Magnocellular Neuroendocrine Cells Research Articles

Tonic NMDAR Currents of NR2A-Containing NMDARs Represent Altered Ambient Glutamate Concentration in the Supraoptic Nucleus.

NMDA receptors (NMDARs) modulate glutamatergic excitatory tone in the brain via two complementary modalities: a phasic excitatory postsynaptic current and a tonic extrasynaptic modality. Here, we demonstrated that the tonic NMDAR-current (I NMDA) mediated by NR2A-containing NMDARs is an efficient biosensor detecting the altered ambient glutamate level in the supraoptic nucleus (SON). I NMDA of magnocellular neurosecretory cells (MNCs) measured by nonselective NMDARs antagonist, AP5, at holding potential (V holding) -70 mV in low concentration of ECF Mg2+ ([Mg2+]o) was transiently but significantly increased 1-week post induction of a DOCA salt hypertensive model rat which was compatible with that induced by a NR2A-selective antagonist, PEAQX (I PEAQX) in both DOCA-H2O and DOCA-salt groups. In agreement, NR2B antagonist, ifenprodil, or NR2C/D antagonist, PPDA, did not affect the holding current (I holding) at V holding -70 mV. Increased ambient glutamate by exogenous glutamate (10 mM) or excitatory amino acid transporters (EAATs) antagonist (TBOA, 50 mM) abolished the I PEAQX difference between two groups, suggesting that attenuated EAATs activity increased ambient glutamate concentration, leading to the larger I PEAQX in DOCA-salt rats. In contrast, only ifenprodil but not PEAQX and PPDA uncovered I NMDA at V holding +40 mV under 1.2 mM [Mg2+]o condition. I ifenprodil was not different in DOCA-H2O and DOCA-salt groups. Finally, NR2A, NR2B, and NR2D protein expression were not different in the SON of the two groups. Taken together, NR2A-containing NMDARs efficiently detected the increased ambient glutamate concentration in the SON of DOCA-salt hypertensive rats due to attenuated EAATs activity.

Astroglial Regulation of Magnocellular Neuroendocrine Cell Activities in the Supraoptic Nucleus.

Studies on the interactions between astrocytes and neurons in the hypothalamo-neurohypophysial system have significantly facilitated our understanding of the regulation of neural activities. This has been exemplified in the interactions between astrocytes and magnocellular neuroendocrine cells (MNCs) in the supraoptic nucleus (SON), specifically during osmotic stimulation and lactation. In response to changes in neurochemical environment in the SON, astrocytic morphology and functions change significantly, which further modulates MNC activity and the secretion of vasopressin and oxytocin. In osmotic regulation, short-term dehydration or water overload causes transient retraction or expansion of astrocytic processes, which increases or decreases the activity of SON neurons, respectively. Prolonged osmotic stimulation causes adaptive change in astrocytic plasticity in the SON, which allows osmosensory neurons to reserve osmosensitivity at new levels. During lactation, changes in neurochemical environment cause retraction of astrocytic processes around oxytocin neurons, which increases MNC's ability to secrete oxytocin. During suckling by a baby/pup, astrocytic processes in the mother/dams exhibit alternative retraction and expansion around oxytocin neurons, which mirrors intermittently synchronized activation of oxytocin neurons and the post-excitation inhibition, respectively. The morphological and functional plasticities of astrocytes depend on a series of cellular events involving glial fibrillary acidic protein, aquaporin 4, volume regulated anion channels, transporters and other astrocytic functional molecules. This review further explores mechanisms underlying astroglial regulation of the neuroendocrine neuronal activities in acute processes based on the knowledge from studies on the SON.

Reconstruction of the Hypothalamo-Neurohypophysial System and Functional Dissection of Magnocellular Oxytocin Neurons in the Brain

The hypothalamo-neurohypophysial system (HNS), comprising hypothalamic magnocellular neuroendocrine cells (MNCs) and the neurohypophysis, plays a pivotal role in regulating reproduction and fluid homeostasis by releasing oxytocin and vasopressin into the bloodstream. However, its structure and contribution to the central actions of oxytocin and vasopressin remain incompletely understood. Using viral tracing and whole-brain imaging, we reconstruct the three-dimensional architecture of the HNS and observe collaterals of MNCs within the brain. By dual viral tracing, we further uncover that subsets of MNCs collaterally project to multiple extrahypothalamic regions. Selective activation of magnocellular oxytocin neurons promote peripheral oxytocin release and facilitate central oxytocin-mediated social interactions, whereas inhibition of these neurons elicit opposing effects. Our work reveals the previously unrecognized complexity of the HNS and provides structural and functional evidence for MNCs in coordinating both peripheral and central oxytocin-mediated actions, which will shed light on the mechanistic understanding of oxytocin-related psychiatric diseases.

