Supraoptic Nucleus Neurons Research Articles

Electrophysiological Effects of Kainic Acid on Vasopressin-Enhanced Green Fluorescent Protein and Oxytocin-Monomeric Red Fluorescent Protein 1 Neurones Isolated from the Supraoptic Nucleus in Transgenic Rats

The supraoptic nucleus (SON) contains two types of magnocellular neurosecretory cells: arginine vasopressin (AVP)-producing and oxytocin (OXT)-producing cells. We recently generated and characterised two transgenic rat lines: one expressing an AVP-enhanced green fluorescent protein (eGFP) and the other expressing an OXT-monomeric red fluorescent protein 1 (mRFP1). These transgenic rats enable the visualisation of AVP or OXT neurones in the SON. In the present study, we compared the electrophysiological responses of AVP-eGFP and OXT-mRFP1 neurones to glutamic acid in SON primary cultures. Glutamate mediates fast synaptic transmission through three classes of ionotrophic receptors: the NMDA, AMPA and kainate receptors. We investigated the contributions of the three classes of ionotrophic receptors in glutamate-induced currents. Three different antagonists were used, each predominantly selective for one of the classes of ionotrophic receptor. Next, we focused on the kainate receptors (KARs). We examined the electrophysiological effects of kainic acid (KA) on AVP-eGFP and OXT-mRFP1 neurones. In current clamp mode, KA induced depolarisation and increased firing rates. These KA-induced responses were inhibited by the non-NMDA ionotrophic receptor antagonist 6-cyano-7-nitroquinoxaline-2,3(1H4H)-dione in both AVP-eGFP and OXT-mRFP1 neurones. In voltage clamp mode, the application of KA evoked inward currents in a dose-dependent manner. The KA-induced currents were significantly larger in OXT-mRFP1 neurones than in AVP-eGFP neurones. This significant difference in KA-induced currents was abolished by the GluK1-containing KAR antagonist UBP302. At high concentrations (250-500 μm), the specific GluK1-containing KAR agonist (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) induced significantly larger currents in OXT-mRFP1 neurones than in AVP-eGFP neurones. Furthermore, the difference between the AVP-eGFP and OXT-mRFP1 neurones in the ATPA currents was approximately equal to the difference in the KA currents. These findings suggest that the GluK1-containing KARs may be more highly expressed in OXT neurones than in AVP neurones. These results may provide new insight into the physiology and synaptic plasticity of SON neurones.

The inhibitory effect of anandamide on oxytocin and vasopressin secretion from neurohypophysis is mediated by nitric oxide

The neurohypophyseal hormones oxytocin (OT) and vasopressin (VP) are involved in behavioral, autonomic and neuroendocrine functions. Both peptides are synthesized in magnocellular neurons of paraventricular and supraoptic nuclei at hypothalamic level whose axons terminate in the neurohypophysis (NH), from where OT and VP are released into the systemic circulation. NH contains abundant nitric oxide (NO) synthase suggesting that NO plays a role in the release of these neuropeptides. The endocannabinoid system is present in magnocellular neurons of the hypothalamic neurohypophyseal system, and we have previously demonstrated that endocannabinoids modulate OT secretion at hypothalamic level.In the present work, we investigated the in vitro effect of the endocannabinoid anandamide (AEA) on OT and VP release from NH of untreated adult male rats and the involvement of NO in this action.Our results showed that AEA decreased OT and VP secretion from NH. AEA action was mediated by NO, since the inhibition of NO synthesis completely blocked this inhibitory effect. We found that cannabinoid receptor type 2 (CB2) and transient receptor potential cation channel subfamily V member 1 (TRPV1) are involved in the inhibitory effect of AEA because AM630 and capsazepine, CB2 and TRPV1 antagonists respectively, but not AM251, a CB1 antagonist, blocked AEA effect at neurohypophyseal level.These findings revealed an interaction between endocannabinoid, nitric oxide and oxytocin/vasopressin systems that could be involved in the modulation of homeostatic, behavioral and reproductive processes.

