Ionic mechanisms underlying differential vulnerability to ischemia in striatal neurons
Introduction
It is well established that certain brain regions and specific neuronal cell types show different sensitivity to hypoxic-ischemic insults (Pulsinelli, 1985, Leblond and Krnjevic, 1989, Haddad and Jiang, 1993a, Haddad and Jiang, 1993b, Martin et al., 1994). When compared to other brain areas, in fact, neostriatum, hippocampal field CA1 and neocortical layers 3, 5 and 6 appear to be particularly prone to develop neuronal damage under ischemic condition (Brierley, 1976, Pulsinelli et al., 1982, Simon et al., 1984a, Hawker and Lang, 1990, Choi, 1990, White et al., 1993). In recent years, basic experimental studies substantially contributed to elucidate the mechanisms underlying the neuronal responses to oxygen and glucose deprivation. In particular, the development of the in vitro single neuron physiological recordings allowed to detect the membrane property changes occurring during metabolic stress and to correlate these changes with the particular susceptibility expressed by neurons to the in vivo ischemia. Importantly, this approach can identify both protective and injurious physiological mechanisms displayed by single neurons during this pathological condition, critical for planning neuroprotective pharmacological strategies during cerebrovascular insults in humans.
For several years, the so called ‘excitotoxic hypothesis’ of neuronal death was claimed to explain, at least partially, the differential vulnerability to ischemia expressed by the various neuronal subtypes. This hypothesis postulates that the excitatory transmitter glutamate plays a major role in the generation of the irreversible membrane potential changes observed during energy deprivation, implying that those cells highly sensitive to this transmitter are more susceptible to the ischemic insults (Rothman and Olney, 1986, Choi and Rothman, 1990). However, the recent experimental work did not provide conclusive evidence on this issue. In fact, although some authors described a neuroprotective effect exerted by the antagonists of both N-methyl-d-aspartate (NMDA) and non-NMDA glutamate receptors on the ischemia-induced cellular damage (Goldberg et al., 1988, Marcoux et al., 1988, Sheardown et al., 1990, Demerle-Pallardy et al., 1991), other studies have shown that specific glutamatergic antagonists failed to affect the neuronal damage caused by ischemia (Block and Pulsinelli, 1987, Wieloch et al., 1988, Albers et al., 1989, Buchan and Pulsinelli, 1990). Moreover, Globus et al. (1991) found, utilizing brain microdialysis, that the glutamate release following the ischemic insult was almost uniform in all the studied brain regions, while the histopathologic lesions caused by energy deprivation were different in single cerebral areas. Taken together, all these studies strongly suggest that ischemic neuronal damage cannot solely result from an excitotoxic mechanism. Other factors must be considered to explain the differential neuronal vulnerability to ischemia shown by distinct brain structures and specific neuronal subtypes. It is now increasingly clear that, among these factors, a major role is played by differential ionic changes produced by the combined oxygen and glucose deprivation at the single neuron level. In this case the differential response to energy failure would be caused by peculiar changes of the intrinsic membrane properties of the neurons due to their different expression of ion channels and transporters, activated or inhibited by the rapid loss of ATP production.
Here we will present recent advances on the understanding of the ionic mechanisms activated by oxygen and glucose deprivation in two different central neurons recorded in vitro: neostriatal ‘spiny’ neurons and neostriatal ‘aspiny’ neurons, exhibiting, respectively, one of the highest sensitivity and resistance to cerebral ischemia and other neurological disorders such as Huntington's disease (Brierley, 1976, Francis and Pulsinelli, 1982, Ferrante et al., 1985, Pulsinelli, 1985, Beal et al., 1986, Xu, 1995, Chasselet et al., 1990, Beal, 1992, Beal, 1995). Neostriatal spiny neurons are medium-sized GABAergic projecting cells representing about 95% of the total neuronal population of this nucleus, conversely neostriatal aspiny neurons are large cholinergic interneurons accounting for only 2–3% of neostriatal cells (Bolam et al., 1984, Wilson et al., 1990, Kawaguchi, 1992, Kawaguchi et al., 1995). During in vitro neurophysiological recordings these two neuronal subtypes can be easily distinguished by means of electrophysiological analysis. In fact, they exhibit extremely different membrane properties, such as resting membrane potential, input resistance, current–voltage relationship, presence or absence of peculiar membrane conductances. These features allow an unequivocal interpretation of the experimental data when the effect of ischemia is studied in these neuronal subtypes.
