Toshihisa Nagatomo

Transient outward current in adult rat supraoptic neurones with slice patch‐clamp technique: inhibition by angiotensin II.

1. Outward potassium currents were recorded from microscopically identified supraoptic neurones of adult Wistar male rats using the whole‐cell patch‐clamp technique in thin‐slice preparations. The basic characteristics of transient outward current (IA or A‐current) and the effects of angiotensin II (AII) on the currents were studied. 2. IA was isolated by subtracting outward currents elicited by stepping from two different holding potentials to a test potential or by applying 4‐aminopyridine (4‐AP) at 5 mM. The isolated IA had a threshold for activation between ‐55 and ‐65 mV and was characterized by fast activation and inactivation. Values of the time to peak and the inactivation time constants for current decay at different test potentials were voltage dependent. 3. Normalized currents for activation and steady‐state inactivation of IA were fitted to the Boltzmann function. The mid‐points and the slope factors were, respectively, ‐35.0 and ‐14.3 +/‐ 0.40 mV (n = 5) for the activation curve, and ‐72.0 and 7.0 +/‐ 0.68 mV (n = 5) for the inactivation curve. 4. The time course of recovery from inactivation was best fitted to a single exponential function with the time constant of 37.8 +/‐ 6.6 ms (n = 6). 5. The effects of AII on IA and delayed rectifier current (IK) were investigated. According to their responses to AII, cells were classified into two groups, sensitive and low‐sensitive. Bolus injection of AII (10 microM, 100 microliters) decreased the IA amplitude by 25.1 +/‐ 2.4% in seven (53.8%) of the thirteen neurones tested (sensitive group), whereas the other six neurones (low‐sensitive group) changed by only 2.2 +/‐ 0.8%. Perfusion of AII (0.1 microM) decreased the IA amplitude by 21.3 +/‐ 3.1% in six (54.5%) of eleven neurones tested (sensitive group), whereas the other five neurones (low‐sensitive group) changed only by 1.7 +/‐ 0.8%. Bolus injection of AII (10 microM, 100 microliters) decreased the IK amplitude 9.6 +/‐1.6% mV in five (45.5%) of the eleven neurones tested (sensitive group), whereas the other six neurones (low‐sensitive group) changed only by 0.46 +/‐ 0.27%. In the sensitive groups, the reduction of IA by AII was significantly larger than that of IK (P < 0.05). 6. Application of saralasin at 1 microM, an AII antagonist, blocked the effects of AII on IA. 7. These results suggest that the excitatory action of AII on supraoptic neurosecretory cells is mediated at least in part through suppression of IA.

Kappa-selective agonists decrease postsynaptic potentials and calcium components of action potentials in the supraoptic nucleus of rat hypothalamus in vitro.

To investigate the effects of the endogenous kappa-receptor agonists dynorphin and leumorphin on neurons of the supraoptic nucleus in the rat hypothalamus, intracellular recordings were made from 62 supraoptic neurons in slice preparations. Bath application of dynorphin and leumorphin at 10(-7) M to 3 x 10(-6) M decreased the spontaneous firing rate with slight hyperpolarization of the membrane potential (-3.8 +/- 0.5 mV, mean +/- S.E.M.) but did not detectably change input resistance. The inhibitory effects were blocked by the relatively selective kappa-antagonist MR-2266. The synthetic kappa-receptor agonist U-50,488H had similar inhibitory effects on supraoptic neurons. Postsynaptic potentials evoked by electrical stimulation dorsal or dorsolateral to the supraoptic nucleus were suppressed by dynorphin and leumorphin. Morphine and [D-Ala, D-Leu]enkephalin, which are relatively selective to mu- and delta-receptors, respectively, influenced the postsynaptic potentials less. Dynorphin and leumorphin also decreased the duration of action potentials that were prolonged by either bath application of tetraethylammonium chloride at 5-10 mM or intracellular injection of Cs ions from the recording electrodes which were filled with 3 M cesium citrate. The prolongation was blocked by 1 mM MnCl2 and 2 mM CoCl2, which suggested that the components were due to voltage-dependent Ca2+ influx. The results suggest that endogenous kappa-receptor agonists inhibit neurosecretory cells of the supraoptic nucleus to suppress synaptic events and Ca2+ components of action potentials.

