Ischemia But Not Anoxia Evokes Vesicular and Ca²⁺-Independent Glutamate Release In the Dorsal Vagal Complex In Vitro

Abstract

Whole cell recordings of fura-2 dialyzed vagal neurons of brain stem slices were used to monitor interstitial glutamate accumulation within the dorsal vagal complex. Anoxia produced a sustained outward current (60 pA) and a moderate [Ca(2+)](i) rise (40 nM). These responses were neither mimicked by [1S,3R]-1-aminocyclo-pentane-1, 3-dicarboxylic acid nor affected by Ca(2+)-free solution, 6-cyano-7-nitroquino-xaline-2,3-dione (CNQX), 2-amino-5-phosphonovalerate (APV), or tetrodotoxin. Anoxia or cyanide in glucose-free saline (in vitro ischemia) as well as ouabain or iodoacetate elicited an initial anoxia-like [Ca(2+)](i) increase that turned after several minutes into a prominent Ca(2+) transient (0.9 microM) and inward current (-1.8 nA). APV plus CNQX (plus methoxyverapamil) inhibited this inward current as well as accompanying spontaneous synaptic activity, and reduced the secondary [Ca(2+)](i) rise to values similar to those during anoxia. Each of the latter drugs delayed onset of both ischemic current and prominent [Ca(2+)](i) rise by several minutes and attenuated their magnitudes by up to 40%. Ca(2+)-free solution induced a twofold delay of the ischemic inward current and suppressed the prominent Ca(2+) increase but not the initial moderate [Ca(2+)](i) rise. Cyclopiazonic acid or arachidonic acid in Ca(2+)-free saline delayed further the ischemic current, whereas neither inhibitors of glutamate uptake (dihydrokainate, D,L-threo-beta-hydroxyaspartate, L-transpyrrolidone-2,4-dicarboxylate) nor the Cl(-) channel blocker 5-nitro-2-(3-phenylpropyl-amino) benzoic acid had any effect. In summary, the response to metabolic arrest is due to activation of ionotropic glutamate receptors causing Ca(2+) entry via N-methyl-D-aspartate receptors and voltage-activated Ca(2+) channels. An early Ca(2+)-dependent exocytotic phase of ischemic glutamate release is followed by nonvesicular release, not mediated by reversed glutamate uptake or Cl(-) channels. The results also show that glycolysis prevents glutamate release during anoxia.

Early Ischemia Enhances Action Potential-Dependent, Spontaneous Glutamatergic Responses in CA1 Neurons

“…Spontaneous release includes both AP-dependent and AP-independent components (Figure 1), with AP-dependent components comprising larger amplitudes than the AP-independent miniatures. In this paper, we first show that H/H caused an increase in the frequency, but not the amplitude, of AP-independent release (Figure 2), as shown by others in CA1 pyramidal neurons (Katchman and Hershkowitz, 1993), neocortical neurons (Fleidervish et al, 2001), and in brainstem cells (Kulik et al, 2000). Although it is possible that there are large-amplitude, TTX-resistant miniatures that contain several presynaptic vesicles, due to the sum of several quanta (Llano et al, 2000), we did not observe large-amplitude sEPSCs in TTX experiments.…”

“…The resting potential was À64.3±5.6 mV in the control and À63.0±5.3 mV during H/H; AP threshold was À42.2±1.1 mV in the control and À40.3 ± 3.0 mV in H/H; AP amplitude was 83.4±8.1 mV in the control and 83.4±4.1 mV in H/H. Although changes in input resistance could occur during longer (up to 6 mins) hypoxic/ischemic episodes (Zhang and Krnjevic, 1993;Fleidervish et al, 2001;Kulik et al, 2000), we did not observe any significant change in the input resistance (178.8±49.4 MO in the control and 183.1±51.1 MO in H/H, P > 0.05; Figure 4C) during the short H/H challenge. Therefore, the enhanced glutamatergic response during transient ischemia was not due to intrinsic property changes in the patched postsynaptic neurons.…”

Glutamate release by an Na+ load and oxidative stress in nerve terminals: relevance to ischemia/reperfusion

“…Real time glutamate measurement in an in vitro model of ischemia showed that glutamate uptake is inhibited within a few minutes of the onset of ischemia (Jabaudou et al 2000). The Na + -dependent glutamate transporter of the plasma membrane fails to remove glutamate under this condition, instead, by a reverse operation, it mediates the release of glutamate (Nicholls and Attwell 1990;Kulik et al 2000;Rossi et al 2000). The driving force for the reverse operation of the glutamate transporter is the increased intracellular Na + concentration resulting mainly from a persistent depolarization and Na + entry via non-inactivating Na + channels (see Urenjak and Obrenovitch 1996).…”

Involvement of Platelet-Derived Growth Factor–BB and Its Receptor-β in Hypoxia-Induced Depression of Excitatory Synaptic Transmission in the Nucleus Tractus Solitarius of Mice

“…Oxygen deprivation may activate cellular mechanisms that allow neurons to adapt to metabolic stress and to survive for a certain period. A shift in metabolic pathways (16), expression of cell-protective proteins (17), and changes in neuronal excitability through modulating ion channels (13,14) or neurotransmitter releases (18) are some of the survival strategies. This study suggests that the PDGF-BB / PDGFR-β signal is one neuroprotective strategy for cell survival against hypoxia by depressing the excitatory synaptic transmission in the NTS neurons, resulting in modulation of the hypoxic ventilatory response.…”