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Characteristics of subfornical organ (SFO) neurons were examined by measuring the cytosolic Ca 2+ concentration ([Ca 2+ ] i ) in acutely dissociated neurons of the rat. SFO neurons, defined by the responsiveness to 50 mM K + (n = 67) responded to glutamate (86%), angiotensin II (AII) (50%), arginine vasopressin (AVP) (66%) and/or carbachol (CCh) (61%), at their maximal concentrations, with marked increases in [Ca 2+ ] i . More than a half (174/307) of SFO neurons examined exhibited spontaneous Ca 2+ oscillations, while the remainder showed a relatively stable baseline under unstimulated conditions. Spontaneous Ca 2+ oscillations were suppressed when extracellular Ca 2+ was removed and were inhibited when extracellular Na + was replaced with equimolar N-methyl-D-glucamine. Ca 2+ oscillations were unaffected by the inhibitor of Ca 2+ -dependent ATPases cyclopiazonic acid, the N-type Ca 2+ channel blocker ω-conotoxin GVIA and the P/Q-type Ca 2+ channel blocker ω-agatoxin IVA, but significantly inhibited by the high-voltage-activated Ca 2+ channel blocker Cd 2+ and the L-type Ca 2+ channel blocker nicardipine. Ca 2+ oscillations were also completely arrested by the voltage-gated Na + channel blocker tetrodotoxin in 50% of SFO neurons but only partially in the remaining neurons. These results suggest that SFO neurons exhibit spontaneous membrane Ca 2+ oscillations that are dependent in part on Ca 2+ entry through L-type Ca 2+ channels, whose activation may result from burst firing. Moreover, AII at picomolar concentrations induced Ca 2+ oscillations in neurons showing no spontaneous Ca 2+ oscillations, while spontaneous Ca 2+ oscillations were arrested by gamma-aminobutyric acid (10 μM), suggesting that rises in [Ca 2+ ] i during Ca 2+ oscillations may play an important role in the modulation of SFO neuron function., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Transient receptor potential vanilloid type 1 (TRPV1) is a polymodal receptor channel that responds to multiple types of stimuli, such as heat, acid, mechanical pressure and some vanilloids. Capsaicin is the most commonly used vanilloid to stimulate TRPV1. TRPV1 channels are expressed in dorsal root ganglion neurons that extend to Aδ- and C-fibers and have a role in the transduction of noxious inputs to the skin into the electrical signals of the sensory nerve. Although noradrenergic nervous systems, including the descending antinociceptive system and the sympathetic nervous system, are known to modulate pain sensation, the functional association between TRPV1 and noradrenaline in primary sensory neurons has rarely been examined. In the present study, we examined the effects of noradrenaline on capsaicin-evoked currents in cultured dorsal root ganglion neurons of the rat by the whole-cell voltage clamp method. Noradrenaline at concentrations higher than 0.1 pM significantly reduced the amplitudes of the inward capsaicin currents recorded at -60 mV holding potential. This inhibitory action was reversed by either yohimbine (an α2 antagonist, 10 nM) or propranolol (a β antagonist, 10 nM). The α2 agonists, clonidine (1 pM) and dexmedetomidine (1 pM) inhibited capsaicin currents, and yohimbine (1 nM) reversed the effects of clonidine. The inhibitory action of noradrenaline was not seen in the neurons pretreated with pertussis toxin (100 μg/ml for 24 h) and the neurons dialyzed intracellularly with guanosine 5'- [β-thio] diphosphate (GDPβS, 200 μM), the catalytic subunit of protein kinase A (250 U/ml) or okadaic acid (1 μM). These results suggest that noradrenaline directly acts on dorsal root ganglion neurons to inhibit the activity of TRPV1 depending on the activation of α2-adrenoceptors followed by the inhibition of the adenylate cyclase/cAMP/protein kinase A pathway.

Ten pairs of protrusions, called accessory lobes (ALs), exist at the lateral sides of the avian lumbosacral spinal cord. Histological evidence indicates that neuron-like cells gather in the ALs, and behavioral evidence suggests that the ALs act as a sensory organ of equilibrium during bipedal walking. Recently, using an electrophysiological method, we reported that cells showing Na + currents and action potentials exist among cells that were dissociated from the ALs. However, it was unclear which isoforms of the voltage-gated sodium channel (VGSC) are expressed in the ALs and whether cells having neuronal morphology in the ALs express VGSCs. To elucidate these points, RT-PCR and immunohistochemical experiments were performed. In RT-PCR analysis, PCR products for Na v 1.1-1.7 were detected in the ALs. The signal intensities of the Na v 1.1 and 1.6 isoforms were stronger than those of the other isoforms. We confirmed that an antibody raised against an epitope peptide of the rat VGSC had cross-reactivity to chick tissues by Western blotting, and we performed immunofluorescence staining using the antibody. The AL contained cells having neuron-like morphology and VGSC-like immunoreactivity at their cytoplasm and/or cell membranes. Filament-like structures showing GFAP-like immunoreactivity infilled intercellular spaces. The VGSC- and GFAP-like immunoreactivities did not overlap. These results indicate that the neuronal isoforms of the VGSC are mainly expressed in the AL and that the neuron-like cells in the ALs express VGSCs. Our findings indicate that AL neurons generate action potentials and send sensory information to the motor systems on the contralateral side of the spinal segment.

Accessory lobes are protrusions located at the lateral sides of the spinal cord of chicks and it has been proposed that they play a role as a sensory organ for equilibrium during walking. We have reported that functional neurons exist in the accessory lobe. As there is histological evidence that synaptic terminals of cholinergic nerves exist near the somata of accessory lobe neurons, we examined the effects of acetylcholine on changes in intracellular Ca2+ concentrations ([Ca2+]i), as an index of cellular activities. Acetylcholine (0.1-100 µM) caused a transient rise in the [Ca2+]i. Acetylcholine-evoked [Ca2+]i rises were observed in the absence of extracellular Ca2+, and they were abolished in the presence of cyclopiazonic acid, an inhibitor of Ca2+-ATPase of intracellular Ca2+ stores or atropine, a muscarinic receptor antagonist. mRNAs coding M3 and M5 isoforms of the muscarinic receptors were detected in accessory lobes by the RT-PCR. These results indicate that chick accessory lobe neurons express functional muscarinic acetylcholine receptors, and that acetylcholine stimulates Ca2+ mobilization from intracellular Ca2+ stores, which elevates the [Ca2+]i in the somata of accessory lobe neurons, through activation of these receptors. Cholinergic synaptic transmission to the accessory lobe neurons may regulate some cellular functions through muscarinic receptors.