Sodium nitroprusside modulates NMDA response in the rat supraoptic neurons in vitro

The modulatory effects of NO on N-methyl-D-aspartate (NMDA)-induced response in neurons of the supraoptic nucleus (SON) were studied by intracellular recording and radioimmunoassay of cyclic nucleotides using the rat brain slice preparation. Depolarization induced by 100 microM NMDA was reduced by application of 1 to 3 mM of the NO-donors, sodium nitroprusside, and isosorbide dinitrate in all 8 neurons and in 6 of 10 neurons, respectively. The scavenger for NO, hemoglobin, and the inhibitor of NO synthase, NG-nitro-L-arginine (LNNA) enhanced the NMDA-induced depolarization in four neurons and two of three neurons, respectively. Intracellular cGMP accumulation induced by NMDA was significantly diminished by LNNA. However, NMDA-induced depolarization was not affected by either the protein kinase inhibitor, N-[2-(methylamino)ethyl]-5- isoquinolinesulfonamide dihydrochloride (H-8), or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). These results indicate that NO reduces NMDA-induced depolarization in a manner that is independent of cGMP and may control the activity of the SON neurons through NMDA receptors.

Inward sodium current involvement in regenerative bursting activity of rat magnocellular supraoptic neurones in vitro.

1. The rat hypothalamic slice preparation was used to investigate the involvement of inward Na+ currents as well as inward Ca2+ currents in the generation of bursting activity by supraoptic (SON) neurones. Intracellular records were made from thirty‐two SON neurones which showed regenerative bursting activity. The bursting activity consisted of spontaneous, intermittent bursts of action potentials with subsequent silent periods. During the bursts, plateau potentials on which action potentials were superimposed were frequently observed. 2. Perfusion of a low‐Na+ medium, a tetrodotoxin (TTX)‐containing medium or a Ca(2+)‐free medium suppressed the regenerative bursting activity. 3. Addition of 3‐10 microM veratridine to Ca(2+)‐free medium elicited regenerative bursting activity and spontaneous plateau potentials. The veratridine‐induced regenerative bursting activity and plateau potentials were blocked by 1 microM TTX. Addition of 5 mM TEA allowed regenerative bursting activity to persist in Ca(2+)‐free medium. 4. These results suggest that TTX‐sensitive Na+ inward currents as well as Ca2+ inward currents contribute to the generation of bursting activity in rat SON cells.

OSMOTIC MODULATION IN GLUTAMATERGIC EXCITATORY SYNAPTIC INPUTS TO NEURONS IN THE SUPRAOPTIC NUCLEUS OF RAT HYPOTHALAMUS IN VITRO

To clarify influence of osmotic stimulation on the excitatory synaptic inputs to the neurosecretory cells of the supraoptic nucleus (SON), the blind patch technique was used in rat hypothalamic slice preparations. Stable whole‐cell recordings were made from 22 neurons in the SON. To observe spontaneous excitatory postsynaptic currents (sEPSCs) in the SON neurons, membrane potentials were clamped between −50 and −90 mV. The effects of hypertonic stimulation on the frequency of the sEPSCs were tested in 18 SON neurons. Bath application of mannitol 30 or 60 mM increased the frequency of the sEPSCs. During the application of mannitol (60 mM), the frequency of the sEPSCs increased in 12 of 15 neurons without a change in amplitude. Hypertonic stimulation with NaCl (30 mM) had similar effects to that of mannitol. The increased frequency of miniature EPSCs (mEPSCs) during mannitol application persisted in the presence of TTX in all 8 SON neurons tested with no change in amplitude. Both the non‐NMDA antagonist CNQX at 10–30 μM (n=6) and the non‐selective glutamate antagonist kynurenic acid at 1 mM (n=3) almost completely blocked the EPSCs while the NMDA antagonist AP‐5 at 10 μM had no effect on the frequency of the EPSCs in the 4 neurons tested. During application of CNQX, mannitol (60 mM) was added to the perfusion medium in 3 SON neurons. Under these conditions, mannitol had no effect on the frequency of EPSCs. We conclude that hypertonic stimulation directly influences glutamatergic inputs to the neurosecretory cells of the SON by an action on the presynaptic terminals and enhances the excitatory synaptic events.