Variation in Progesterone Receptors and GnRH Expression in the Hypothalamus of the Pregnant South American Plains Vizcacha, Lagostomus maximus (Mammalia, Rodentia)1

In mammals, elevated levels of progesterone (P4) throughout gestation maintain a negative feedback over the hypothalamic-hypophyseal-gonadal (H-H-G) axis, avoiding preovulatory follicular growth and preventing ovulation. Recent studies showed that in the South American plains vizcacha (Lagostomus maximus) folliculogenesis progresses to preovulatory stages during gestation, and an ovulatory process seems to occur at midgestation. The aim of this work was to analyze hypothalamic gonadotropin-releasing hormone (GnRH) and P4 receptors (PR) expression and luteinizing hormone (LH) secretion and correlate these with the functional state of the ovary in nonovulating and ovulating females and gestating females with special emphasis in the supposedly ovulating females at midgestation. We investigated P4 and LH serum levels as well as the distribution, localization, and expression of PR and GnRH in the hypothalamus of L. maximus at different time points during gestation and in nongestating, ovulating and nonovulating, females. A significant increment in GnRH, P4, and LH was detected in midpregnant vizcachas with respect to early-pregnant and to ovulating females. PR was also significantly increased in midpregnant animals. PR was detected in neurons of the preoptic and hypothalamic areas. Coexistence of both PR and GnRH in neurons of medial preoptic area and supraoptic nucleus was detected. Midpregnant animals showed increased number of PR immunoreactive cells at median eminence, localized adjacently to GnRH immunoreactive fibers. High expression of hypothalamic GnRH and PR, despite an increased level of P4, was correlated with the presence of antral, preovulatory follicles, and luteinized unruptured follicles at midgestation that suggest a possible role of the H-H-G axis in the modulation of ovulation during gestation in L. maximus.

Oxytocin in Brattleboro Rats: Increased Synthesis is Contrasted by Blunted Intrahypothalamic Release From Supraoptic Nucleus Neurones

Adult male Brattleboro rats were used to investigate the impact of the congenital absence of vasopressin on the release pattern of oxytocin (OXT) within the hypothalamic supraoptic nucleus (SON) in response to a 10-min forced swimming session and osmotic stimulation. Both immunohistochemical and in situ hybridisation data suggest that vasopressin-deficient animals have more oxytocin-synthesising neurones in the SON than homozygous wild-type controls. Unexpectedly, both forced swimming and peripheral osmotic stimulation resulted in a blunted release profile of oxytocin within the SON of vasopressin-deficient rats compared to controls. A similar intranuclear OXT response to direct osmotic stimulation of the SON by retrodialysis with hypertonic Ringer's solution in both genotypes confirmed the capability of SON neurones to locally release oxytocin in vasopressin-deficient rats, indicating an altered processing of information originating from multisynaptic inputs rather than a deficit in release capacity. Taken together with data obtained in previous studies, the present findings provide evidence suggesting that autocrine and paracrine signalling of magnocellular neurones differs within the paraventricular nucleus and the SON. Thus, significant alterations in intra-SON oxytocin mRNA levels cannot easily be extrapolated to intranuclear release profiles and the local signal intensity of this neuropeptide after physiological stimulation.

Expressions of epithelial Na+ channel subunits in the supraoptic nucleus of the hypothalamus in the male rat in response to dietary NaCl intake

All three epithelial Na+ channel (ENaC) subunits (α, β, and γ) were demonstrated in vasopressin (VP) synthesizing magnocellular neurons (MNCs) in the hypothalamic supraoptic (SON) and paraventricular nuclei. Our recent findings showed that ENaC mediates a Na+ leak current that changes the frequency of action potentials in MNCs. In the present study, we assessed the effect of dietary NaCl intake on ENaC subunits in the SON. Male rats were fed either a 8% NaCl diet, a NaCl deficient, or a control diet for two weeks. Then, relative changes in the subunit mRNAs in the SON and kidney were measured by real‐time RT‐PCR. The 8% NaCl diet caused significant increase in β‐ and γ‐ENaC mRNAs in the SON, while expression of α‐ENaC did not change. In contrast, the 8% NaCl diet did not cause any marked changes in expressions of the subunits in the kidney, while a deficiency in dietary NaCl caused a significant rise in α‐ENaC mRNA. Change in the subcellular distribution of α‐ENaC immunoreactivety in MNCs was also observed using confocal microscopy. The labeling in the MNCs from the 8% NaCl diet group was distinctively concentrated towards the plasma membrane in all 5 rats tested, while those from the NaCl deficient and control groups were mainly dispersed in the cytoplasm with no distinct labeling at the plasma membrane. These findings imply that dietary NaCl affects ENaC activity in VP MNCs, which, in turn, affects VP secretion.NIH grant: R21 HL093728