Neurones in the supraoptic nucleus (SON) of the hypothalamus possess intrinsic osmosensing mechanisms, which are lost in transient receptor potential vanilloid 1 (Trpv1)-knock-out mice. The molecular nature of the osmosensory mechanism in SON neurones is believed to be associated with the N-terminal splice variant of Trpv1, although their entire molecular structures have not been hitherto identified. In this study, we sought for TRPV1-related molecules and their function in the rat SON. We performed RT-PCR and immunohistochemistry to detect TRPV1-related molecules in the SON, and patch-clamp and imaging of the cytosolic Ca(2+) concentration ([Ca(2+)]i) to measure responses to osmolality changes and TRPV-related drugs in acutely dissociated SON neurones of rats. RT-PCR analysis revealed full-length Trpv1 and a new N-terminal splice variant, Trpv1_SON (LC008303) in the SON. Positive immunostaining was observed using an antibody against the N-terminal portion of TRPV1 in arginine vasopressin (AVP)-immunoreactive neurones, but not in oxytocin (OT)-immunoreactive neurones. Approximately 20% of SON neurones responded to mannitol (50 mM) with increased action potential firing, inward currents, and [Ca(2+)]i mobilization. Mannitol-induced responses were observed in AVP neurones isolated from AVP-eGFP transgenic rats and identified by GFP fluorescence, but not in OT neurones isolated from OT-mRFP transgenic rats and identified by RFP fluorescence. The mannitol-induced [Ca(2+)]i responses were reversibly blocked by the non-selective TRPV antagonist, ruthenium red (10 μM) and the TRPV1 antagonists, capsazepine (10 μM) and BCTC (10 μM). Although the TRPV1 agonist, capsaicin (100 nM) evoked no response at room temperature, it triggered cationic currents and [Ca(2+)]i elevation when the temperature was increased to 36°C. These results suggest that AVP neurones in the rat SON possess functional full-length TRPV1. Moreover, differences between the responses to capsaicin or hyperosmolality obtained in rat SON neurones and those obtained from dorsal root ganglion neurones or TRPV1-expressing cells indicate that the osmoreceptor expressed in the SON may be a heteromultimer in which TRPV1 is co-assembled with some other, yet unidentified, molecules., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

While magnocellular neurons in the supraoptic nucleus (SON) possess rich Gi/o-mediated mechanisms, molecular and cellular properties of G-protein-activated inwardly rectifying K(+) (GIRK) channels have been controversial. Here, properties of GIRK channels are examined by RT-PCR and whole-cell patch-clamp techniques in rat SON neurons. Patch clamp experiments showed that the selective GABAB agonist, baclofen, enhanced currents in a high K(+) condition. The baclofen-enhanced currents exhibited evident inward rectification and were blocked by the selective GABAB antagonist, CGP55845A, the IRK channel blocker, Ba(2+), and the selective GIRK channel blocker, tertiapin, indicating that baclofen activates GIRK channels via GABAB receptors. The GIRK currents were abolished by N-ethylmaleimide pretreatment, and prolonged by GTPγS inclusion in the patch pipette, suggesting that Gi/o proteins are involved. RT-PCR analysis revealed mRNAs for all four GIRK 1-4 channels and for both GABABR1 and GABABR2 receptors in rat SON. However, the concentration-dependency of the baclofen-induced activation of GIRK currents had an EC50 of 110 µM, which is about 100 times higher than that of baclofen-induced inhibition of voltage-dependent Ca(2+) channels. Moreover, baclofen caused no significant changes in the membrane potential and the firing rate. These results suggest that although GIRK channels can be activated by GABAB receptors via the Gi/o pathway, this occurs at high agonist concentrations, and thus may not be a physiological mechanism regulating the function of SON neurons. This property that the membrane potential receives little influence from GIRK currents seems to be uncommon for CNS neurons possessing rich Gi/o-coupled receptors, and could be a special feature of rat SON neurons., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Birds have ten pairs of protrusions, "accessory lobes", on the lateral sides of the lumbosacral spinal cord. It has been proposed that accessory lobes act as a sensory organ of equilibrium and neurons in accessory lobes transmit sensory information to the motor center. We have reported that cells in chick accessory lobes express functional voltage-gated Na(+) and K(+) channels and generate action potentials. In this study, we examined properties of voltage-gated Ca(2+) channels (VGCCs). The amplitude of voltage-gated Ca(2+) channel currents carried by Ca(2+) and Ba(2+) increased gradually during 10 min rather than showing the usual run-down. The current-voltage relationship of Ba(2+) currents was consistent with that of the high-voltage-activated Ca(2+) channel. The proportion of Ba(2+) currents inhibited by ω-conotoxin GVIA was larger than 80%, indicating that the major subtype is N type. Amplitudes of tail currents of Ca(2+) currents evoked by repetitive pulses at 50 Hz are stable for 1 s. If the major subtype of VGCCs at synaptic terminals is also N type, this property may contribute to the establishment of stable synaptic connections between accessory lobe neurons, which are reported to fire at frequencies higher than 15 Hz, and postsynaptic neurons in the spinal cord.

