Eiji Shigetomi

Action Potential-Independent Release of Glutamate by Ca2+ Entry through Presynaptic P2X Receptors Elicits Postsynaptic Firing in the Brainstem Autonomic Network

P2X receptors are ATP-gated channels permeable to cations including Ca2+. In acute slices containing the nucleus of the solitary tract, in which neuronal ATP release and ATP-elicited physiological responses are demonstrated in vivo, we recorded spontaneous action potential-independent EPSCs [miniature EPSCs (mEPSCs)]. Activation of presynaptic P2X receptors with α,β-methylene ATP (αβmATP) triggered Ca2+-dependent glutamate release that was resistant to blockade of voltage-dependent calcium channels but abolished by P2X receptor antagonists. mEPSCs elicited with αβmATP were of larger amplitude than basal mEPSCs and resulted in postsynaptic firing caused by temporal summation of miniature events. The large-amplitude mEPSCs provoked by αβmATP were likely to result from highly synchronized multivesicular release of glutamate at single release sites. Neither αβmATP nor ATP facilitated GABA release. We conclude that this facilitated release and consequent postsynaptic firing underlie the profound autonomic responses to activation of P2X receptors observed in vivo.

Distinct modulation of evoked and spontaneous EPSCs by purinoceptors in the nucleus tractus solitarii of the rat

1 Whole‐cell transmembrane currents of second‐order neurones in the caudal part of the nucleus tractus solitarii (cNTS) of brainstem slices of the rat were recorded to analyse the effects of adenosine 5′‐triphosphate (ATP) on: (1) EPSCs evoked by the solitary tract stimulation (eEPSCs) and (2) spontaneous EPSCs (sEPSCs). 2 ATP (10−6 to 10−4 m) significantly reduced the amplitude of eEPSCs to 46·6 ± 7·4 % and increased the frequency of sEPSCs to 268·0 ± 71·5 % of the control without significant changes in sEPSC amplitude. These opposite effects of ATP on eEPSCs and sEPSCs were concurrently observed in about 80 % of cNTS neurones recorded. 3 The reduction of eEPSC amplitude by ATP was similarly observed with the addition of an equimolar solution of adenosine but not with α,β‐methylene ATP and was suppressed by 8‐cyclopentyltheophylline (CPT) and 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX). Addition of pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) did not affect the reduction of eEPSC amplitude by ATP. 4 The increase in sEPSC frequency by ATP remained under tetrodotoxin addition but was abolished in the presence of PPADS. 5 It is suggested that ATP activates: (1) presynaptic adenosine A1 receptors, after being hydrolysed to adenosine, reducing evoked release of glutamate from the primary afferent terminals and (2) presynaptic P2X receptors on the axon terminals of intrinsic excitatory cNTS neurones facilitating spontaneous release of glutamate. This is the first evidence that ATP modulates excitatory synaptic inputs arising from distinct origins and converging on a single postsynaptic neurone in diametrically opposite directions through activation of distinct presynaptic purinoceptors.

Facilitation of spontaneous glycine release by anoxia potentiates NMDA receptor current in the hypoglossal motor neurons of the rat

Deficiency in energy supply, such as occurs during hypoxia, anoxia, metabolic stress and mitochondrial failure, strongly affects the excitability of central neurons. Such lowered energy supply evokes various changes in spontaneous synaptic input to the hippocampal and cortical neurons. However, how this energy deprivation affects synaptic input to motor neurons, which are also vulnerable to energy deprivation, has never been addressed. Here we report for the first time the effect of metabolic stress on synaptic input to motor neurons by recording postsynaptic currents in the hypoglossal nucleus. Chemical anoxia with NaCN (1 mm) and anoxia with 95% N2 induced a persistent inward current and a marked and robust increase in action potential‐independent synaptic input. This increase was abolished by strychnine, but not by picrotoxin, CNQX or MK‐801, indicating glycine release facilitation. Blockade of voltage‐dependent Ca2+ channels and extracellular Ca2+ deprivation strongly attenuated this facilitation. The amplitude of inward currents evoked by local application of NMDA to the motor neurons in the presence of strychnine was significantly increased during NaCN application. A saturating concentration of d‐serine occluded this potentiation, suggesting that released glycine activated the glycine‐binding sites of NMDA receptors. By contrast, neurons in the dorsal motor nucleus of the vagus showed no detectable change in synaptic input in response to NaCN. These data suggest that increase in synaptically released glycine in response to metabolic stress may play an exacerbating role in NMDA receptor‐mediated excitotoxicity in motor neurons.

Differential increases in P2X receptor levels in rat vagal efferent neurones following a vagal nerve section.

Extracellular ATP can influence cells via activation of P2X purinoceptors, the distribution of which can be altered in the central and peripheral nervous systems following injury or tissue damage. Here we have investigated the effect of a unilateral section of the cervical vagus nerve on the distribution of P2X(1), P2X(2), P2X(3), P2X(4) and P2X(7) receptor subunit immunoreactivity (R-IR) in the dorsal vagal motor nucleus (DVN) and the nucleus ambiguus (NA) in the medulla oblongata. As early as 2 days, and followed up to 14 days, there was a dramatic ipsilateral increase in P2X(1), P2X(2) and P2X(4)R-IR in the cell soma of vagal efferent neurones in the DVN following the nerve section, but not the NA. There were no changes in P2X(3) and P2X(7)R-IR in either nuclei. To test for possible functional consequences of increased P2X receptor levels, whole-cell patch-clamp recordings were made from DVN cells in brainstem slices 4 days following unilateral vagotomy. Application of ATP revealed large cell-to-cell variance in the current amplitude in neurones from both sectioned and control DVN. However, when ATP responses were compared to those elicited by the nicotinic acetylcholine receptor agonist carbachol, the mean ratio of the peak ATP-evoked current to the peak carbachol-evoked current was significantly larger in DVN neurones ipsilateral to the section. Thus the increase in P2XR levels in DVN cells ipsilateral to a nerve section are likely to reflect an increase in expression of functional P2XRs on the cell surface.