Advances in the neurophysiology of magnocellular neuroendocrine cells.

Hypothalamic magnocellular neuroendocrine cells have unique electrical properties and a remarkable capacity for morphological and synaptic plasticity. Their large somatic size, their relatively uniform and dense clustering in the supraoptic and paraventricular nuclei, and their large axon terminals in the neurohypophysis make them an attractive target for direct electrophysiological interrogation. Here, we provide a brief review of significant recent findings in the neuroplasticity and neurophysiological properties of these neurones that were presented at the symposium "Electrophysiology of Magnocellular Neurons" during the 13th World Congress on Neurohypophysial Hormones in Ein Gedi, Israel in April 2019. Magnocellular vasopressin (VP) neurones respond directly to hypertonic stimulation with membrane depolarisation, which is triggered by cell shrinkage-induced opening of an N-terminal-truncated variant of transient receptor potential vanilloid type-1 (TRPV1) channels. New findings indicate that this mechanotransduction depends on actin and microtubule cytoskeletal networks, and that direct coupling of the TRPV1 channels to microtubules is responsible for mechanical gating of the channels. Vasopressin neurones also respond to osmostimulation by activation of epithelial Na+ channels (ENaC). It was shown recently that changes in ENaC activity modulate magnocellular neurone basal firing by generating tonic changes in membrane potential. Both oxytocin and VP neurones also undergo robust excitatory synapse plasticity during chronic osmotic stimulation. Recent findings indicate that new glutamate synapses induced during chronic salt loading express highly labile Ca2+ -permeable GluA1 receptors requiring continuous dendritic protein synthesis for synapse maintenance. Finally, recordings from the uniquely tractable neurohypophysial terminals recently revealed an unexpected property of activity-dependent neuropeptide release. A significant fraction of the voltage-dependent neurohypophysial neurosecretion was found to be independent of Ca2+ influx through voltage-gated Ca2+ channels. Together, these findings provide a snapshot of significant new advances in the electrophysiological signalling mechanisms and neuroplasticity of the hypothalamic-neurohypophysial system, a system that continues to make important contributions to the field of neurophysiology.

Labile Calcium-Permeable AMPA Receptors Constitute New Glutamate Synapses Formed in Hypothalamic Neuroendocrine Cells during Salt Loading.

Magnocellular neuroendocrine cells (MNCs) of the hypothalamus play a critical role in the regulation of fluid and electrolyte homeostasis. They undergo a dramatic structural and functional plasticity under sustained hyperosmotic conditions, including an increase in afferent glutamatergic synaptic innervation. We tested for a postulated increase in glutamate AMPA receptor expression and signaling in magnocellular neurons of the male rat hypothalamic supraoptic nucleus (SON) induced by chronic salt loading. While without effect on GluA1-4 subunit mRNA, salt loading with 2% saline for 5-7 d resulted in a selective increase in AMPA receptor GluA1 protein expression in the SON, with no change in GluA2-4 protein expression, suggesting an increase in the ratio of GluA1 to GluA2 subunits. Salt loading induced a corresponding increase in EPSCs in both oxytocin (OT) and vasopressin (VP) neurons, with properties characteristic of calcium-permeable AMPA receptor-mediated currents. Unexpectedly, the emergent AMPA synaptic currents were silenced by blocking protein synthesis and mammalian target of rapamycin (mTOR) activity in the slices, suggesting that the new glutamate synapses induced by salt loading require continuous dendritic protein synthesis for maintenance. These findings indicate that chronic salt loading leads to the induction of highly labile glutamate synapses in OT and VP neurons that are comprised of calcium-permeable homomeric GluA1 AMPA receptors. The glutamate-induced calcium influx via calcium-permeable AMPA receptors would be expected to play a key role in the induction and/or maintenance of activity-dependent synaptic plasticity that occurs in the magnocellular neurons during chronic osmotic stimulation.