Ten pairs of protrusions, called accessory lobes (ALs), exist at the lateral sides of avian lumbosacral spinal cords. Histological and behavioral evidence suggests that neurons are present in ALs and the AL acts as a sensory organ of equilibrium during walking. Neurons in the outer layer of the AL consistently show glutamate-like immunoreactivity and neurons in the central region of the AL show glutamate receptor-like immunoreactivity. However, it is unknown how glutamate acts on the functional activity of AL neurons. In this study, we examined the effects of glutamate on the electrical activities of AL neurons using the patch clamp technique. There are two types of neurons among isolated AL neurons: spontaneously firing and silent neurons. Among silent neurons, 42 % of neurons responded to glutamate and generated repetitive firing. Kainate and glutamate in combination with the NMDA receptor antagonist, MK-801, also induced firing and evoked an inward current. On the other hand, the application of AMPA, NMDA or glutamate in combination with the non-NMDA receptor antagonist, CNQX, did not. These results indicate that chick AL neurons express functional kainate receptors to respond to glutamate and suggest that the glutamatergic transmission plays a role in excitatory regulation of AL neurons of the chick.

Adult rat dorsal root ganglion (DRG) neurons cultured in the presence of 100-ng/mL NGF were reported to show spontaneous action potentials in the cell-attached recording. In this study, underlying mechanisms were examined in the whole-cell and outside-out voltage clamp recording. In 75% neurons with on-cell firing, transient inward current spikes were repetitively recorded in the voltage clamp mode at -50 mV in the whole-cell configuration (named "Isp"). Isp with stable amplitudes occurred in an all-or-none fashion, and was abolished by TTX (< 100 nM), lidocaine (< 1 mM) and a reduction of extracellular Na(+) (154 to 100 mM) in an all-or-none fashion, suggesting that Isp reflects spontaneous dicharges occurring at the loosely voltage-clamped regions. Isp was also observed in the excised outside-out patches and the kinetics and the sensitivity to TTX and lidocaine resembled those in the whole-cell. Spontaneous action potentials were also recorded in the current clamp mode. Small subthreshold spikes often preceded the action potentials. When the localized discharge affected a whole-somatic membrane potential to overcome a threshold, the action potential generated. These results indicate that the triggering sources of the action potential exist in the somatic membrane itself in NGF-treated DRG neurons.

It has been hypothesized that chick accessory lobes (ALs) contain functional neurons and act as a sensory organ of equilibrium. It was reported that neurons located in an outer layer of ALs showed γ-aminobutyric acid (GABA)- and glutamic acid decarboxylase (GAD)-like immunoreactivity more strongly than centrally located neurons, which were surrounded by the GAD-immunoreactive terminals. We investigated effects of GABA on the electrical activity of AL neurons. About 50% of embryonic AL neurons exhibited spontaneous firing. In the on-cell recording, GABA, muscimol, and GABA in combination with CGP35348 inhibited this firing. In whole-cell voltage clamp recordings, GABA and muscimol evoked a transient current. The mean reversal potential of GABA-evoked currents was close to the theoretical reversal potential of Cl⁻. These results indicate that GABA exerts the inhibitory effect on the firing through the activation of GABA(A) receptors. In addition, the intracellular concentration of Cl⁻ was estimated to be about 16 mM in measurements with the gramicidin-perforated configuration, indicating the physiological reversal potential of the GABA current was about -60 mV. In conclusion, AL neurons have an intrinsic mechanism to evoke the spontaneous firing, which can be arrested by the inhibitory mechanism through the activation of the GABA(A) receptors.

Multiple mechanisms that maintain Ca(2+) homeostasis and provide for Ca(2+) signalling operate in the somatas and neurohypophysial nerve terminals of supraoptic nucleus (SON) neurones. Here, we examined the Ca(2+) clearance mechanisms of SON neurones from adult rats by monitoring the effects of the selective inhibition of different Ca(2+) homeostatic molecules on cytosolic Ca(2+) ([Ca(2+)](i)) transients in isolated SON neurones. In addition, we measured somatodendritic vasopressin (AVP) release from intact SON tissue in an attempt to correlate it with [Ca(2+)](i) dynamics. When bathing the cells in a Na(+)-free extracellular solution, thapsigargin, cyclopiazonic acid (CPA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the inhibitor of plasma membrane Ca(2+)-ATPase (PMCA), La(3+), all significantly slowed down the recovery of depolarisation (50 mM KCl)-induced [Ca(2+)](i) transients. The release of AVP was stimulated by 50 mM KCl, and the decline in the peptide release was slowed by Ca(2+) transport inhibitors. In contrast to previous reports, our results show that in the fully mature adult rats: (i) all four Ca(2+) homeostatic pathways, the Na(+)/Ca(2+) exchanger, the endoplasmic reticulum Ca(2+) pump, the plasmalemmal Ca(2+) pump and mitochondria, are complementary in actively clearing Ca(2+) from SON neurones; (ii) somatodendritic AVP release closely correlates with intracellular [Ca(2+)](i) dynamics; (iii) there is (are) Ca(2+) clearance mechanism(s) distinct from the four outlined above; and (iv) Ca(2+) homeostatic systems in the somatas of SON neurones differ from those expressed in their terminals., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Adult rat dorsal root ganglion (DRG) neurons cultured in the presence of 100 ng/ml NGF show spontaneous action potentials and fluctuations in their cytosolic Ca(2+) concentrations ([Ca(2+)](i)). In the present study, the Ca(2+) sources of the [Ca(2+)](i) fluctuations and the types of neurons whose excitability was affected by NGF were examined. In the subpopulation of NGF-treated neurons, obvious fluctuations of [Ca(2+)](i) were observed. The [Ca(2+)](i) fluctuations were inhibited by Ca(2+) removal or inhibitors of voltage-gated Ca(2+) channels. Regardless of the treatment with NGF, about half of the neurons responded to capsaicin and 10% of the neurons responded to icilin, and almost all icilin-responding neurons also responded to capsaicin. Fluctuations of [Ca(2+)](i) with large amplitudes were observed in 12 out of 131 NGF-treated neurons. Among these 12 neurons, 10 neurons responded to both capsaicin and icilin. The degree of the [Ca(2+)](i) fluctuations in the NGF-treated neurons responding to both capsaicin and icilin was significantly larger than in other neurons. These results suggest that neurons expressing both capsaicin- and icilin-sensitive TRP channels are susceptible to NGF and become hyperexcitable and that Ca(2+) influx through voltage-gated Ca(2+) channels is the major source contributing to the [Ca(2+)](i) fluctuations. Since such DRG neurons could play a physiological role as nociceptors, the NGF-induced spontaneous activity of DRG neurons may be the underlying mechanism of neuropathic pain.