Nav1.7: Stress-Induced Changes in Immunoreactivity within Magnocellular Neurosecretory Neurons of the Supraoptic Nucleus

BackgroundNaV1.7 is preferentially expressed, at relatively high levels, in peripheral neurons, and is often referred to as a “peripheral” sodium channel, and NaV1.7-specific blockers are under study as potential pain therapeutics which might be expected to have minimal CNS side effects. However, occasional reports of patients with NaV1.7 gain-of-function mutations and apparent hypothalamic dysfunction have appeared. The two sodium channels previously studied within the rat hypothalamic supraoptic nucleus, NaV1.2 and NaV1.6, display up-regulated expression in response to osmotic stress.ResultsHere we show that NaV1.7 is present within vasopressin-producing neurons and oxytocin-producing neurons within the rat hypothalamus, and demonstrate that the level of Nav1.7 immunoreactivity is increased in these cells in response to osmotic stress.ConclusionsNaV1.7 is present within neurosecretory neurons of rat supraoptic nucleus, where the level of immunoreactivity is dynamic, increasing in response to osmotic stress. Whether NaV1.7 levels are up-regulated within the human hypothalamus in response to environmental factors or stress, and whether NaV1.7 plays a functional role in human hypothalamus, is not yet known. Until these questions are resolved, the present findings suggest the need for careful assessment of hypothalamic function in patients with NaV1.7 mutations, especially when subjected to stress, and for monitoring of hypothalamic function as NaV1.7 blocking agents are studied.

Selective up-regulation of JunD transcript and protein expression in vasopressinergic supraoptic nucleus neurones in water-deprived rats.

The magnocellular neurones (MCN) of the supraoptic nucleus (SON) undergo reversible changes during dehydration. We hypothesise that alterations in steady-state transcript levels might be partially responsible for this plasticity. In turn, regulation of transcript abundance might be mediated by transcription factors. We have previously used microarrays to identify changes in the expression of mRNAs encoding transcription factors in response to water deprivation. We observed down-regulation of 11 and up-regulation of 31 transcription factor transcripts, including members of the activator protein-1 gene family, namely c-fos, c-jun, fosl1 and junD. Because JunD expression and regulation within the SON has not been previously described, we have used in situ hybridisation and the quantitative reverse transcriptase-polymerase chain reaction to confirm the array results, demonstrating a significant increase in JunD mRNA levels following 24 and 72h of water deprivation. Western blot and immunohistochemistry revealed a significant increase in JunD protein expression following dehydration. Double-staining fluorescence immunohistochemistry with a neurone-specific marker (NeuN) demonstrated that JunD staining is predominantly neuronal. Additionally, JunD immunoreactivity is observed primarily in vasopressin-containing neurones with markedly less staining seen in oxytocin-containing MCNs. Furthermore, JunD is highly co-expressed with c-Fos in MCNs of the SON following dehydration. These results suggest that JunD plays a role in the regulation of gene expression within MCNs of the SON in association with other Fos and Jun family members.