SUN-472 Labile Ca-Permeable AMPA Receptors Comprise New Synapses Following Salt Loading-Induced Plasticity in Hypothalamic Magnocellular Neurons

Magnocellular neuroendocrine cells of the hypothalamus play an important role in the regulation of fluid and electrolyte homeostasis. They undergo a dramatic structural plasticity under sustained physiological activation, including an increase in glutamatergic synaptic innervation. We tested for plasticity in the glutamate AMPA receptor expression in magnocellular neurons of the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) induced by sustained activation by chronic salt loading-induced dehydration. We found that salt loading by with 2% saline drinking water for 5-7 days resulted in a significant selective increase in GluA1 protein expression in the rat SON, while no change in the mRNA expression or membrane localization of any of the AMPA receptor subunits was observed, suggesting a post-transcriptional up regulation of GluA1 at excitatory synapses on SON neurons. We performed whole-cell recordings of excitatory postsynaptic currents (EPSCs) to determine the contribution of Ca2+ -permeable AMPA receptors (CP-AMPARs) to synaptic transmission by the EPSC rectification index and the sensitivity of EPSCs to the selective antagonist of CP-AMPA receptors, 1-naphthyl-acetyl-spermine (NAS). We found that neurons from the salt-loaded group showed a significantly greater inward rectification and an increased sensitivity to NAS compared to the control group. To further determine the role of rapid protein turnover in the AMPAR plasticity, we preincubated slices for over 60 min in the translational inhibitor cycloheximide to block local protein synthesis, which. Blocking protein synthesis reverted the Ca permeability and reduced the EPSC frequency and EPSC rectification in SON neurons from in the salt-loaded group to the levels of controls. These findings together suggest that salt loading induces highly labile glutamate synapses in magnocellular neurons that are comprised almost exclusively of CP-AMPARs, and indicate an essential role for rapid protein synthesis in the maintenance of the new synapses. This glutamate receptor plasticity should result in an increase in glutamate-induced calcium influx, which could play a key role in the activity-dependent neuronal-glial remodeling of the magnocellular neurons that occurs during chronic dehydration. Supported by NIH R01 and NSF IOS grants.

Cell signaling dependence of rapid glucocorticoid-induced endocannabinoid synthesis in hypothalamic neuroendocrine cells

Glucocorticoids induce a rapid synthesis of endocannabinoid in hypothalamic neuroendocrine cells by activation of a putative membrane receptor. Somato-dendritically released endocannabinoid acts as a retrograde messenger to suppress excitatory synaptic inputs to corticotropin-releasing hormone-, oxytocin-, and vasopressin-secreting cells. The non-genomic signaling mechanism responsible for rapid endocannabinoid synthesis by glucocorticoids has yet to be fully characterized. Here we manipulated cell signaling molecules pharmacologically using an intracellular approach to elucidate the signaling pathway activated by the membrane glucocorticoid receptor in hypothalamic neuroendocrine cells. We found that rapid glucocorticoid-induced endocannabinoid synthesis in magnocellular neuroendocrine cells requires the sequential activation of multiple kinases, phospholipase C, and intracellular calcium mobilization. While there remain gaps in our understanding, our findings reveal many of the critical players in the rapid glucocorticoid signaling that culminates in the retrograde endocannabinoid modulation of excitatory synaptic transmission.

Modulatory effect of dopamine receptor 5 on the neurosecretory Dahlgren cells of the olive flounder, Paralichthys olivaceus

A neuromodulatory role for dopamine has been reported for magnocellular neuroendocrine cells in the mammalian hypothalamus. We examined its potential role as a local intercellular messenger in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) of the euryhaline flounder Paralichthys olivaceus. In vitro application of dopamine (DA) caused an increase in electrical activity (firing frequency, recorded extracellularly) of Dahlgren cells, recruitment of previously silent cells, together with a greater proportion of cells showing phasic (irregular) activity. The dopamine precursor, levodopa (L-DOPA), also increased firing frequency, cell recruitment and enhanced bursting and tonic activity. The effect of dopamine was blocked by the D1, D5 receptor antagonist SCH23390, but not by the D2, D3, D4 receptor antagonist amisulpride. Transcriptome sequencing revealed that all DA receptors (D1, D2, D3, D4, and D5) were present in the flounder CNSS. However, quantitative RT-PCR revealed that D5 receptor mRNA expression was significantly increased in the CNSS following dopamine superfusion. These findings suggest that dopamine may modulate CNSS activity in vivo, and therefore neurosecretory output, through D5 receptors.