Ten pairs of protrusions, called accessory lobes (ALs), exist at the lateral sides of avian lumbosacral spinal cords. Histological evidence has shown that neurons are present in AL and behavioral evidence suggests that AL acts as a sensory organ of equilibrium during bipedal walking. However, there is little functional evidence to indicate that cells in AL have neuronal functions. To elucidate this point, we developed a method to dissociate cells from chick AL and made electrophysiological recordings with the whole-cell patch clamp technique. Cells dissociated by enzymatic digestion from chick AL contained two major types of cells. One was round with clear cytosol and the other had a round cell body, rich cytosolic structures and some processes. Rapidly activating inward currents and slowly activating outward currents were recorded in response to depolarizing pulses to -10 mV under the voltage clamp configuration only from the latter type of cells. TTX at 100 nM inhibited the inward current by 85%, indicating the functional expression of TTX-sensitive voltage-gated Na(+) channel (VGSC). Activation and inactivation kinetics of the inward currents in AL cells were similar to those of mammalian VGSC. The VGSC-expressing AL cells generated action potentials in response to depolarization under the current clamp configuration. These results clearly indicate that functional neurons expressing fast inactivating and TTXsensitive VGSC which generate action potentials exist in the AL of the chick. These lines of cellular evidence clearly indicate that functional neurons exist in ALs and further support the proposal that the chick ALs function as the sensory organ of equilibrium.

Acute estradiol (E2) can potentiate the excitatory responses of hypothalamic ventromedial nucleus (VMN) neurons to neurotransmitters. To investigate the mechanism(s) underlying the potentiation, the whole-cell patch voltage clamp technique was used to study VMN neurons in hypothalamic slices prepared from female juvenile (3-5 weeks) rats. A voltage step and/or ramp was applied every 5 min to evoke whole-cell currents before, during and after a treatment with E2 (10 nM), corticosterone (10 nM) or vehicle for up to 20 min. Acute E2 increased inward currents in 38% of neurons tested. Their average peak inward current amplitudes started to increase within 5 min and reached the maximum of 163% of pretreatment level (Pre) at 20 min of treatment before recovering toward Pre. These increases are significantly greater than the Pre and corresponding vehicle controls and non-responsive neurons. Outward currents were decreased significantly by E2 in 27% of E2-treated cells, down to 60% of Pre levels. E2 also appeared to affect the kinetics of the inward and outward currents of estrogen-responsive neurons. Whenever observed, the effects of acute E2 were reversible after a 5- to 10-min washing. Probability analysis indicates that E2 affected the inward and the outward currents independently. The E2 effects are specific in that they were not produced by similar treatment with vehicle or corticosterone. Pharmacological characterizations using ion replacement and channel blockers showed that the inward currents were mediated practically all by Na(+) and the outward currents mainly by K(+). Thus, acute E2 can enhance inward Na(+) and attenuate outward K(+) currents. Since both effects will lead to an increase in neuronal excitability, they may explain our previous observation that E2 potentiates the excitation of VMN neurons.

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor (PAC1 receptor) in regulating hypothalamic supraoptic neurones was investigated using PAC1 receptor-deficient male mice (PAC1-/-). The effects of PACAP on [Ca2+]i were investigated in freshly dissociated supraoptic neurones and on the somatodendritic release of vasopressin and oxytocin, examined on intact supraoptic nuclei. In supraoptic neurones from wild-type mice (PAC1+/+), 100 nm PACAP induced an increase in [Ca2+]i and release of vasopressin and oxytocin, whereas in heterozygous (PAC1+/-) and null-mutant mice (PAC1-/-), PACAP was much less effective. PACAP had no effect on these two parameters when applied to isolated neurohypophysial nerve terminals of PAC1+/+ and PAC1-/- mice, and rats. In conclusion, the PAC1 receptor is solely responsible for the PACAP-induced [Ca2+]i signalling and secretion of vasopressin and oxytocin in the somatodendritic region of supraoptic neurones.

We have reported that supraoptic nucleus (SON) neurones are excited by prostaglandin E2 (PGE2) presumably via dual postsynaptic PG receptors, FP receptors and unidentified EP receptors, and that presynaptic EP receptors may also be involved in the excitation. In the present study, to clarify the receptor mechanism of the PGE2-mediated actions on SON neurones, we studied the pre- and postsynaptic effects of four newly developed EP agonists that are selective for each of the four EP receptors, EP1-4, on rat SON neurones using extracellular recording and whole-cell patch-clamp techniques. The EP4 agonist ONO-AE1-329 mimicked the excitatory effects of PGE2, whereas the EP1 agonist ONO-DI-004, the EP2 agonist ONO-AE1-257 and the EP3 agonist ONO-AE-248 had little or no effect. The effects of ONO-AE1-329 were unaffected by the EP1/FP/TP antagonist, ONO-NT-012, which potently suppressed the excitation caused by the FP agonist fluprostenol and PGE2. ONO-AE1-329 caused marked excitation when responses to fluprostenol were desensitized by repeated applications of fluprostenol. Patch-clamp analysis in SON neurones showed that ONO-AE1-329 induced inward currents at a holding potential of -70 mV and the reversal potential of the currents was -35.1 +/- 2.3 mV. On the other hand, the frequency of spontaneous inhibitory postsynaptic currents recorded from SON slice preparations was suppressed by ONO-AE-248, but unaffected by the other three EP agonists. These results suggest that SON neurones possess postsynaptic EP4 receptors and that gamma-aminobutyric acid neurones innervating SON neurones possess presynaptic EP3 receptors in their terminals. Activation of the two EP receptors may be involved in the excitatory regulation of SON neurones by PGE2.