Taurine Release by Astrocytes Modulates Osmosensitive Glycine Receptor Tone and Excitability in the Adult Supraoptic Nucleus

Cells can release the free amino acid taurine through volume-regulated anion channels (VRACs), and it has been hypothesized that taurine released from glial cells is capable of inhibiting action potential (AP) firing by activating neuronal glycine receptors (GlyRs) (Hussy et al., 1997). Although an inhibitory GlyR tone is widely observed in the brain, it remains unknown whether this specifically reflects gliotransmission because most neurons also express VRACs and other endogenous molecules can activate GlyRs. We found that VRACs are absent in neurons of the rat supraoptic nucleus (SON), suggesting that glial cells are the exclusive source of taurine in this nucleus. Application of strychnine to rat hypothalamic explants caused a depolarization of SON neurons associated with a decrease of chloride conductance and could excite these cells in the absence of fast synaptic transmission. This inhibitory GlyR tone was eliminated by pharmacological blockade of VRACs, by cellular taurine depletion, by metabolic inactivation of glia with fluorocitrate, and after retraction of astrocytic processes that intercalate neuronal somata and dendrites. Finally, GlyR tone varied inversely with extracellular fluid tonicity to mediate the osmotic control of AP firing by SON neurons. These findings establish taurine as a physiological gliotransmitter and show that gliotransmission is a spatially constrained process that can be modulated by the morphological rearrangement of astrocytes.

Role of Bcl-2 in the regulation of CREB activity and vasopressin expression in hypothalamic neurons

For the last years, data have appears that indicate that functions of the antiapoptotic protein Bcl-2 are not restricted by its role in prevention of apoptosis. Earlier we showed that Bcl-2 participated in regulation of synthesis and secretion of vasopressin by neurons of magnocellular hypothalamic nuclei. Based on our previous works about effect of administration of the Bcl-2 blocker HA-14-1 on activities of the ERK1/2 cascade and of the transcriptional factor CREB in hypothalamic vasopressinergic neurons, we formulated hypothesis about the mediated action of Bcl-2 on vasopressin biosynthesis by transcription factor CREB and/or the ERK1/2 signal cascade. In the presented work we studied effects of Bcl-2 inactivation on vasopressin expression and activity of the ERK1/2 cascade and CREB in experiments on briefly incubated hypothalamus slides containing supraoptic nuclei. We have shown that inactivation of Bcl-2 in vitro by the blocker HA14-1 led to a decrease in the CREB activity, reduced significantly the vasopressin mRNA level, and increased the vasopressin content in the supraoptic nucleus neurons, with the ERK1/2 cascade activity being increased. Effect of Bcl-2 on the vasopressin synthesis activity did not depend on activity of the ERK1/2 signal cascade. Thus, Bcl-2 has been shown to play an activating role in control of vasopressin neurons of the hypothalamus supraoptic nucleus. The presented data confirm that effect of Bcl-2 on vasopressin synthesis by the supraoptic nucleus neurons is mediated by its ability to control activity of the transcriptional factor CREB.

Dp71 gene disruption alters the composition of the dystrophin-associated protein complex and neuronal nitric oxide synthase expression in the hypothalamic supraoptic and paraventricular nuclei

DP71 is the major cerebral dystrophin isoform and exerts its multiple functions via the dystrophin-associated protein complex (DAPC), also comprised of β-dystroglycan (β-DG) and α1-syntrophin (α1-Syn). Since DP71 disruption leads to impairment in the central control of the osmoregulatory axis, we investigated: 1) the DAPC composition in the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) of Dp71-null mice; and 2) the expression and activity of neuronal nitric oxide synthase (nNOS), because it is a potential partner of the DAPC and a functional index of osmoregulatory axis activity. In wild-type mice, dystrophins and their autosomal homologs the utrophins, β-DG, and α1-Syn were localized in astrocyte end feet. In Dp71-null mice, the levels of β-DG and α1-Syn were lower and utrophin expression did not change. The location of the DAPC in astrocytic end feet suggests that it could be involved in hypothalamic osmosensitivity, which adapts the osmotic response. The altered composition of the DAPC in Dp71-null mice could thus explain why these mice manifest an hypo-osmolar status. In the SON and PVN neurons of Dp71-null mice, nNOS expression and activity were increased. Although we previously established that DP140 is expressed de novo in these neurons, the DAPC remained incomplete due to the low levels of β-DG and α1-Syn produced in these cells. Our data reveal the importance of DP71 for the constitution of a functional DAPC in the hypothalamus. Such DAPC disorganization may lead to modification of the microenvironment of the SON and PVN neurons and thus may result in a perturbed osmoregulation.