Section snippets
Effects of ischemia on neostriatal spiny neurons
As shown in Fig. 1, ischemia causes opposite membrane potential changes in the two populations of neostriatal neurons. Glucose and oxygen deprivation induces a membrane depolarization in striatal spiny neurons. The amplitude of the ischemia-induced membrane depolarization is time-dependent (Fig. 1B). Ischemia-induced membrane changes start 30–60 s after the interruption of the oxygen- and glucose-containing solution (Fig. 1Ca) and increase progressively during the whole period of ischemia. When
Effects of ischemia on neostriatal aspiny neurons
Unlike spiny neurons, striatal aspiny interneurons are hyperpolarized by glucose and oxygen deprivation (Fig. 1Cb) (Calabresi et al., 1997a, Pisani et al., 1999). This hyperpolarization is coupled with a reduction of the apparent input resistance as detected by the decreased amplitude of hyperpolarizing pulses of long duration (1–3 s). Ischemia-induced membrane hyperpolarization starts 30–60 s after the interruption of the physiological solution and increases progressively during the whole
Role of glutamate receptor activation, voltage-dependent Na+ channels, Na+ and Ca2+ entry in the ischemia-induced membrane depolarization of striatal spiny neurons
The role of the activation of glutamate receptors (and of other putative neurotransmitters), of voltage-dependent Na+ channels and of Ca2+ entry in the generation of the ischemia-induced membrane depolarization of striatal vulnerable neurons has been studied in different ways and provided interesting results. TTX, a blocker of the Na+ channels mediating action potential discharge (and, consequently, neurotransmitter release) in neurons, produces no detectable changes on the membrane
Role of transmitter release, K+ and Ca2+ conductances in the ischemia-induced membrane hyperpolarization of striatal aspiny neurons
As seen for the membrane potential changes induced by energy deprivation in striatal spiny neurons, the blockade of synaptic transmission by TTX fails to produce changes in the aglycemia- and ischemia-induced membrane hyperpolarization of aspiny neurons (Fig. 4B) (Calabresi et al., 1997a, Pisani et al., 1999), ruling out the contribution of a putative hyperpolarizing transmitter in this phenomenon. Interestingly, also antagonists of adenosine receptors fail to attenuate the hyperpolarization of
Effects of the pharmacological blockade of ATP-dependent K+ channels and of the Na+/Ca2+ exchanger on the ischemic depolarization of striatal spiny neurons
As seen, Na+ influx plays a major role in the membrane depolarization induced by energetic failure in striatal spiny neurons. This ionic event possibly contributes to explain the high sensitivity of this neuronal subtype to ischemic insults in humans. However, when deprived of both oxygen and glucose, other ionic adjustments occur in striatal spiny neurons to limit the membrane depolarization of the cell and the damage that invariably follows the loss of ionic homeostasis. A critical event is
Effects of neuroprotective drugs on the electrophysiological effects produced by in vitro ischemia in striatal neurons
It has been proposed that antiepileptic and neuroprotective drugs may protect central neurons during ischemic/hypoxic insults since it has been shown that these drugs inhibit voltage-dependent Na+ and Ca2+ channels, and also depress excitatory synaptic transmission (Benoit and Escande, 1991, Pratt et al., 1992, Martin et al., 1993, Bensimon et al., 1994, Harden, 1994, Upton, 1994, Lynch et al., 1995, Schachter, 1995, Koroshetz and Moskowitz, 1996, Meldrum, 1996, Centonze et al., 1998, Calabresi
Summary and conclusion
Ischemia induces a membrane depolarization in spiny neurons. Periods of ischemia <6 min cause reversible membrane potential changes while longer periods determine irreversible membrane depolarization in these cells. Although the pathogenesis of ischemic neuronal damage was first attributed to excitatory amino acids (Kass and Lipton, 1982, Rothman, 1983, Rothman, 1984, Simon et al., 1984a, Simon et al., 1984b), intracellular studies on hypoglossal and vagal motor neurons have shown that
Acknowledgements
We wish to thank Massimo Tolu for the excellent technical assistance. This study was supported by a Ministero della Sanità (Progetto Finalizzato S. Lucia) grant and a CNR (Invecchiamento) grant to PC. The study was also supported by a MURST/CNR (legge 95/95) grant and a MURST/COFIN (1998) grant to GB.
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