Abstracts

Rationale: Hypothermia has been beneficial in the treatment of severe neurological injuries that include hypoxic ischemic injury/stroke, traumatic brain injury, and status epilepticus. Many of the therapeutic mechanisms have been attributed to neuroprotection with hypometabolism, but hypothermia has many effects on CNS function and circuit activity. Little is known about how hypothermia affects drug actions and what pharmacological agents will maximize its benefits and minimize epileptiform hyperexcitablity with re-warming. Methods: Here we explore hypothermia actions on synaptic inhibition using visualized whole-cell patch-clamp of dentate gyrus granule cells in hippocampal slices. Mean-variance analysis and receptor-kinetic computational models of GABA-A receptors at synaptic sites were optimized to fit experimentally measured IPSCs before and after temperature lowering and exposure to GABAergic agents such as barbiturates. Results: We find that lowering temperature from 34 deg C to 21-26 deg C has dramatic effects on miniature IPSCs with an increase in the decay-time (11.7 +/- 4.0 to 21.2 +/- 6.4 ms; p<.05), Area-Under-the-Curve (-705 +/- 85 to -1170 +/- 331 pA ms; p<.05), and 10-90% rise-time (.36 +/- .05 to .54 +/- .14 ms; p<.05). A peak amplitude change from -61.9 +/- 13.2 to -56.0 +/- 10.2 pA at lower temperatures was not significant. A decrease in miniature IPSC frequency from 3.4 +/- 1.0 to 1.6 +/- .4 Hz suggests presynaptic actions of hypothermia as well. Hypothermia also modifies the effectiveness of pharmacological agents such as barbiturates with phenobarbital (200uM) having a modest 41% and 27% increase in the decay-time and AUC, respectively, during hypothermia, but a strongly significant 180% and 165% increase in decay-time and AUC under more normothermic conditions. Preliminary results using a 7-state GABA-A receptor kinetic computational model suggests that hypothermia has effects on kinetic parameters including a prolongation of open state duration. Because barbiturates also prolong GABA-A receptor open-state duration, the reduced effectiveness of phenobarbital with hypothermia may represent an occlusive interaction between the two types of therapy. Conclusions: Hypothermia has pronounced effects on synaptic inhibition, with direct effects on GABA-A receptor kinetics that may be important at extrasynaptic sites and tonic inhibition as well. The enhancement of inhibition with hypothermia may be an important therapeutic mechanism. Computational optimization of a combination of non-pharmacological with pharmacological therapies should improve treatment effectiveness and neurological outcome, while minimizing untoward effects with re-warming.