Nicotinic acetylcholine receptors (nAchRs) in supraoptic nucleus (SON) neurons: Potential role in regulation of food intake

Nicotine induces anorexia. nAchRs are expressed in SON of the hypothalamus, where oxytocin (OT) and vasopressin (VP) are synthesized. Nicotine stimulates release of VP as well as OT, an anorexic agent. Deficits in OT are associated with human obesity. Thus, nicotine may reduce food intake, in part, via modulation of OT neurons in SON. In our current studies, live‐cell calcium imaging was used to determine if acetylcholine (Ach) or nicotine altered intracellular calcium ([Ca++]i)signaling in SON neurons. Hypothalamic explants that include SON were loaded with the calcium sensitive dye, Fura‐2AM. Changes in [Ca++]i were monitored by changes in the 340/380 nm emission. A short application of Ach (5 mM) to the bath caused an increase in [Ca++]i in a subset of SON neurons, in the presence or absence of PNU‐120596 (10 μM), a positive allosteric modulator of α7 nAchRs. Ca++ response to Ach was reduced but still existed in the presence of atropine (10 μM) to block muscarinic AchRs. Nicotine (20–100 μM) also induced [Ca++]i increase in a portion of SON neurons. These findings are consistent with activation of nAchRs in SON neurons. Future studies will further characterize the specific subtypes of nAchRs in SON and identify the peptidergic phenotype of responsive neurons. Supported by AHA 0735329N.

Co‐localization of Dynorphin with inducible cAMP element repressor (ICER) in the supraoptic nucleus

The endogenous kappa (κ) opioid agonist, Dynorphin, and its receptors have been shown to be present at the supraoptic nucleus (SON) as well as co‐localized within vasopressin secretory vesicles. Central administration of U50, κ agonist, increases ICER expression at the level of the SON, which may play a role in modulating vasopressin release. This study examined central κ‐mediated changes in ICER expression in SON neurons expressing Dynorphin. Rats were implanted with an ICV cannula for microinjection of drug/vehicle, and each was acclimated to a metabolic cage for collection of urine during the experimental protocol. Tissue sections through SON were processed for ICER using commercially available polyclonal anti‐ICER and Dynorphin antibodies. U50 (1μg/5μl) significantly increased water free diuresis and ICER staining in the SON as compared to control (all P < 0.05). ICER‐ir staining significantly increased in Dynorphin positive cells of SON. These results raise the possibility that ICER activation could be an important substrate through which κ opioids mediate magnocellular neuron inhibition to suppress vasopressin secretion. Understanding the role of the ICER transcription factor in regulating SON neuronal activity could provide new and potentially important insight into mechanisms of water and electrolyte homeostasis in health and disease. Supported by 1SC2 HL104639 (HBG).

Regulation of TRPV2 in Magnocellular Neurons of the Supraoptic Nucleus in Rat

Transient receptor potential vanilloid channel 2 (TRPV2) is activated by hypoosmolality in expression systems. Our objective was to study the role TRPV2 in the magnocellular cells (MNC) of the supraoptic nucleus (SON) in a bile duct ligated rat (BDL) model of hepatic cirrhosis associated with inappropriate vasopressin (AVP) release. We hypothesized that changes in TRPV2 channel expression in AVP, but not oxytocin (OXY) neurons, may contribute to inappropriate AVP release in BDL rats. We used laser capture microdissection (LCM) to capture 7–10 MNC AVP neurons for real‐time RT‐PCR to measure TRPV2 mRNA. The 2−ΔΔCt for TRPV2 mRNA in the LCM AVP neurons of SON in BDL rats averaged 2.5 ± 0.5 vs. 1.0 ± 0.3 in shams. Western blot analysis indicated that TRPV2 protein also was increased in SON by BDL. Following BDL, TRPV2 immunostaining and TRPV2/AVP colocalization was significantly increased in the SON (SHAM TRPV2, 41 ± 2; BDL TRPV2, 52 ± 4; SHAM AVP/TRPV2, 23 ± 1; BDL AVP/TRPV2, 35 ± 2). However, TRPV2 and OXY colocalization was not affected (SHAM OXY/TRPV2, 12 ± 1; BDL OXY/TRPV2, 8.75 ± 1.3). Increased TRPV2 mRNA and protein expression in AVP SON neurons of BDL rats could contribute to changes in osmosensitivity related to inappropriate AVP release. R01 HL062579.