GABAergic inhibitory inputs to subfornical organ neurons in rat slice preparations

To investigate GABAergic inhibitory inputs to neurons of the subfornical organ (SFO), intracellular recordings were made in rat brain slice preparations. Inhibitory postsynaptic potentials, which occurred spontaneously or were evoked by focal electric stimulation, had reversal potentials of approximately -60 mV, and were almost totally abolished by the GABAA antagonists bicuculline at 3-100 microM or picrotoxin at 50 microM. Following the application of bicuculline or picrotoxin, the resting membrane potentials were decreased by 4-8 mV. GABA at 10-100 microM and the GABAA agonist muscimol at 1-100 microM decreased the membrane resistance and the firing rate in all neurons tested. The reversal potential of the response to muscimol was similar to that for inhibitory postsynaptic potentials. The actions of muscimol persisted in the presence of 1 microM tetrodotoxin, implying that muscimol must act directly on the recorded neurons. These results suggest that there is a tonic inhibitory GABAergic input to SFO neurons which are mainly mediated through GABAA receptors.

Allyl isothiocyanates and cinnamaldehyde potentiate miniature excitatory postsynaptic inputs in the supraoptic nucleus in rats

Allyl isothiocyanates (AITC) and cinnamaldehyde are pungent compounds present in mustard oil and cinnamon oil, respectively. These compounds are well known as transient receptor potential ankyrin 1 (TRPA1) agonists. TRPA1 is activated by low temperature stimuli, mechanosensation and pungent irritants such as AITC and cinnamaldehyde. TRPA1 is often co-expressed in TRPV1. Recent study showed that hypertonic solution activated TRPA1 as well as TRPV1. TRPV1 is involved in excitatory synaptic inputs to the magnocellular neurosecretory cells (MNCs) that produce vasopressin in the supraoptic nucleus (SON). However, it remains unclear whether TRPA1 may be involved in this activation. In the present study, we examined the role of TRPA1 on the synaptic inputs to the MNCs in in vitro rat brain slice preparations, using whole-cell patch-clamp recordings. In the presence of tetrodotoxin, AITC (50μM) and cinnamaldehyde (30μM) increased the frequency of miniature excitatory postsynaptic currents without affecting the amplitude. This effect was significantly attenuated by previous exposure to ruthenium red (10μM), non-specific TRP channels blocker, high concentration of menthol (300μM) and HC-030031 (10μM), which are known to antagonize the effects of TRPA1 agonists. These results suggest that TRPA1 may exist at presynaptic terminals to the MNCs and enhance glutamate release in the SON.

TRPV1 Gene Deficiency Attenuates Miniature EPSC Potentiation Induced by Mannitol and Angiotensin II in Supraoptic Magnocellular Neurons