Modulatory effect of glutamate GluR2 receptor on the caudal neurosecretory Dahlgren cells of the olive flounder, Paralichthys olivaceus

A neuromodulatory role for glutamate has been reported for magnocellular neuroendocrine cells in mammalian hypothalamus. We examined the potential role of glutamate as a local intercellular messenger in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) in the euryhaline flounder Paralichthys olivaceus. In pharmacological experiments in vitro, glutamate (Glu) caused an increase in electrical activity of Dahlgren cells, recruitment of previously silent cells, together with a greater proportion of cells showing phasic (irregular) activity. The glutamate substrate, glutamine (Gln), led to increased firing frequency, cell recruitment and enhanced bursting activity. The glutamate effect was not blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, or the GluR1/GluR3 (AMPA) receptor antagonist IEm1795-2HBr, but was blocked by the broad-spectrum α-amino-3-hydroxy- 5- methyl-4-isoxazo-lepropionic acid (AMPA) receptor antagonist ZK200775. Our transcriptome sequencing study revealed three AMPA receptor (GluR1, GluR2 and GluR3) in the olive flounder CNSS. Quantitative RT-PCR revealed that GluR2 receptor mRNA expression was significant increased following dose-dependent superfusion with glutamate in the CNSS. GluR1 and GluR3 receptor mRNA expression were decreased following superfusion with glutamate. L-type Ca2+ channel mRNA expression had a significant dose-dependent decrease following superfusion with glutamate, compared to the control. In the salinity challenge experiment, acute transfer from SW to FW, GluR2 receptor mRNA expression was significantly higher than the control at 2 h. These findings suggest that GluR2 is one of the mechanisms which can medicate glutamate action within the CNSS, enhancing electrical activity and hence secretory output.

Nongenomic Glucocorticoid Suppression of a Postsynaptic Potassium Current via Emergent Autocrine Endocannabinoid Signaling in Hypothalamic Neuroendocrine Cells following Chronic Dehydration.

Glucocorticoids rapidly stimulate endocannabinoid synthesis and modulation of synaptic transmission in hypothalamic neuroendocrine cells via a nongenomic signaling mechanism. The endocannabinoid actions are synapse-constrained by astrocyte restriction of extracellular spatial domains. Exogenous cannabinoids have been shown to modulate postsynaptic potassium currents, including the A-type potassium current (IA), in different cell types. The activity of magnocellular neuroendocrine cells is shaped by a prominent IA. We tested for a rapid glucocorticoid modulation of the postsynaptic IK and IA in magnocellular neuroendocrine cells of the hypothalamic paraventricular nucleus (PVN) using whole-cell recordings in rat brain slices. Application of the synthetic glucocorticoid dexamethasone (Dex) had no rapid effect on the IK or IA amplitude, voltage dependence, or kinetics in magnocellular neurons in slices from untreated rats. In magnocellular neurons from salt-loaded rats, however, Dex application caused a rapid suppression of the IA and a depolarizing shift in IA voltage dependence. Exogenously applied endocannabinoids mimicked the rapid Dex modulation of the IA, and CB1 receptor antagonists and agonists blocked and occluded the Dex-induced changes in the IA, respectively, suggesting an endocannabinoid dependence of the rapid glucocorticoid effect. Preincubation of control slices in a gliotoxin resulted in the partial recapitulation of the glucocorticoid-induced rapid suppression of the IA. These findings demonstrate a glucocorticoid suppression of the postsynaptic IA in PVN magnocellular neurons via an autocrine endocannabinoid-dependent mechanism following chronic dehydration, and suggest a possible role for astrocytes in the control of the autocrine endocannabinoid actions.

