Real-time imaging of intrinsic optical signals during early excitotoxicity evoked by domoic acid in the rat hippocampal slice

Abstract

Excitotoxicity involves neuronal depolarization and eventual cell death primarily through excess activation of glutamate receptors. Neuronal cell swelling is considered an early excitotoxic event mediated by ionic influx (mainly Na+ and Cl-) followed by water. Changes in the intrinsic optical signals of nerve tissue correlate with neuronal activity such that light transmittance (LT) increases across the brain slice as cells swell. The present study examined the effects of domoic acid, a potent excitotoxic food contaminant and glutamate analogue, on intrinsic optical signals in the rat hippocampal slice. A brief 1-min exposure to 10 microM domoate at 22 degrees C elevated LT by 58% in the apical dendritic region of CA1 and to a lesser extent in the molecular layer of the upper dentate gyrus. The responses peaked by 5 min and slowly reversed during a 30-min wash. The same responses were evoked by a 1-min application of 10 microM alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) at 22 degrees C. Minor changes were observed in the CA3 region and the lower blade of the dentate gyrus. At 37 degrees C, exposure to 10 microM domoate for 10 min resulted in apparent irreversible neuronal damage in the CA1 and upper dentate regions. The Na+ channel blocker tetrodotoxin (1 microM) eliminated the evoked CA1 population spike but not the LT increase, indicating that the domoate signal is not associated with action potential discharge pre- or post-synaptically. However, the response to domoate at 22 degrees C was reversibly blocked by the nonspecific glutamate receptor antagonist kynurenate and the non-N-methyl-D-aspartate (non-NMDA) receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). The response was not blocked by the NMDA receptor antagonist 2-amino-5-phosphonovaleate (AP-5) nor the kainate receptor blocker gamma-D-glutamylaminomethyl sulfonate (GAMS). Relative tissue resistance (RREL) measured across the CA1 dendritic region increased rapidly in response to domoate and fell slowly over 30 min, which paralleled the LT response described above. The increase in RREL was blocked by kynurenate. We propose that domoate binding to AMPA receptors opens channels mediating ionic influx, presumably Na+ followed passively by Cl-. Water follows, producing prolonged postsynaptic swelling in the CA1 and dentate regions where AMPA receptors are most abundant. At higher temperature this swelling can progress to permanent neuronal injury. Imaging intrinsic optical signals allows a real-time view of early excitotoxic events and may prove useful in assessing potentially therapeutic agents that reduce damage induced by excitotoxic agents or ischemia.