Circadian Modulation of Osmoregulated Firing in Rat Supraoptic Nucleus Neurones

The antidiuretic hormone vasopressin (VP) promotes water reabsorption from the kidney and levels of circulating VP are normally related linearly to plasma osmolality, aiming to maintain the latter close to a predetermined set point. Interestingly, VP levels rise also in the absence of an increase in osmolality during late sleep in various mammals, including rats and humans. This circadian rhythm is functionally important because the absence of a late night VP surge results in polyuria and disrupts sleep in humans. Previous work has indicated that the VP surge may be caused by facilitation of the central processes mediating the osmotic control of VP release, and the mechanism by which this occurs was recently studied in angled slices of rat hypothalamus that preserve intact network interactions between the suprachiasmatic nucleus (SCN; the biological clock), the organum vasculosum lamina terminalis (OVLT; the central osmosensory nucleus) and the supraoptic nucleus (SON; which contains VP-releasing neurohypophysial neurones). These studies confirmed that the electrical activity of SCN clock neurones is higher during the middle sleep period (MSP) than during the late sleep period (LSP). Moreover, they revealed that the excitation of SON neurones caused by hyperosmotic stimulation of the OVLT was greater during the LSP than during the MSP. Activation of clock neurones by repetitive electrical stimulation, or by injection of glutamate into the SCN, caused a presynaptic inhibition of glutamatergic synapses made between the axon terminals of OVLT neurones and SON neurones. Consistent with this effect, activation of clock neurones with glutamate also reduced the excitation of SON neurones caused by hyperosmotic stimulation of the OVLT. These results suggest that clock neurones in the SCN can mediate an increase in VP release through a disinhibition of excitatory synapses between the OVLT and the SON during the LSP.

Endothelin‐Mediated Calcium Responses in Supraoptic Nucleus Astrocytes Influence Magnocellular Neurosecretory Firing Activity

In addition to their peripheral vasoactive effects, accumulating evidence supports an important role for endothelins (ETs) in the regulation of the hypothalamic magnocellular neurosecretory system, which produces and releases the neurohormones vasopressin (VP) and oxytocin (OT). Still, the precise cellular substrates, loci and mechanisms underlying the actions of ETs on the magnocellular system are poorly understood. In the present study, we combined patch-clamp electrophysiology, confocal Ca(2+) imaging and immunohistochemistry to study the actions of ETs on supraoptic nucleus (SON) magnocellular neurosecretory neurones and astrocytes. Our studies show that ET-1 evoked rises in [Ca(2+) ](i) levels in SON astrocytes (but not neurones), an effect largely mediated by the activation of ET(B) receptors and mobilisation of thapsigargin-sensitive Ca(2+) stores. The presence of ET(B) receptors in SON astrocytes was also verified immunohistochemically. ET(B) receptor activation either increased (75%) or decreased (25%) SON firing activity, both in VP and putative OT neurones, and these effects were prevented when slices were preincubated in glutamate receptor blockers or nitric oxide synthase blockers, respectively. Moreover, ET(B) -mediated effects in SON neurones were also prevented by a gliotoxin compound, and when changes in [Ca(2+) ](i) were prevented with bath-applied BAPTA-AM or thapsigargin. Conversely, intracellular Ca(2+) chelation in the recorded SON neurones failed to block ET(B) -mediated effects. In summary, our results indicate that ET(B) receptor activation in SON astrocytes induces the mobilisation of [Ca(2+) ](i) , likely resulting in the activation of glutamate and nitric oxide signalling pathways, evoking in turn excitatory and inhibitory SON neuronal responses, respectively. Taken together, our study supports an important role for astrocytes in mediating the actions of ETs on the magnocellular neurosecretory system.