Perinatal exposure to organohalogen pollutants decreases vasopressin content and its mRNA expression in magnocellular neuroendocrine cells activated by osmotic stress in adult rats

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are environmental pollutants that produce neurotoxicity and neuroendocrine disruption. They affect the vasopressinergic system but their disruptive mechanisms are not well understood. Our group reported that rats perinatally exposed to Aroclor-1254 (A1254) and DE-71 (commercial mixtures of PCBs and PBDEs) decrease somatodendritic vasopressin (AVP) release while increasing plasma AVP responses to osmotic activation, potentially emptying AVP reserves required for body-water balance. The aim of this research was to evaluate the effects of perinatal exposure to A1254 or DE-71 (30mgkg/day) on AVP transcription and protein content in the paraventricular and supraoptic hypothalamic nuclei, of male and female rats, by in situ hybridization and immunohistochemistry. cFOS mRNA expression was evaluated in order to determine neuroendocrine cells activation due to osmotic stimulation. Animal groups were: vehicle (control); exposed to either A1254 or DE-71; both, control and exposed, subjected to osmotic challenge. The results confirmed a physiological increase in AVP-immunoreactivity (AVP-IR) and gene expression in response to osmotic challenge as reported elsewhere. In contrast, the exposed groups did not show this response to osmotic activation, they showed significant reduction in AVP-IR neurons, and AVP mRNA expression as compared to the hyperosmotic controls. cFOS mRNA expression increased in A1254 dehydrated groups, suggesting that the AVP-IR decrease was not due to a lack of the response to the osmotic activation. Therefore, A1254 may interfere with the activation of AVP mRNA transcript levels and protein, causing a central dysfunction of vasopressinergic system.

Permanently compromised NADPH-diaphorase activity within the osmotically activated supraoptic nucleus after in utero but not adult exposure to Aroclor 1254

Stimulated vasopressin (VP) release from magnocellular neuroendocrine cells in the supraoptic nucleus (SON) of hyperosmotic rats is inhibited by treatment with the industrial polychlorinated biphenyl (PCB) mixture, Aroclor 1254. Because VP responses to hyperosmotic stimulation are regulated by nitric oxide (NO) signaling, we studied NO synthase (NOS) activity in the SON of hyperosmotic rats as potential target of PCB-induced disruption of neuroendocrine processes necessary for osmoregulation. To examine PCB-induced changes in NOS activity under normosmotic and hyperosmotic conditions, male Sprague-Dawley rats were exposed to Aroclor 1254 (30mg/kg/day) in utero and NADPH-diaphorase (NADPH-d) activity was assessed in SON sections at three ages: postnatal day 10, early adult (3–5 months) or late adult (14–16 months). Hyperosmotic treatment increased mean NADPH-d staining density of oil hyperosmotic controls by 19.9% in early adults and 58% in late adulthood vs normosmotic controls. In utero exposure to PCBs reduced hyperosmotic-induced upregulation of NADPH-d activity to control levels in early adults and by 28% in late adults. Basal NADPH-d was reduced in postnatal rats. Rats receiving PCB exposure as early adults orally for 14 days displayed normal responses. Our findings show that developmental but not adult exposure to PCBs significantly reduces NOS responses to hyperosmolality in neuroendocrine cells. Moreover, reduced NADPH-d activity produced by in utero exposure persisted in stimulated late adult rats concomitant with reduced osmoregulatory capacity vs oil controls (375±9 vs 349±5mOsm/L). These findings suggest that developmental PCBs permanently compromise NOS signaling in the activated neuroendocrine hypothalamus with potential osmoregulatory consequences.

TRPV1 channel activity triggered by hyperosmotic stimulation increases phosphorylated NOS activity in rat supraotic slices (1182.10)

