Hypoxic Spreading Depression (HSD) in the Substantia Nigra: An in vitro comparison with the hippocampus and implications for Vascular Parkinsonism

Abstract

Cerebral ischemia often results in sudden neurological symptoms, re ecting the acute pathophysiological processes initiated by metabolic compromise. The severity of ischemic brain injury is correlated with the early occurrence of hypoxic spreading depression (HSD), a profound and rapid depolarization of neurons and glia, which propagates as a wavefront across susceptible brain regions. This phenomenon has been well characterised in ischemia-sensitive cortical areas, including the CA1 hippocampal region, particularly in response to oxygen and glucose deprivation (OGD), an in vitro model of brain ischemia. However, little is known of the early ischemic events induced in the Substantia Nigra (SN), a ventral midbrain nucleus involved in movement control and reward prediction. Dopamine-producing Substantia Nigra pars compacta (SNc) neurons are thought to have a metabolically vulnerable phenotype; therefore, we hypothesised that the dopaminergic region of the SN would quickly develop HSD and an associated cascade of pathophysiological events during OGD. We also thought that the sudden-onset movement de cits reported in patients with vascular parkinsonism (VP) could be initiated by HSD in the SN. A short period of OGD (10 min) evoked HSD in the Substantia Nigra of submerged coronal midbrain slices obtained from P21-23 Wistar rats, resembling responses recorded in the CA1 hippocampal region. In the SN, HSD occurred within 4.2 0.1 min of OGD, indicated by a negative extracellular DC potential shift and increased tissue light transmittance, re ecting synchronized cell depolarization and swelling. The event propagated in a lateral to medial direction across the Substantia Nigra pars reticulata (SNr), but did not involve the immediately adjacent SNc region. In the SNr, HSD `ignition' was delayed after blocking ionotropic glutamate receptors, but the response amplitude and propagation velocity were una ected. This suggests that glutamatergic excitation during the early period of OGD contributes to HSD initiation but does not a ect its propagation in the SNr region, similar to the e ects seen in the CA1 hippocampal region. HSD occurrence in the SNr correlated with functional and morphological impairments. A sudden burst of ring was recorded in non-dopaminergic SNr neurons immediately prior to HSD onset, followed by persistent inactivity. These events are attributed to rapid and irreversible cell membrane depolarization, similar to the electrophysiological responses recorded during HSD onset in pyramidal neurons of the CA1 hippocampal region. Furthermore, SNr neurons had swollen somata and beaded dendrites when assessed following 10 min of reperfusion after OGD (i.e. return of O2 and glucose). Those morphological changes re ect acute structural injury, also observed in CA1 hippocampal neurons. In contrast, OGD inhibited SNc neuron ring, prior to the occurrence of HSD in the adjacent SNr region. The SNc neuron response involved a prolonged cell membrane hyperpolarization, which turned to slow depolarization after the HSD wave had passed through the SN. Remarkably, unlike SNr and pyramidal CA1 neurons, dopaminergic SNc neurons repolarized during reperfusion, with many resuming spontaneous ring. This functional recovery was also associated with morphological intactness. Nigral HSD was associated with profound energy de cits and ion dysregulation, further resembling the well-characterised events in the CA1 hippocampal region. Despite similar mitochondrial depolarization in SNr and SNc regions during OGD (indicated by increased rhodamine 123 uorescence), ATP energy levels fell suddenly in the SNr during HSD wavefront propagation. In contrast, the adenylate energy charge decreased more slowly in the SNc. Regional ATP, ADP and AMP content was assessed in situ with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The SNr region also developed rapid extracellular hyperkalemia and irreversible neuronal [Ca2+]i elevation, unlike slower-developing and more recoverable [K+]o and [Ca2+]i changes in the HSD-resistant SNc. Our results suggest that the HSD-resistance of SNc neurons relates to the cell membrane hyperpolarization and inhibition of ring, which develops during the early period of OGD. This cell silencing persisted despite block of inhibitory neurotransmission with antagonists of GABAA, adenosine A1 and dopamine D2 receptors, or block of channels that are classically associated with coupling membrane potential and neuronal ring to metabolism (ATP-sensitive K+ channels and BK/SK Ca+-sensitive K+ channels). However, the inhibitory e ect seen in SNc neurons was abolished with non-speci c K+ channel blockers (Ba2+ or high concentration of TEA). In the presence of such blockers, OGD led to sudden and irreversible cell membrane depolarization, swollen somata and beaded dendrites. These results indicate that a yet unidenti ed K+ conductance is crucial for the HSD-resistance of SNc neurons. Interestingly, lowering the extracellular Na+ concentration delayed the OGD-evoked hyperpolarization and silencing of SNc neurons, suggesting that the critical resistance to HSD is initiated, at least in part, by Na+ (possibly involving KNa channels). Acute glial responses to OGD were also investigated. Glial cells in the SN (recorded from the border region between the SNc and SNr) depolarized in a manner which re ected the OGD-evoked extracellular K+ rise, resembling the response recorded from glial cells in CA1 hippocampal region. However, unlike hippocampal glia, those cells in the SN continued to depolarize during reperfusion and showed a loss of immunoreactivity for glial brillary acidic protein (GFAP). These ndings suggest that glial cells in the SN are unusually sensitive to ischemia-reperfusion. This study demonstrates that dopaminergic neurons of the SNc region are relatively resistant to HSD due to critical K+ channel activation in the early period of OGD. In contrast to the initial hypothesis, OGD did not lead to acute injury of these neurons; therefore, our results suggest that nigral HSD is not a direct cause of vascular parkinsonism. Instead, adjacent SNr neurons and glia appear particularly vulnerable to OGD and reperfusion. The mechanisms of diminished SNr neuron function and withdrawal of glial support require further studies, as these factors may facilitate delayed SNc neuron injury following brainstem ischemia.