Absence seizures afflict a significant percentage of children with epilepsy. Attempts to understand the molecular mechanisms of absence seizures may be facilitated by studying animal models. Our earlier studies using the lethargic (lh/lh) mutant mouse model of absence seizures showed: i) an apparent requirement for GABAB receptors in absence seizures; ii) an increased number of GABAB receptors in neocortex and thalamic nuclei compared to control (+/+) mice; iii) no change in electrophysiologic responses mediated by postsynaptic GABAB receptors in thalamic neurons in lh/lh compared to +/+ mice; and iv) alterations in biochemical functions mediated by presynaptic GABAB receptors in neocortex and thalamus in lh/lh compared to +/+ mice. The short-term goals of this proposal are to seek physiologic correlates to our findings regarding GABAB responses in lh/lh and +/+ mice, and to determine if these responses cause absence seizures. We will perform 6 specific aims to answer the following questions.
First (aims 1 -3)), what is the physiologic correlate of our evidence that presynaptic GABAB receptor-mediated function is altered in thalamic and neocortical neurons from lh/lh mice? Second (aims 4 and 5), is the altered function mediated by presynaptic GABAB receptors in lh/lh mice caused by the increased numbers of GABAB receptors in these mice? Third (aim 6), are absence seizures in lh/lh mice caused by the increased numbers of GABAB receptors in these mice? In the long-term, the answers forthcoming will increase our understanding of the physiologic and pathophysiologic roles of GABAB receptor-mediated function. More importantly, this information may lead to better therapies for patients with absence seizures.
In specific aims 1 and 2 we will use Cs+-filled microelectrodes and whole- cell voltage-clamp techniques in ventrobasal (VB) thalamic neurons (aim 1) or neocortical pyramidal neurons (aim 2) of lh/lh and +/_ slices to measure the effect of presynaptic GABAB receptor activation on IPSCs (autoreceptor function) and EPSCs (heteroreceptor function) evoked by local stimulation. We will activate presynaptic GABAB receptors by: i) applying the GABAB receptor agonist (-) baclofen to the bath; and ii) measuring paired-pulse and homosynaptic depression.
In specific aim 3 we will use Cs+ filled microelectrodes and whole-cell voltage clamp techniques in VB thalamic neurons to measure the effect of presynaptic GABAB receptor activation on spindles evoked by stimulation of nucleus reticularis thalami (NRT).
In specific aim 4 we will down-regulate GABAB receptors in lh/lh mice by chronic administration of GABAB receptor agonists. We will then measure the effect of presynaptic GABAB receptors on [3H]-GABA release in thalamic and neocortical synaptosomes from these mice and from """"""""normal"""""""" lh/lh mice.
In specific aim 5 we will use whole-cell voltage-clamp techniques in VB thalamic neurons of down-regulated lh/lh mice to measure the effect of presynaptic GABAB receptors on spindles evoked by stimulation of NRT.
In specific aim 6 we will implant bipolar recording electrodes into neocortex of +/+ and lh/lh mice. After up-regulating GABAB receptors by chronic administration of GABAB antagonists to half of the +/+ mice, we will use EEG recordings to compare absence seizure frequency in the groups.