Magnocellular neuroendocrine cells (MNCs) in the supraoptic (SON) and paraventricular nucleus (PVN) of the hypothalamus release vasopressin (VP) from axons and somata/dendrites in response to hyperosmotic stimulation. Transient receptor potential vanilloid 1 (TRPV1) channels mediate osmosensory transduction and axonal neurosecretion in SON MNCs. Our lab has shown that osmotic‐stimulated somatodendritic VP responses occur in a TRPV1‐ and NOS‐dependent manner. Since TRPV1 activation may result in NO production, our current study examined the interaction between TRPV1 and NO synthase (NOS) under hyperosmotic conditions. Acutely dissected SON slices were prepared from adult male Holtzman rats perfused with sucrose‐enriched aCSF under deep anesthesia. Slices were treated with hyperosmotic stimulation in the presence and absence of TRPV1 antagonist SB366791 (1.5 μM) and later post‐fixed. Using double‐label immunohistochemistry for phosphorylated stimulatory residue of neuronal NOS (p‐nNOS ser1412) and combined VP‐neurophysin (PS41) and oxytocin‐neurophysin (PS38) we found elevated p‐nNOS immunoreactivity in MNCs under hyperosmotic conditions but not always in the presence of SB366791, suggesting that increased NO signaling may occur downstream of TRPV1 channel activity. By analogy to other neuroendocrine systems p‐nNOS ser1412 may participate in neuropeptide secretion perhaps by coupling with NMDAr on MNCs. We also immunoprobed for downstream signaling partners of NOS and found expression of sGC (beta subunit), an enzyme that converts GTP to cGMP.We conclude that hyperosmotic stimulation activates TRPV1 channels that produce stimulatory phosphorylation of nNOS leading to somatodendritic release of VP from supraoptic MNCs.Grant Funding Source: Supported by American Physiological Society

Astrocytic localization of supraoptic serine racemase and its potential participation in NMDA‐stimulated vasopressin responses in hypothalamo‐neurohypophysial explants (1182.9)

Our lab has previously shown that vasopressinergic magnocellular neuroendocrine cells (MNCs) express various NMDA receptor subunits including the obligatory NR1. Other reports show that NMDA receptors (NMDAr) mediate osmotically‐induced vasopressin (VP) release from the hypothalamo‐neurohypophysial system (HNS). Recently, Panatier et al 2006 revealed that D‐serine is the endogenous coagonist for NMDAr in MNCs of the supraoptic nucleus (SON). Therefore, we investigated the role of D‐serine in NMDAr‐mediated VP release from acutely dissected HNS explants taken from adult male C57BL/6 mice and incubated in a perifusion chamber from which samples were analyzed for vasopressin (VP) using EIA (Gregg & Sladek 1984). After a 4 hr incubation, explants were treated with 5‐100 uM NMDA for 1 hr in the presence and absence of SR inhibitor, L‐Aspartic acid β‐hydroxamate (LA‐βH; 3 mM). We found that NMDAr‐mediated VP release was reduced by LA‐βH suggesting that SR activity and D‐serine signaling are required. In immunohistochemical experiments using adult SON sections, we found marked colocalization of green SR immunoreactivity (ir) with either of two astrocyte specific markers, glial fibrillary acidic protein (GFAP) and glial excitatory amino acid transporter GLT‐1 labeled red. In other experiments animals were injected with Evans Blue (EB), which retrogradely labels VP and oxytocin‐producing MNCs projecting to the posterior pituitary with red fluorescence (excitation/emission maxima ~540/680 nm). SR ir was examined under two osmotic states: hyperosmotic (3.5M NaCl) or normosmotic (0.9% NaCl; 0.6cc/100g bw, i.p.). EB‐positive MNCs showed little or no SR ir, which was redistributed in hyperosmotic animals. We hypothesize that a significant proportion of SR is expressed in astrocytes and that D‐serine gliotransmission contributes to VP responses in SON MNCs.Grant Funding Source: Supported by APS STRIDE summer fellowship (JV), APS Porter fellowship (MV), and MARC (GG, RF, RG).

Glial Control of Endocannabinoid Heterosynaptic Modulation in Hypothalamic Magnocellular Neuroendocrine Cells