Magnocellular neurones in the supraoptic nucleus and paraventricular nucleus express mRNA for nitric oxide synthase (NOS) and the expression becomes more prominent when the release of vasopressin or oxytocin is stimulated. It has also been reported that NO donors inhibit the electrical activity of supraoptic nucleus neurones, but the mechanism involved in the inhibition remains unclear. In the present study, to know whether modulation of synaptic inputs into supraoptic neurones is involved in the inhibitory effect of NO, we measured spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) from rat supraoptic nucleus neurones in slice preparations identified under a microscope using the whole-cell mode of the slice-patch-clamp technique. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), reversibly increased the frequency of spontaneous IPSCs mediated by GABAA receptors, without affecting the amplitude, indicating that NO potentiated IPSCs via a presynaptic mechanism. The NO scavenger, haemoglobin, suppressed the potentiation of IPSCs by SNAP. On the other hand, SNAP did not cause significant effects on EPSCs mediated by non-NMDA glutamate receptors. The membrane permeable analogue of cGMP, 8-bromo cGMP, caused a significant reduction in the frequency and amplitude of both IPSCs and EPSCs. The results suggest that NO preferentially potentiates the inhibitory synaptic inputs into supraoptic nucleus neurones by acting on GABA terminals in the supraoptic nucleus, possibly via a cGMP-independent mechanism. The potentiation may, at least in part, account for the inhibitory action of NO on the neural activity of supraoptic neurones.

The release of vasopressin and oxytocin is regulated by the electrical activity of magnocellular neurosecretory cells in the supraoptic and paraventricular nuclei, which is under the control of a great variety of neurotransmitters and neuromodulators. The major neural signals to the supraoptic nucleus are from excitatory glutamate inputs and inhibitory GABA inputs. In recent studies, the voltage-clamp mode of the whole-cell patch-clamp technique has been applied to slice preparations from rat hypothalamus to monitor synaptic inputs to supraoptic neurones. Spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) are abolished by CNQX and picrotoxin, respectively, but are insensitive to tetrodotoxin, indicating that they represent quantal release of glutamate and GABA, respectively, from nerve terminals of presynaptic neurones. GABA and glutamate show remarkable suppressive effects on both EPSCs and IPSCs via presynaptic GABA(B) and mGlu receptors, respectively. Noradrenaline, which excites supraoptic neurones via postsynaptic alpha1-receptors, also suppresses IPSCs and potentiates EPSCs. On the other hand, prostaglandin E2, which excites supraoptic neurones via postsynaptic prostaglandin E2 (EP) receptors of the EP4 subclass, also suppresses IPSCs via EP3 receptors but has little effect on EPSCs. Thus pre- and postsynaptic mechanisms may act cooperatively to excite supraoptic neurones. Nitric oxide, which inhibits supraoptic neurones, potentiates IPSCs without affecting EPSCs. This provides another example for the preferential modulation of IPSCs of supraoptic neurones. On the other hand, PACAP, which causes a long-lasting increase in the firing frequency via the postsynaptic receptors, has no effect on EPSCs and IPSCs, suggesting that some ligands act only at postsynaptic receptors. Thus multiple patterns for pre- and postsynaptic modulation are present in the supraoptic nucleus, and the electrical activity of supraoptic neurones is regulated via complex mechanisms at both pre- and postsynaptic sites.

Prostaglandin E2 (PGE2) has been implicated in the excitatory regulation of magnocellular neurones in the supraoptic nucleus (SON). We have recently reported that PGE2 excited SON neurones by directly activating postsynaptic PGE2 receptors (EP receptors) of a subclass other than EP1-3, but did not affect excitatory postsynaptic currents (EPSCs). In the present study, we examined presynaptic effects of PGE2 on rat SON neurones by measuring spontaneous inhibitory postsynaptic currents (IPSCs) by a slice patch-clamp technique. PGE2 inhibited spontaneous IPSCs in a dose-dependent and reversible manner. PGE2 selectively suppressed the frequency of IPSCs without affecting the amplitude of IPSCs in the presence of tetrodotoxin, a blocker of Na+ channels, indicating that the effects were presynaptic. The inhibitory effects of PGE2 on the frequency of IPSCs were mimicked by the EP1/EP3 agonists, 17PT-PGE2 and sulprostone, and the EP2/EP3 agonist, misoprostol, whereas the EP2 agonist, butaprost, or the FP agonist, fluprostenol, had little effect. The effects of PGE2 on IPSCs were unaffected by the selective EP1 antagonist, SC-51322. They were unaffected also by antagonists of GABAB and alpha2 adrenergic receptors, which are present at presynaptic terminals of GABA neurones in the SON and cause suppression of spontaneous IPSCs. The inhibitor of PG synthesis, indomethacin, had little effect on spontaneous IPSCs and on the inhibitory effects of PGE2 as well as of the GABAB agonist, baclofen, and noradrenaline. These results suggest that PGE2 inhibits release of GABA from the GABAergic terminals innervating SON neurones by activating presynaptic EP receptors, presumably of the EP3 subclass, and that such a presynaptic mechanism may play a role in the excitatory regulation of SON neurones by PGE2.

