MELATONIN PROTECTS PRIMARY CULTURES OF CEREBELLAR GRANULE NEURONS FROM KAINATE BUT NOT FROM N-METHYL-D-ASPARTATE EXCITOTOXICITY

The antiexcitotoxic efficacy of melatonin, a putative endogenous hydro xyl radical scavenger, was studied in primary cultures of rat cerebell ar granule neurons. Excitotoxicity was induced in 7- to 9-day-old cult ures by an exposure to glutamate (15 min in the absence of magnesium) or to glutamate receptor agonists, kainate (30 min), and N-methyl-D-as partate (60 min in the absence of magnesium). Thereafter, cultures wer e returned to the culture-conditioned medium for 18 h at the end of wh ich time viability was assessed by quantitative staining with 3-(4,5-d imethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. Cotreatment wit h melatonin (500 mu M) protected the neurons completely from the toxic ity of kainate (up to 1 mM) and shifted the ED(50) for glutamate from 55 +/- 2.6 to 97 +/- 3.6 mu M. Melatonin cotreatment was ineffective i n protecting the neurons from N-methyl-D-aspartate toxicity. When mela tonin was added to the cultures only before or after kainate treatment , there was no resultant protection from kainate toxicity. The neuropr otective effect of melatonin does not appear to be related to the dire ct action of melatonin on ionotropic glutamate receptors. That is, the kainate-stimulated inward currents measured by a patch-clamp techniqu e in voltage clamped neurons and the kainate-stimulated increase in fr ee cytosolic calcium measured at the single cell level using digital i maging fluorescent microscopy with fura-a were not affected by melaton in. Moreover, the binding of [H-3]glutamate to rat cerebellar membrane s was not competed off by melatonin. Further studies are needed to eva luate the pharmacologic relevance of the neuroprotective action of mel atonin. (C) 1995 Academic Press, Inc