Immunohistochemical investigation of bcl-2 and p53 levels in rat hypothalamus after sleep deprivation

On Wistar rats in view of electrophysiological parameters after sleep deprivation (SD; awake by gentle handling method) and the subsequent postdeprivative sleep (PDS) immunohistochemical investigation of Bcl-2 and p53 peptides optical density levels in neurons of paraventricular (PVN), supraoptic (SON) and median (MnPN) hypothalamus nuclei was carried out. The Bcl-2 was increased in all nuclei both after SD and PDS. The level of p53 was increased in PVN and SON after SD and PDS, but in MnPN only on PDS. Any morphological attributes of apoptosis in the nuclei was not revealed. Obtained data testify an active role of p53 and Bcl-2 peptides in regulation of neuronal activity in hypothalamus at change of a cycle wakefulness-sleep.

Brain-Derived Neurotrophic Factor-Tyrosine Kinase B Pathway Mediates NMDA Receptor NR2B Subunit Phosphorylation in the Supraoptic Nuclei Following Progressive Dehydration

We studied the effects of water deprivation (WD) on the phosphorylation of tyrosine kinase B (TrkB) and NMDA receptor subunits in the supraoptic nucleus (SON) of the rat. Laser capture microdissection and quantitative reverse transcriptase polymerase chain reaction was used to demonstrate brain-derived neurotrophic factor (BDNF) and TrkB gene expression in vasopressin SON neurones. Immunohistochemistry confirmed BDNF staining in vasopressin neurones, whereas staining for phosphorylated TrkB was increased following WD. Western blot analysis of brain punches containing the SON revealed that tyrosine phosphorylation of TrkB (pTrkBY(515)), serine phosphorylation of NR1 (pNR1S(866) or pNR1) and tyrosine phosphorylation of NR2B subunits (pNR2BY(1472) or pNR2B) were significantly increased in WD animals compared to controls. Access to water for 2 h reduced pTrkBY(515) content to control levels without affecting pNR1 or pNR2B. Four hours of rehydration was needed to reduce pNR1 and pNR2B to control levels. To test whether increased phosphorylation of TrkB in the present study is mediated by BDNF, a group of animals were instrumented with right SON cannula coupled to mini-osmotic pumps filled with vehicle or TrkB-Fc fusion protein, which prevents BDNF binding to TrkB. In the left SON contralateral to the cannula, TrkB phosphorylation was significantly enhanced following WD. Separate analysis of the right SON, which received TrkB-Fc, showed that the TrkB receptor phosphorylation following WD was significantly attenuated. Although increased pNR1S(866) following WD was not affected by local infusion of TrkB-Fc, pNR2BY(1472) was significantly reduced. Co-immunoprecipitation revealed an increased physical interaction between Fyn kinase and NR2B and TrkB in the SON following WD. Thus, activation of TrkB in the SON following WD may affect cellular excitability through the phosphorylation of NR2B subunits.