1. The expression, distribution and function of P2X purinoceptors in the supraoptic nucleus (SON) were investigated by reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and Ca2+-imaging and whole-cell patch-clamp techniques, respectively. 2. RT-PCR analysis of all seven known P2X receptor mRNAs in circular punches of the SON revealed that mRNAs for P2X2, P2X3, P2X4, P2X6 and P2X7 receptors were expressed in the SON, and mRNAs for P2X3, P2X4 and P2X7 were predominant. 3. In situ hybridization histochemistry for P2X3 and P2X4 receptor mRNAs showed that both mRNAs were expressed throughout the SON and in the paraventricular nucleus (PVN). 4. ATP caused an increase in [Ca2+]i in a dose-dependent manner with an ED50 of 1.7 x 10-5 M. The effects of ATP were mimicked by ATPgammaS and 2-methylthio ATP (2MeSATP), but not by AMP, adenosine, UTP or UDP. alphabeta-Methylene ATP (alphabetaMeATP) and ADP caused a small increase in [Ca2+]i in a subset of SON neurones. 5. The P2X7 agonist 2'- & 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) at 10-4 M increased [Ca2+]i, but the potency of BzATP was lower than that of ATP. In contrast, BzATP caused a more prominent [Ca2+]i increase than ATP in non-neuronal cells in the SON. 6. The effects of ATP were abolished by extracellular Ca2+ removal or by the P2 antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), and inhibited by extracellular Na+ replacement or another P2 antagonist, suramin, but were unaffected by the P2X7 antagonist oxidized ATP, and the inhibitor of Ca2+-ATPase in intracellular Ca2+ stores cyclopiazonic acid. 7. Two patterns of desensitization were observed in the [Ca2+]i response to repeated applications of ATP: some neurones showed little or moderate desensitization, while others showed strong desensitization. 8. Whole-cell patch-clamp analysis showed that ATP induced cationic currents with marked inward rectification. The ATP-induced currents exhibited two patterns of desensitization similar to those observed in the [Ca2+]i response. 9. The results suggest that multiple P2X receptors, including P2X3, are functionally expressed in SON neurones, and that activation of these receptors induces cationic currents and Ca2+ entry. Such ionic and Ca2+-signalling mechanisms triggered by ATP may play an important role in the regulation of SON neurosecretory cells.

Prostaglandins (PGs) have been implicated in the regulation of vasopressin (VP) and oxytocin (OT) release in response to various stimuli. To examine the site and mechanism of actions of PGs, we studied effects of PGE2 and PG-receptor agonists on supraoptic nucleus (SON) neurones of rat hypothalamic slice preparations using extracellular recording and whole-cell patch-clamp techniques. PGE2 modulated the electrical activity of more than 80% of the neurones studied. The effects of PGE2 on both phasic and non-phasic neurones were mostly excitatory, and dose-dependent. The effects of PGE2 were mimicked by PGF2alpha or the FP agonist, fluprostenol, whereas PGD2 or the selective EP, IP or TP agonist was less effective or had no effect. The effects of PGE2 were unaffected by the EP1 antagonist, SC-51322, but reduced to 80% of control by the EP1/FP/TP antagonist, ONO-NT-012, which reduced the effects of fluprostenol to 32% of control. Moreover, some neurones responsive to PGE2 did not respond to fluprostenol. Patch-clamp analysis in SON slice preparations revealed that PGE2 at 10(-6) M depolarized the membrane potential by 3.9+/-0.3 mV from the resting membrane potential of -58.4+/-2.2 mV in the current-clamp mode. In the voltage-clamp mode, PGE2 induced inward currents at a holding potential of -70 or -80 mV, while it did not affect spontaneous excitatory postsynaptic currents. PGE2 induced currents also in dissociated SON neurones and the reversal potential of the currents was -35.5+/-0.9 mV, which was similar to that of currents induced by fluprostenol. These results suggest that SON neurones possess at least two types of PG receptors, FP receptors and EP receptors of a subclass different from EP1, EP2, or EP3, and that activation of these receptors leads to the opening of nonselective cation channels, membrane depolarization and increase of the action potential discharge.

It has been shown that noradrenergic activation has great influence on the activities of hypothalamic supraoptic neurons. No direct evidence has been reported on the presynaptic effects of adrenoceptors in the actions of noradrenaline on supraoptic neurons, although postsynaptic mechanisms have been studied extensively. In the present study, we explored presynaptic effects of noradrenaline on the supraoptic neurons by measuring spontaneous inhibitory postsynaptic currents (IPSC) with the whole-cell patch-clamp technique. Noradrenaline reduced the frequency of IPSCs in a dose-dependent (10(-9) to 10(-3) M) and reversible manner. Noradrenaline did not affect the amplitude of IPSCs at concentrations of 10(-9) to 10(-5) M, but reduced the amplitude of IPSCs at high concentrations (10(-4) and 10(-3) M). The inhibitory effects of noradrenaline were mimicked by the alpha2-agonist clonidine (10(-4) M), but not by the alpha1-agonist methoxamine (10(-4) M) nor by the beta-agonist isoproterenol (10(-4) M). Moreover, the inhibitory effects of noradrenaline on IPSCs were blocked by the non-selective alpha antagonist phentolamine (10(-4) M) or the selective alpha2-antagonist yohimbine (10(-4) M), but not by the alpha1-antagonist prazosin (10(-4) M). These results suggest that noradrena-line inhibits release of GABA from the presynaptic GABAergic terminals of the supraoptic neurons by activating presynaptic alpha2-adrenoceptors and such presynaptic mechanisms may play a role in the excitatory control of SON neurons by noradrenergic neurons., (Copyright 1998 Elsevier Science B.V.)

In neurosecretory cells of the supraoptic nucleus (SON) of rats, pituitary adenylate cyclase activating polypeptide (PACAP) causes an increase in [Ca2+]i, and stimulates somatodendritic vasopressin (VP) release. In this report, to elucidate the ionic mechanism of the action of PACAP, membrane potentials and ionic currents were measured from SON neurones in slice preparations or from dissociated SON neurones. In the current clamp mode, PACAP depolarized membrane potentials of both phasic and non-phasic neurones and increased the firing rate. Moreover, simultaneous measurements of membrane potentials and [Ca2+]i revealed that the membrane depolarization correlated well with increases in [Ca2+]i. In the voltage-clamp mode, PACAP induced inward currents at a holding potential of -70 or -80 mV in a dose-dependent manner and the time course of the currents was similar to that of the PACAP-induced membrane depolarization. The averaged reversal potential of the PACAP-induced currents obtained from dissociated SON neurones was -33 mV, which was close to the reversal potential of non-selective cation currents in SON neurones. The currents were rapidly and reversibly inhibited by a cation-channel blocker, gadolinium. Analysis of synaptic inputs into SON neurones in slice preparations revealed that PACAP had little or no effects on the frequency of spontaneous excitatory and inhibitory postsynaptic currents. These results suggest that pituitary adenylate cyclase activating polypeptide (PACAP) activates PACAP receptors in the postsynaptic membrane of the supraoptic nucleus (SON) neurones, and that the activation of PACAP receptors leads to opening of non-selective cation channels, depolarization of the membrane potential, and increase in the firing rate in SON neurones. Such mechanisms may account for the PACAP-induced increase in [Ca2+]i and vasopressin (VP) release observed in SON neurones.