Blocker-resistant presynaptic voltage-dependent Ca2+ channels underlying glutamate release in mice nucleus tractus solitarii

The visceral sensory information from the internal organs is conveyed via the vagus and glossopharyngeal primary afferent fibers and transmitted to the second-order neurons in the nucleus of the solitary tract (NTS). The glutamate release from the solitary tract (TS) axons to the second-order NTS neurons remains even in the presence of toxins that block N- and P/Q-type voltage-dependent Ca(2+) channels (VDCCs). The presynaptic VDCC playing the major role at this synapse remains unidentified. To address this issue, we examined two hypotheses in this study. First, we examined whether the remaining large component occurs through activation of a omega-conotoxin GVIA (omega-CgTX)-insensitive variant of N-type VDCC by using the mice genetically lacking its pore-forming subunit alpha(1B). Second, we examined whether R-type VDCCs are involved in transmitter release at the TS-NTS synapse. The EPSCs evoked by stimulation of the TS were recorded in medullary slices from young mice. omega-Agatoxin IVA (omega-AgaIVA; 200 nM) did not significantly affect the EPSC amplitude in the mice genetically lacking N-type VDCC. SNX-482 (500 nM) and Ni(2+) (100 microM) did not significantly reduce EPSC amplitude in ICR mice. These results indicate that, unlike in most of the brain synapses identified to date, the largest part of the glutamate release at the TS-NTS synapse in mice occurs through activation of non-L, non-P/Q, non-R, non-T and non-N (including its posttranslational variants) VDCCs at least according to their pharmacological properties identified to date.

A dual-role played by extracellular ATP in frequency-filtering of the nucleus Tractus solitarii network.

ATP is now identified to be an important signaling molecule in the CNS1, 2, 3. However, there are only few brain regions in which the function of ATP-mediated signaling is demonstrated from the molecular to whole animal levels. The caudal part of the nucleus of the solitary tract (cNTS) is such a rare structure. In the cNTS, neuronal ATP release4,5, hypoxia-induced increase in purine concentration6, abundant expression of P2X and P2Y receptors7, 8, 9, 10, 11, 12, extracellular hydrolysis of ATP to adenosine4,13, and rich expression of adenosine transporters14 have been demonstrated. In addition, a microinjection of agonists for P2X and adenosine receptors into cNTS in anesthetized rats exerts profound cardiorespiratory effects15, 16, 17. Taken together, ATP, in tandem with its extracellular metabolite adenosine, is thought to be involved in the neuronal signaling in the cNTS, where various visceral signals including those from pulmonary stretch receptors and peripheral chemoreceptor converge.

S13-3. Glial dysfunction and eplileptogenicity

Temporal lobe epilepsy (TLE) is the most common type of drug-resistant epilepsy, partly characterized by hippocampus sclerosis. Recent accumulating evidence show that changes in glial cells are obvious in TEL, but an involvement of glial changes in TLE is still a matter of debate. Here, we show that status epilepticus (SE) induced activation of microglia, followed by induction of “epileptogenic astrocytes” in the hippocampus. Pilocarpine (Pilo) was used to induce SE in male adult B6 mice. Morphological and functional changes in glia were assessed by immunohistochemical analysis and Ca2+ imaging in the hippocampal slices, respectively. Pilo increased a fast and transient microglial activation (1–3 days after SE), which was followed by sustained activation of astrocytes in the hippocampus (7–28 days after SW). Twenty-eight days after SE, the mice showed susceptibility to Pilo, suggesting induction of epileptogenicity. At this time point, reactive astrocytes displayed excess Ca2+ excitability, reduction of which resulted in inhibition of epileotogenicity. Taken together, after SE, astrocytes become “epileptogenic astrocytes”, whose aberrant Ca2+ excitability should be a cause of epileptogenicity.

Visualization of diversity of calcium signals in astrocytes.

新 し い グ リ ア 細 胞 機 能 : 新 規 可 視 化 ・ 操 作 法 に よ り わ か っ た グ リ ア の 新 機 能 3 要約:アストロサイトは,中枢神経系全般に存在し, シナプスや血管と密接に接触することから神経活動に 必要な栄養を供給し,またその栄養を運ぶ血管を制御 して血流を調節すると考えられている.最近の研究に より,アストロサイトは,神経構造を維持するのみな らず,積極的に神経構造及び神経活動に影響を与える ことが明らかになりつつある.アストロサイトは神経 細胞と異なり顕著な電気的なシグナルを発生しないが, Caシグナルなどの化学的なシグナルを生じて活動 すると考えられている.過去 20 年以上の研究の歴史 の中で,アトロサイトは様々な刺激に応じて活動依存 的にCaシグナルを生じ,これによってほかの脳細胞 と情報伝達することが示されてきた.近年開発された 可視化技術により,Caシグナルがより詳細に解析さ れ,その驚くべき多様性が示されてきた.さらに覚醒 下行動中の動物を用いた解析により,アストロサイト の Caシグナルと動物の行動との関連性が明らか となりつつある.病態時においてはアストロサイト Caシグナルが劇的に変調しこれが病態の一因とな る可能性も示されてきている.本稿では,近年開発さ れたアストロサイト Caシグナルの可視化技術に焦 点をあて,アストロサイトのCaシグナルが脳の健康 と病態にどう関係しているのか,その最新の知見を紹 介したい.