Effects of chloride transport inhibition and chloride substitution on neuron function and on hypoxic spreading-depression-like depolarization in rat hippocampal slices.

Abstract Chloride fluxes play a crucial role in synaptic inhibition, cell pH regulation, as well as in cell volume control. In many neuropathological processes, cell swelling is a pivotal parameter, since cell volume changes and the dimension of the interstitial space critically modulate synchronized neuronal activity as well as the tissue's susceptibility to seizures or spreading depression. This study therefore focuses on the effects of different Cl− transport inhibitors and Cl− substitution on neuronal function and hypoxia-induced changes in rat hippocampal tissue slices. Orthodromically evoked focal excitatory postsynaptic potentials were depressed by furosemide (2 mM), 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (1 mM) and Cl− substitution by methylsulfate, but were enhanced by 4,4′-dinitrostilbene-2,2′-disulfonic acid (1 mM). All four treatments induced multiple population spike firing in response to single orthodromic volleys, suggesting reduced synaptic inhibition. Antidromic population spikes increased following Cl− withdrawal, were unaffected in the presence of furosemide and 4,4′-dinitrostilbene-2,2′-disulfonic acid, but were abolished by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid. The amplitude of the hypoxic spreading-depression-like extracellular potential shift was reduced by furosemide, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and Cl− withdrawal, i.e. by the same treatments that depressed orthodromically evoked postsynaptic potentials. Furosemide prolonged the time to onset and the duration of the spreading-depression-like extracellular potential shift, while 4,4′-dinitrostilbene-2,2′-disulfonic acid shortened the time to onset. Spreading-depression-related cell swelling was recorded as the shrinkage of relative interstitial space, which was measured as tetramethylammonium-chloride space. Neither the Cl− transport inhibitors nor Cl− withdrawal had any detectable effect on spreading-depression-related cell swelling. CA1 pyramidal neurons usually hyperpolarized during drug application and their input resistance decreased. Cl− withdrawal increased their input resistance and caused spontaneous burst firing. Hypoxia caused the expected spreading-depression-like rapid, near complete depolarization of single pyramidal neurons and drastically reduced their input resistance. The three Cl− transport inhibitors and Cl− withdrawal delayed the onset of the hypoxic depolarization. In low Cl− solutions, the apparent threshold potential at which spreading depression was triggered shifted to more positive membrane potentials. The final voltage of the hypoxic depolarization was, however, not affected. It appears from these results that the reduction in the hypoxic spreading-depression-like extracellular potential shifts by Cl− transport inhibitors is at least partially attributable to desynchronization of depolarization, not to decreased depolarization in individual cells. Other contributing factors could be changes in recording conditions, depression of swelling-induced amino acid release from glial cells and unspecific side-effects of the applied drugs. Desynchronization could also account for the delayed spreading-depression onset. It is concluded that Cl− fluxes play a role in the triggering of spreading depression, but the spreading-depression-like depolarization itself or its self-regenerative character is not mediated by Cl−.