Abstracts

Over the past decade there has been great interest in the therapeutic potential of brain cooling for epilepsy and other neurological diseases ([italic]Epilepsy Currents[/italic] 3:153 -156, 2003). Because we have found that hypothermia can rapidly reduce synaptic potentials, we decided to directly test the hypothesis that cooling blocks central neurotransmitter release. We used conventional, submerged rat entorhinal-hippocampal slices and recorded the field potential in the stratum radiatum. A Peltier device embedded in the bottom of the slice chamber directly contacted the slice and enabled us to determine the effects of rapid cooling (latency less than 5 sec) on the evoked synaptic response. We cooled other slices with a bath perfusion system to determine the effect of slow cooling on synaptic responses. In a second set of experiments, we microinjected a portion of the CA1 region with the fluorescent vesicular label FM1-43, which was taken up by presynaptic terminals in a stimulation-dependent manner. We then used 2-photon microscopy to monitor the effect of cooling on evoked transmitter release. One Hz pulses were applied, and Z-stacks (5 image planes) were obtained every minute and analyzed to quantify loss of fluorescence. Over the temperature range 33[deg]- 20[deg] C, rapid cooling over several seconds caused a 49% reduction in the evoked synaptic response. When we perfusion cooled over the same temperature range, which required 30 minutes for temperature equilibration, we observed a similar reduction in synaptic response. Interestingly, however, at 28[deg] C, there was a transient increase in the size of the synaptic potential, indicating some differences in the effects of slow and fast cooling. When we loaded slices with FM1-43, we found very little loss of dye under control, unstimulated conditions for up to 60 minutes ([tau] = .015 sec^-1). When we stimulated (1 Hz; 50 [mu]sec) in stratum radiatum, there was an exponential loss of FM1-43 fluorescence ([tau] = 0.16 sec^-1 ). When the slices were cooled to 22[deg] C and then stimulated in an identical manner, the rate of fluorescence decay was dramatically reduced ([tau] = .027 sec^-1). Both slow and rapid cooling diminish synaptic currents, although the former can induce a paradoxical increase in current over a narrow temperature range. This may be the result of pump inhibition and elevated neuronal excitability. In either case, the FM1-43 experiments suggest that the dominant effect of cooling is a reduction in evoked neurotransmitter release. The temperatures required to reduce transmitter release parallel temperatures that abort seizures in our epilepsy models. These temperatures are, however, considerably lower than temperatures associated with neuroprotection in stroke models, suggesting that diminished neurotransmitter release cannot account for this observation. (Supported by NINDS (R01 NS 42936 and R21 NS 045652 to SMR))