Abstracts

Neurod/beta2 is a basic helix loop helix transcription factor required for granule cell determination and differentiation. Anatomically, homozygous neurod/beta2 (ND) null mice show a complete lack of granule cells in the dentate gyrus and a neurological phenotype of spontaneous limbic epilepsy. While studying the mechanisms of neuronal plasticity caused by this striking defect, we observed that these mice are resistant to all but lethal doses of pilocarpine, while displaying normal sensitivity to kainate-induced seizures. This phenotype has also been described in mice deficient in the gene for the m1 muscarinic receptor, the molecular target of pilocarpine (Hamilton et al., 1997). We therefore investigated the cholinergic innervation of the ND hippocampus to determine whether the loss of granule cells, an important target of hippocampal cholinergic synapses, resulted in a decrease in m1 receptor expression. Homozygous adult ND and +/+ mice were injected with either kainate (40-45mg/kg) or pilocarpine (250-370 mg/kg) and monitored behaviorally and with EEG recordings. M1 receptor (Alomone, Israel), VAChT (R Edwards, UCSF), CAT (Chemicon, CA), BrdU (Boehringer Mannheim, IN) immunohistochemistry were performed using specific antibodies. Septal cholinergic neurons were injected with DiI (Molecular Probes, OR) and terminal innervation patterns were examined by microscopy. ND mice showed typical status epilepticus when injected with kainate at doses effective in +/+ mice. Pilocarpine in doses up to 370 mg/kg i.p. failed to initiate [italic]status epilepticus[/italic]. M1 receptor immunohistochemistry revealed apparently normal levels of receptor expression in hippocampal pyramidal cell layers, neocortex, and other brain regions. DiI tracing studies show that cholinergic axons make some direct contacts onto cells in the CAP region. Although the number of cholinergic neurons in medial septum was normal, there was a large overall decrease in hippocampal terminal innervation. Despite the presence of M1 receptors, ND mice are resistant to pilocarpine-induced [italic]status epilepticus[/italic]. This strongly implies that pilocarpine-induced activation of the CA3 pyramidal cells alone is insufficient to produce prolonged seizures, and that intact hippocampal circuitry, including cholinergic innervation of a normal dentate granule cell-CA3 pathway is necessary for sustaining the prolonged seizures triggered by this muscarinic agonist. (Supported by National Institutes of Health (NIH) grants 29709 (JLN) and FAPESP (MM).)