1. Voltage-dependent Ca2+ currents of dissociated rat supraoptic nucleus (SON) neurones were measured using the whole-cell configuration of the patch-clamp technique to examine direct postsynaptic effects of GABAB receptor activation on SON magnocellular neurones. 2. The selective GABAB agonist baclofen reversibly inhibited voltage-dependent Ca2+ currents elicited by voltage steps from a holding potential of -80 mV to depolarized potentials in a dose-dependent manner. The ED50 of baclofen for inhibiting Ca2+ currents was 1.4 x 10-6 M. Baclofen did not inhibit low threshold Ca2+ currents elicited by voltage steps from -120 to -40 mV. 3. Inhibition of high threshold Ca2+ currents by baclofen was rapidly and completely reversed by the selective GABAB antagonists, CGP 35348 and CGP 55845A, when the antagonists were added at the molar ratio vs. baclofen of 10 : 1 and 0.01 : 1, respectively. It was also reversed by a prepulse to +150 mV lasting for 100 ms. 4. The inhibition of Ca2+ currents was abolished when the cells were pretreated with pertussis toxin for longer than 20 h or with N-ethylmaleimide for 2 min. It was also abolished when GDPbetaS was included in the patch pipette. When GTPgammaS was included in the patch pipette, baclofen produced irreversible inhibition of Ca2+ currents and this inhibition was again reversed by the prepulse procedure. 5. The inhibition of N-, P/Q-, L- and R-type Ca2+ channels by baclofen (10-5 M) was 24.1, 10.5, 3.1 and 3. 6 %, respectively, of the total Ca2+ currents. Only the inhibition of N- and P/Q-types was significant. 6. These results suggest that GABAB receptors exist in the postsynaptic sites of the SON magnocellular neurones and mediate selective inhibitory actions on voltage-dependent Ca2+ channels of N- and P/Q-types via pertussis toxin-sensitive G proteins, and that such inhibitory mechanisms may play a role in the regulation of SON neurones by the GABA neurones.

In the present study, the effects of glutamate and of agonists for ionotropic and metabotropic glutamate receptors on intracellular Ca2+ concentration ([Ca2+]i) were investigated in neurons of the rat supraoptic nucleus (SON). We used the intracellular Ca2+ imaging technique with fura-2, in single magnocellular neurons dissociated from the SON of rats. Glutamate (10(-6)-10(-4) M) evoked a dose-dependent increase in [Ca2+]i. The glutamate agonists exerted similar effects, although with some differences in the characteristics of their responses. The [Ca2+]i response to NMDA was smaller than those of glutamate or the non-NMDA receptor agonists, AMPA and kainate, but was significantly enhanced by the removal of extracellular Mg2+. Glutamate, as well as quisqualate, an agonist for both ionotropic and metabotropic glutamate receptors, evoked a [Ca2+]i increase in a Ca2+-free condition, suggesting Ca2+ release from intracellular Ca2+ stores. This was further evidenced by [Ca2+]i increases in response to a more selective metabotropic glutamate receptor agonist, t-ACPD, in the absence of extracellular Ca2+. Furthermore, the quisqualate-induced Ca2+ release was abolished by the selective metabotropic glutamate receptor antagonist, (S)-4-carboxyphenylglycine. The results suggest that metabotropic glutamate receptors as well as non-NMDA and NMDA receptors are present in the SON neurons, and that activation of the first leads to Ca2+ release from intracellular Ca2+ stores and the activation of the latter two types induces Ca2+ entry. These dual mechanisms of Ca2+ signalling may play a role in the regulation of SON neurosecretory cells by glutamate.

Pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity and its receptor mRNA have been reported in the supraoptic and the paraventricular nucleus (SON and PVN, respectively) and PACAP has been implicated in the regulation of magnocellular neurosecretory cell function. To examine the site and the mechanism of the action of PACAP in the neurosecretory cells, we measured AVP release from SON slice preparations and the cytosolic Ca2+ concentration ([Ca2+]i) from single dissociated SON neurons. PACAP at concentrations from 10(-12) to 10(-7) M increased [Ca2+]i in dissociated SON neurons in a dose-dependent manner. The patterns of the PACAP-induced [Ca2+]i increase were either sustained increase or cytosolic Ca2+ oscillations. PACAP (10[-7] M) increased [Ca2+]i in 27 of 27 neurons and glutamate (10[-4] M) increased [Ca2+]i in 19 of 19 SON neurons examined, whereas angiotensin II (10[-7] M) increased [Ca2+]i in only 15 of 60 SON neurons examined. PACAP at lower concentrations (10[-10] to 10[-8] M) increased [Ca2+]i in 70-80% of neurons examined. Although the onset and recovery of the PACAP-induced [Ca2+]i increase were slower than those observed with glutamate, the spatial distribution of the [Ca2+]i increases in response to the two ligands were similar: [Ca2+]i increase at the proximal dendrites was larger and faster and that at the center of the soma was smaller and slower. The PACAP-induced [Ca2+]i responses were abolished by extracellular Ca2+ removal, the L-type Ca2+-channel blocker, nicardipine, or by replacement of extracellular Na+ with N-methyl D-glucamine, and were partially inhibited by the Na+-channel blocker, tetrodotoxin. The N-type Ca2+-channel blocker, omega-conotoxin GVIA did not significantly inhibit the PACAP-induced [Ca2+]i responses. Furthermore, PACAP (10[-7] M) as well as glutamate (10[-4] M) increased AVP release from SON slice preparations, and extracellular Ca2+ removal or nicardipine inhibited the AVP release in response to PACAP. These results indicate that PACAP enhances Ca2+ entry via voltage-gated Ca2+ channels and increases [Ca2+]i, which, in turn, stimulates somatodendritic vasopressin release by directly activating PACAP receptors on SON neurons. The results also suggest that PACAP in the SON may play a pivotal role in the control of the neurohypophyseal function at the level of the soma or the dendrites.