Cannabinoid receptors are functionally operant at both glutamate and GABA synapses on hypothalamic magnocellular neuroendocrine cells; however, retrograde endocannabinoid actions are evoked at only glutamate synapses. We tested whether the functional targeting of evoked retrograde endocannabinoid actions to glutamate, and not GABA, synapses on magnocellular neurons is the result of the spatial restriction of extracellular endocannabinoids by astrocytes. Whole-cell GABA synaptic currents were recorded in magnocellular neurons in rat hypothalamic slices following manipulations to reduce glial buffering of extracellular signals. Depolarization- and glucocorticoid-evoked retrograde endocannabinoid suppression of synaptic GABA release was not detected under normal conditions, but occurred in both oxytocin and vasopressin neurons under conditions of attenuated glial coverage and depressed glial metabolic function, suggesting an emergent endocannabinoid modulation of GABA synapses with the loss of astrocyte function. Tonic endocannabinoid suppression of GABA release was insensitive to glial manipulation. Blocking cannabinoid transport mimicked, and increasing the extracellular viscosity reversed, the effect of suppressed glial buffering on the endocannabinoid modulation of GABA release. Evoked, but not tonic, endocannabinoid modulation of GABA synapses was mediated by 2-arachidonoylglycerol. Therefore, depolarization- and glucocorticoid-evoked 2-arachidonoylglycerol release from magnocellular neurons is spatially restricted to glutamate synapses by astrocytes, but spills over onto GABA synapses under conditions of reduced astrocyte buffering; tonic endocannabinoid modulation of GABA release, in contrast, is likely mediated by anandamide and is insensitive to astrocytic buffering. Astrocytes, therefore, provide dynamic control of stimulus-evoked 2-arachidonoylglycerol, but not tonic anandamide, regulation of GABA synaptic inputs to magnocellular neuroendocrine cells under different physiological conditions.

A neuronal population in hypothalamus that dramatically resists acute ischemic injury compared to neocortex

Pyramidal neurons (PyNs) of the cortex are highly susceptible to acute stroke damage, yet "lower" brain regions like hypothalamus and brain stem better survive global ischemia. Here we show for the first time that a "lower" neuron population intrinsically resists acute strokelike injury. In rat brain slices deprived of oxygen and glucose (OGD), we imaged anoxic depolarization (AD) as it propagated through neocortex or hypothalamus. AD, the initial electrophysiological event of stroke, is a front of depolarization that drains residual energy in compromised gray matter. The extent of AD reliably determines ensuing cortical damage, but do all CNS neurons generate a robust AD? During 10 min of OGD, PyNs depolarize without functional recovery. In contrast, magnocellular neuroendocrine cells (MNCs) in hypothalamus under identical stress generate a weak and delayed AD, resist complete depolarization, and rapidly repolarize when oxygen and glucose are restored. They recover their membrane potential, input resistance, and spike amplitude and can survive multiple OGD exposures. Two-photon microscopy in slices derived from a fluorescent mouse line confirms this protection, revealing PyN swelling and dendritic beading after OGD, whereas MNCs are not injured. Exposure to the Na(+)-K(+)-ATPase inhibitor ouabain (100 μM) induces AD similar to OGD in both cell types. Moreover, elevated extracellular K(+) concentration ([K(+)](o)) evokes spreading depression (SD), a milder version of AD, in PyNs but not MNCs. Therefore overriding the pump by OGD, ouabain, or elevated [K(+)](o) evokes a propagating depolarization in higher gray matter but not in MNCs. We suggest that variation in Na(+)-K(+)-ATPase pump efficiency during ischemia injury determines whether a neuronal type succumbs to or resists stroke.

TRPV1 channels contribute to hyperosmotic‐induced release of vasopressin from somata and dendrites of magnocellular neuroendocrine cells in supraoptic punches

Magnocellular neuroendocrine cells (MNCs) in the supraoptic nucleus (SON) of the hypothalamus release the antidiuretic hormone, vasopressin (VP), in response to hyperosmotic and hypovolemic stimulation. Transient Receptor Potential Vanilloid 1 (TRPV1) receptor channels mediate osmosensitive electrical responses in MNCs and systemic VP release (Naeini et. al., 2006) but the specific mechanism linking TRPV1 channels and secretion of VP has yet to be determined. To examine the role of TRPV1 channels in somatodendritic VP release during hyperosmotic stimulation we treated acutely dissected SON punches in vitro with 350 mOsm/l Locke's solution in the presence and absence of the TRPV1 antagonist, 5‐iodoresiniferatoxin (SB366791). VP values were quantified using enzyme‐linked immunoassay. Hyperosmotic stimulation enhanced VP release and 1.5μM SB366791 attenuated this (p<0.05; n= 39). Mean (±s.e.m.) values for extracellular VP levels in isosmotic, hyperosmotic and hyperosmotic/SB366791 were 7.15±1.7, 11.1±2.8, 3.68±0.7 pg/ml μg protein, respectively. Doses of 0.15 and 10μM also reduced VP levels to 5.5±1.0 and 5.1±1.0, respectively. Immunohistochemistry showed TRPV1 immunoreactivity in SON MNCs. We conclude that TRPV1 channels in SON MNCs are activated during hyperosmotic stimulation and are required for somatodendritic VP secretion during osmotic challenge. (Supported by UC MEXUS)