The release of arginine vasopressin (AVP) from the magnocellular neurosecretory cells (MNCs) in the supraoptic nucleus (SON) is crucial for body fluid homeostasis. The MNC activity is modulated by synaptic inputs and humoral factors. A recent study demonstrated that an N-terminal splice variant of the transient receptor potential vanilloid type 1 (TRPV1) is essential for osmosensory transduction in the SON. In the present study, we examined the effects of mannitol and angiotensin II on miniature EPSCs (mEPSCs) in the supraoptic MNCs using whole-cell patch-clamp recording in in vitro slice preparation. Mannitol (60 mm) and angiotensin II (0.1 μm) increased the frequency of mEPSCs without affecting the amplitude. These effects were attenuated by pre-exposure to a nonspecific TRPV channel blocker, ruthenium red (10 μm) and enhanced by pre-exposure to cannabinoid type1 receptor antagonist, AM251 (2 μm). Mannitol-induced potentiation of mEPSCs was not attenuated by angiotensin II receptor antagonist, losartan (10 μm), indicating independent pathways of mannitol and angiotensin II to the TRPV channels. The potentiation of mEPSCs by mannitol was not mimicked by a TRPV1 agonist, capsaicin, and also not attenuated by TRPV1 blockers, capsazepine (10 μm). PKC was involved in angiotensin II-induced potentiation of mEPSCs. The effects of mannitol and angiotensin II on the supraoptic MNCs in trpv1 knock-out mice were significantly attenuated compared with those in wild-type mice counterparts. The results suggest that hyperosmotic stimulation and angiotensin II independently modulate mEPSCs through capsaicin-insensitive TRPV1 channel in the presynaptic terminals of the SON.

Intracellular EGTA alters phasic firing of neurons in the rat supraoptic nucleus in vitro

To determine the function of intracellular free Ca2+ which is important in generating the phasic firing pattern characteristic of vasopressin neurons in the supraoptic nucleus (SON), we injected the highly specific Ca(2+)-chelating agent ethyleneglycol-bis-(beta-aminoethyl ether) N,N-tetraacetic acid (EGTA) into SON cells in the rat hypothalamic slice preparation. Intracellular recordings from 29 SON neurons which showed phasic firing were analyzed. Of the 29 SON neurons, 21 were recorded with microelectrodes filled with 3 M potassium acetate and 20 of the 21 neurons retained the phasic pattern more than 1 h after penetration by the electrode. Only one neuron lost phasic firing and fired randomly. By contrast, in all 8 neurons which were recorded with microelectrodes filled with 100 mM EGTA/2 M potassium acetate, phasic firing disappeared 10-80 min after penetration of the recording electrode although the neurons still showed spontaneous activity. These neurons also lost the after hyperpolarization and plateau potentials which followed bursting discharges. Our results suggest that intracellular free Ca2+ may play an important role in generating phasic firing.

Ghrelin Potentiates Miniature Excitatory Postsynaptic Currents in Supraoptic Magnocellular Neurones

Ghrelin is an orexigenic peptide discovered in the stomach as a ligand of the orphan G‐protein coupled receptor, and participates in the regulation of growth hormone (GH) release. Previous studies have demonstrated that ghrelin suppressed water intake and stimulated the secretion of arginine vasopressin in rats. We examined the effect of ghrelin on the excitatory synaptic inputs to the magnocellular neurosecretory cells (MNCs) in the supraoptic nucleus (SON) using whole‐cell patch‐clamp recordings in in vitro rat and mouse brain slice preparations. The application of ghrelin (10−7∼10−6 m) caused a significant increase in the frequency of the miniature excitatory postsynaptic currents (mEPSCs) in a dose‐related manner without affecting the amplitude. The increased frequency of the spontaneous EPSCs persisted in the presence of tetrodotoxin (1 μM). Des‐n‐octanoyl ghrelin (10−6 m) did not have a significant effect on the mEPSCs. The ghrelin‐induced potentiation of the mEPSCs was significantly suppressed by previous exposure to the transient receptor potential vanilloid (TRPV) blocker, ruthenium red (10 μM) and GH secretagougue type 1a receptor selective antagonist, BIM28163 (10 μm). The effects of ghrelin on the supraoptic MNCs in trpv1 knockout mice were significantly attenuated compared to those in wild‐type mice counterparts. These results suggest that ghrelin participates in the regulation of synaptic inputs to the MNCs in the SON via interaction with the GH secretagogue type 1a receptor, and that the TRPV1 channel may be involved in ghrelin‐induced potentiation of mEPSCs to the MNCs in the SON.