We investigated the expression of neuronal nitric oxide synthase (nNOS) gene in the paraventricular (PVN) and supraoptic nuclei (SON) of rats with inherited diabetes insipidus (DI), using in situ hybridization histochemistry. The DI rats showed hypo-osmotic polyuria and polydipsia with arginine vasopressin (AVP) deficiency. The expression of nNOS gene in the PVN and SON in homozygous (di/di) rats was significantly increased in comparison to normal Wistar and heterozygous (di/+) rats. nNOS gene-expressing cells were distributed throughout the PVN and SON, including the divisions of AVP and oxytocin gene expressing cells in di/di rats. These results suggest that the expression of nNOS gene is upregulated in the magnocellular neurons in the PVN and SON of inherited DI rats.

1. The function of presynaptic GABA receptors in the regulation of transmitter release in supraoptic nucleus (SON) magnocellular neurons was investigated by recording spontaneous postsynaptic currents from rat magnocellular SON neurons in a slice preparation (150 microns thick, 1.8 mm in diameter) using the whole-cell patch-clamp technique. 2. Both the spontaneous EPSCs and IPSCs were TTX resistant. The EPSCs were abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), whereas the IPSCs were abolished by picrotoxin, suggesting that the EPSCs and IPSCs are synaptic inputs from glutamatergic and GABAergic neurons, respectively. 3. The selective GABAB agonist, baclofen, reduced the frequency of both the EPSCs and IPSCs without affecting the amplitude. The time constant of the decay phase of both the EPSCs and IPSCs remained unchanged after baclofen application. 4. The reduction of the frequency of the synaptic currents by baclofen was dose dependent (10 nM to 100 microM) and the EC50 values were 5.8 and 8.5 microM for the EPSCs and IPSCs, respectively. 5. The effect of baclofen (10 microM) was antagonized by the selective GABAB antagonist, 2-hydroxy-saclofen (2OH-saclofen), at 300 microM. 6. When given alone, 2OH-saclofen (100 microM) increased the frequency of both the EPSCs and IPSCs without affecting their amplitude, suggesting that endogenously released GABA in the slice acts on presynaptic GABAB receptors. 7. The GABAA agonist, muscimol, reduced the frequency of EPSCs, and picrotoxin increased the frequency of the EPSCs, suggesting that GABAA receptors also participate in the presynaptic inhibition of glutamate release. 8. Taken together, these data suggest that GABAB receptors are present on the presynaptic terminals of both GABA and glutamate neurons in the SON, and that these presynaptic GABAB receptors play an important role in the regulation of the neuronal activity in SON magnocellular neurons.

The hypothalamus is known to be an integrative site of cardiovascular, endocrine and autonomic functions. Our previous studies, using extracellular, intracellular and/or whole cell patch-clamp recordings in rat hypothalamic slice preparations, revealed that cardiovascular related peptides such as atrial natriuretic polypeptides (ANP), B-type polypeptides (BNP), endothelin (ET), angiotensin II (AII) and interleukin-1 beta (IL-1 beta) influence the hypothalamic neurons. ANP modulated the firing rates in the supraoptic nucleus (SON). BNP inhibited the SON neurons and these effects were mediated through cGMP and cGMP-dependent protein kinase. ET also inhibited approximately 60% of SON neurons. By using slice patch-clamp techniques, AII inhibited the transient outward potassium current in the SON neurons. IL-1 beta increased the firing rate and depolarized the membrane of the most SON neurons. A new type of transmitter, nitric oxide (NO), identified as an endothelial-derived relaxing factor (EDRF), modulated the glutaminergic inputs of the SON neurons. The results suggest that cardiovascular related peptides and NO modulate the neuronal activity of neurosecretory cells in the SON.

Abstract We have reported that supraoptic nucleus (SON) neurones are excited by prostaglandin E2 (PGE2 ) presumably via dual postsynaptic PG receptors, FP receptors and unidentified EP receptors, and that presynaptic EP receptors may also be involved in the excitation. In the present study, to clarify the receptor mechanism of the PGE2 -mediated actions on SON neurones, we studied the pre- and postsynaptic effects of four newly developed EP agonists that are selective for each of the four EP receptors, EP1-4 , on rat SON neurones using extracellular recording and whole-cell patch-clamp techniques. The EP4 agonist ONO-AE1-329 mimicked the excitatory effects of PGE2 , whereas the EP1 agonist ONO-DI-004, the EP2 agonist ONO-AE1-257 and the EP3 agonist ONO-AE-248 had little or no effect. The effects of ONO-AE1-329 were unaffected by the EP1 /FP/TP antagonist, ONO-NT-012, which potently suppressed the excitation caused by the FP agonist fluprostenol and PGE2 . ONO-AE1-329 caused marked excitation when responses to fluprostenol were desensitized by repeated applications of fluprostenol. Patch-clamp analysis in SON neurones showed that ONO-AE1-329 induced inward currents at a holding potential of -70 mV and the reversal potential of the currents was -35.1 ± 2.3 mV. On the other hand, the frequency of spontaneous inhibitory postsynaptic currents recorded from SON slice preparations was suppressed by ONO-AE-248, but unaffected by the other three EP agonists. These results suggest that SON neurones possess postsynaptic EP4 receptors and that γ-aminobutyric acid neurones innervating SON neurones possess presynaptic EP3 receptors in their terminals. Activation of the two EP receptors may be involved in the excitatory regulation of SON neurones by PGE2 . [ABSTRACT FROM AUTHOR]