Synchronized Network Oscillations in Rat Tuberoinfundibular Dopamine Neurons: Switch to Tonic Discharge by Thyrotropin-Releasing Hormone

The pituitary hormone, prolactin, triggers lactation in nursing mothers. Under nonlactating conditions, prolactin secretion is suppressed by powerful inhibition from hypothalamic tuberoinfundibular dopamine (TIDA) neurons. Although firing pattern has been suggested as integral to neuroendocrine control, the electrical behavior of TIDA cells remains unknown. We demonstrate that rat TIDA neurons discharge rhythmically in a robust 0.05 Hz oscillation. The oscillation is phase locked between neurons, and while it persists during chemical synaptic transmission blockade, it is abolished by gap junction antagonists. Thyrotropin-releasing hormone (TRH) potently stimulates prolactin release, an effect assumed to take place in the pituitary. In TIDA cells, TRH caused a transition from phasic to tonic firing through combined pre- and postsynaptic effects. These findings suggest a model for prolactin regulation where a TIDA network switch from oscillations to sustained discharge converts dopamine from an antagonist at high concentrations to a functional agonist as dopamine output from the network decreases.

Neuropeptidomics of the Supraoptic Rat Nucleus

The mammalian supraoptic nucleus (SON) is a neuroendocrine center in the brain regulating a variety of physiological functions. Within the SON, peptidergic magnocellular neurons that project to the neurohypophysis (posterior pituitary) are involved in controlling osmotic balance, lactation, and parturition, partly through secretion of signaling peptides such as oxytocin and vasopressin into the blood. An improved understanding of SON activity and function requires identification and characterization of the peptides used by the SON. Here, small-volume sample preparation approaches are optimized for neuropeptidomic studies of isolated SON samples ranging from entire nuclei down to single magnocellular neurons. Unlike most previous mammalian peptidome studies, tissues are not immediately heated or microwaved. SON samples are obtained from ex vivo brain slice preparations via tissue punch and the samples processed through sequential steps of peptide extraction. Analyses of the samples via liquid chromatography mass spectrometry and tandem mass spectrometry result in the identification of 85 peptides, including 20 unique peptides from known prohormones. As the sample size is further reduced, the depth of peptide coverage decreases; however, even from individually isolated magnocellular neuroendocrine cells, vasopressin and several other peptides are detected.

Neuroendocrine Regulation of Fluid and Electrolyte Balance By Ovarian Steroids: Contributions From Central Oestrogen Receptors

Like other hormonally mediated mechanisms, maintenance of body fluid osmolality requires integrated responses from multiple signals at various tissue locales, a large number of which are open to modulation by circulating endocrine factors including the ovarian steroid, oestrogens (E(2)). However, the precise mechanism and the site of action of E(2) in regulating fluid osmolality are not properly understood. More importantly, the biological significance of this action is not clear and the physiological circumstances in which this modulation is engaged remain incomplete. The demonstration of oestrogen receptors (ER) in neural tissues that bear no direct relation to reproduction led us to examine and characterise the expression of ER in brain nuclei that are critical for the maintenance of fluid osmolality. In the rat, ERbeta is prominently expressed in the vasopressin magnocellular neuroendocrine cells of the hypothalamus, whereas ERalpha is localised extensively in the sensory circumventricular organ neurones in the basal forebrain. These nuclei are the primary brain sites that are engaged in defense of fluid perturbation, thus providing a neuroendocrine basis for oestrogenic influence on body fluid regulation. Plasticity in receptor expression that accompanies fluid disturbances at these central loci suggests the functional importance of the receptors and implicates E(2) as one of the fluid regulating hormones in water homeostasis.