Kisspeptin Activation of Supraoptic Nucleus Neurons in Vivo

Oxytocin and vasopressin are synthesized by magnocellular neurosecretory cells in the hypothalamic supraoptic and paraventricular nuclei and are released from the posterior pituitary gland into the circulation. Intravenous administration of the ligand for the G protein-coupled receptor 54 receptor, kisspeptin-10, increases plasma oxytocin levels and intracerebroventricular kisspeptin-10 increases vasopressin levels, indicating that kisspeptin might play a role in various physiological functions via stimulation of oxytocin and vasopressin secretion. Because posterior pituitary hormone secretion is dependent on action potential (spike) discharge, we used in vivo extracellular single unit recording to determine the effects of kisspeptin-10 on supraoptic nucleus neurons in urethane-anaesthetized female rats. Intravenous kisspeptin-10 (100 μg) increased the firing rate of oxytocin neurons from 3.7 ± 0.8 to 4.7 ± 0.8 spikes/sec (P = 0.0004), but only a quarter of vasopressin neurons responded to iv kisspeptin-10, showing a short (<3 sec) high-frequency (>15 spikes/sec) burst of firing. By contrast, intracerebroventricular kisspeptin-10 (2 and 40 μg) did not alter oxytocin or vasopressin neuron firing rate. To investigate the pathway involved in the peripheral action of kisspeptin-10, we used i.p. capsaicin to desensitize vagal afferents, which prevented the i.v. kisspeptin-10-induced increase of oxytocin neuron firing rate. This is the first report to show that peripheral, but not central, kisspeptin-10 increases the activity of oxytocin neurons and a proportion of vasopressin neurons and that endogenous kisspeptin regulation of supraoptic nucleus neurons is likely via vagal afferent input, with kisspeptin acting as a hormone rather than as a neuropeptide in this system.

State‐dependent changes in astrocyte regulation of extrasynaptic NMDA receptor signalling in neurosecretory neurons

Despite the long-established presence of glutamate NMDA receptors at extrasynaptic sites (eNMDARs), their functional roles remain poorly understood. Factors influencing the concentration and time course of glutamate in the extrasynaptic space, such as the topography of the neuronal–glial microenvironment, as well as glial glutamate transporters, are expected to affect eNMDAR-mediated signalling strength. In this study, we used in vitro and in vivo electrophysiological recordings to assess the properties, functional relevance and modulation of a persistent excitatory current mediated by activation of eNMDARs in hypothalamic supraoptic nucleus (SON) neurons. We found that ambient glutamate of a non-synaptic origin activates eNMDARs to mediate a persistent excitatory current (termed tonic I(NMDA)), which tonically stimulates neuronal activity. Pharmacological blockade of GLT1 astrocyte glutamate transporters, as well as the gliotoxin α-aminodadipic acid, enhanced tonic I(NMDA) and neuronal activity, supporting an astrocyte regulation of tonic I(NMDA) strength. Dehydration, a physiological challenge known to increase SON firing activity and to induce neuroglial remodelling, including reduced neuronal ensheathment by astrocyte processes, resulted in blunted GLT1 efficacy, enhanced tonic I(NMDA) strength, and increased neuronal activity. Taken together, our studies support the view that glial modulation of tonic I(NMDA) activation contributes to regulation of SON neuronal activity, contributing in turn to neuronal homeostatic responses during a physiological challenge.

Apamin increases post-spike excitability of supraoptic nucleus neurons in anaesthetized morphine-naïve rats and morphine-dependent rats: consequences for morphine withdrawal excitation

Supraoptic nucleus (SON) oxytocin neurons develop morphine dependence when chronically exposed to this opiate and undergo excitation when morphine is subsequently withdrawn. Morphine withdrawal excitation is evident as an increased action potential (spike) firing rate and is associated with an increased post-spike excitability that is consistent with the expression of an enhanced post-spike afterdepolarization (ADP) during withdrawal. Here, we administered apamin (which inhibits the medium afterhyperpolarization [mAHP] in vitro and unmasks an ADP) into the SON of urethane-anaesthetized rats to determine its effects on oxytocin neurons in vivo. As predicted, intra-SON apamin administration increased the propensity to fire a spike soon (<100ms) after each spike (post-spike excitability) more in oxytocin neurons recorded from morphine-treated rats than in morphine-naïve rats. However, intra-SON apamin did not alter the overall firing rate of oxytocin neurons recorded from morphine-treated rats or morphine-naïve rats, indicating that an increase in post-spike excitability alone is not sufficient to trigger withdrawal excitation of oxytocin neurons. Nevertheless, bilateral intra-SON apamin infusion increased oxytocin secretion (which depends on firing pattern as well as firing rate) by 90±46% in morphine-dependent rats (P<0.01 compared to aCSF). Hence, an increase in post-spike excitability does not appear to drive morphine withdrawal-induced increases in oxytocin neuron firing rate, but does contribute to withdrawal-induced hyper-secretion of oxytocin.