobilization stress was simulated by keeping rats in special plastic penal cages for 6 hours daily for 7 consecutive days. Melatonin (Sigma, USA, 99.5% purification degree) at a dose of 0.5 mg/kg, in 1.0 ml of solvent (0.9% ethanol solution on physiologic saline) was injected daily, intraperitoneally.Results. When the animals were kept under the standard light regime, the ultrastructural organization of the hypothalamic nuclei at 2 p.m. indicated their low functional activity in comparison with the studies carried out at 2 a.m. Prolonged exposure of rats to immobilization stress was reflected in a significant rearrangement of the ultrastructural organization of supraoptic nuclei of the hypothalamus. The established changes can be considered as a manifestation of neurosecretory activity suppression, a decrease in neurosecretase production by hypothalamic neurons. Melatonin injections against the background of immobilization stress resulted in relative normalization of ultrastructural state of neurons of supraoptic nuclei of the hypothalamus of animals. In particular, studies at 2 a.m. revealed light neurosecretory cells containing a large nucleus, it was pyknotically altered. Karyolema invaginations, euchromatin dominance in the nucleus were observed. Heterogeneous changes were observed on the part of mitochondria. Enlarged tubules of granular endoplasmic reticulum were seen. At the same time, a small number of ribosomes and few hormonal granules were noticeable in neuroplasm. The mentioned picture of neurosecretory cells reflects a relative improvement in their electron microscopic state, which is evidenced by the appearance of neurosecretory granules. However, the ultrastructure of other organelles of the studied neurons indicates a depleted state caused by prolonged immobilization.Conclusions. 1. In animals under standard photoperiod conditions, the structural organization of supraoptic neurons of the hypothalamic nuclei during the nighttime of the experiment reflects the intensity of intracellular synthesizing processes (at 2 a.m.). A decrease in the activity of the structures under study is noted during the daytime. 2. Under immobilization stress, the ultrastructural organization of the above neurons indicates a pronounced disturbance of reactive nature with the signs of decreased functional ability of the structures and the phenomena of edema and destruction during the period of observation. 3. Melatonin injections against the background of immobilization stress led to a relative improvement in the ultrastructural state of the animals’ hypothalamic nuclei neurons, which is evidenced by the appearance of neurosecretory granules. However, the ultrastructure of other organelles of the studied neurons indicated a depleted state caused by prolonged immobilization.

Morphometric analysis of supraoptic neurons of the rat hypothalamic nuclei under conditions of prolonged illumination

The article reviews the results of studies of the morphofunctional state of neurons of the supraoptic nuclei of the rat hypothalamus under conditions of different duration of light regime. Under standard light regime in rats, a diurnal rhythm of morphofunctional activity of supraoptic nucleus neurons with maximum activity during daytime (before 2 p.m.) is recorded.&#x0D; In animals subjected to prolonged light exposure, more pronounced changes in the morphofunctional state of the supraoptic neurons of the hypothalamus at 2 a.m. than at 2 p.m. were established. Thus, the neuronal nucleus area was 94.08 ± 9.55 μm2 and was significantly greater than that in intact animals. The nucleo-cytoplasmic ratio of supraoptic hypothalamic neuron at 2 a.m. was lower than that in intact animals due to a decrease in specific nucleus volume. In comparison with the day period (2 p.m.), before 2 a.m. there was revealed a decrease of the neuron body area of supraoptic nuclei of hypothalamus due to possible decrease of the area of nucleus and nucleolus of cells. This was the reason for the increase in the nucleo-cytoplasmic ratio in the neurons under observation at night, which was 2.51 ± 0.023 units. Constant light regime did not cause inversion of the rhythm of morphofunctional activity of the neurons under study, the maximum values, as in intact animals, occurred in the daytime observation period.

Plasticity in Intrinsic Excitability of Hypothalamic Magnocellular Neurosecretory Neurons in Late-Pregnant and Lactating Rats.

Oxytocin and vasopressin secretion from the posterior pituitary gland are required for normal pregnancy and lactation. Oxytocin secretion is relatively low and constant under basal conditions but becomes pulsatile during birth and lactation to stimulate episodic contraction of the uterus for delivery of the fetus and milk ejection during suckling. Vasopressin secretion is maintained in pregnancy and lactation despite reduced osmolality (the principal stimulus for vasopressin secretion) to increase water retention to cope with the cardiovascular demands of pregnancy and lactation. Oxytocin and vasopressin secretion are determined by the action potential (spike) firing of magnocellular neurosecretory neurons of the hypothalamic supraoptic and paraventricular nuclei. In addition to synaptic input activity, spike firing depends on intrinsic excitability conferred by the suite of channels expressed by the neurons. Therefore, we analysed oxytocin and vasopressin neuron activity in anaesthetised non-pregnant, late-pregnant, and lactating rats to test the hypothesis that intrinsic excitability of oxytocin and vasopressin neurons is increased in late pregnancy and lactation to promote oxytocin and vasopressin secretion required for successful pregnancy and lactation. Hazard analysis of spike firing revealed a higher incidence of post-spike hyperexcitability immediately following each spike in oxytocin neurons, but not in vasopressin neurons, in late pregnancy and lactation, which is expected to facilitate high frequency firing during bursts. Despite lower osmolality in late-pregnant and lactating rats, vasopressin neuron activity was not different between non-pregnant, late-pregnant, and lactating rats, and blockade of osmosensitive ΔN-TRPV1 channels inhibited vasopressin neurons to a similar extent in non-pregnant, late-pregnant, and lactating rats. Furthermore, supraoptic nucleus ΔN-TRPV1 mRNA expression was not different between non-pregnant and late-pregnant rats, suggesting that sustained activity of ΔN-TRPV1 channels might maintain vasopressin neuron activity to increase water retention during pregnancy and lactation.

Aquaporin 4 differentially modulates osmotic effects on vasopressin neurons in rat supraoptic nucleus.

Glial fibrillary acidic protein (GFAP) molecularly associates with aquaporin 4 (AQP4) in astrocytic plasticity. Here, we further examined how AQP4 modulates osmotic effects on vasopressin (VP) neurons in rat supraoptic nucleus (SON) through interactions with GFAP in astrocytes. Brain slices from adult male rats were kept under osmotic stimulation. Western blot, co-immunoprecipitation, immunohistochemistry and patch-clamp recordings were used for analysis of expressions and interactions between GFAP and AQP4, astrocyte-specific proteins in the SON, as well as their influence on VP neuronal activity. Data were analysed using SPSS software. Hyposmotic challenge (HOC) of acute SON slices caused an early (within 5 minutes) and transient increase in the colocalization of AQP4 with GFAP filaments. This effect was prominent at astrocytic processes surrounding VP neuron somata and was accompanied by inhibition of VP neuronal activity. Similar HOC effect was seen in the SON isolated from rats subjected to in vivo HOC, wherein a transiently increased molecular association between GFAP and AQP4 was detected using co-immunoprecipitation. The late stage rebound excitation (10minutes) of VP neurons in brain slices subjected to HOC and the associated astrocytic GFAP's 'return to normal' were both hampered by 2-(nicotinamide)-1,3,4-thiadiazole, a specific AQP4 channel blocker that itself did not influence VP neuronal activity. Moreover, this agent prevented hyperosmotic stress-evoked excitation of VP neurons and associated reduction in GFAP filaments. These findings indicate that osmotically driven increase in VP neuronal activity requires the activation of AQP4, which determines a retraction of GFAP filaments.