Analysis of voltage-gated and synaptic conductances contributing to network excitability defects in the mutant mouse tottering

1. Intracellular current- and voltage-clamp recordings were carried out in CA3 pyramidal neurons from hippocampal slices of adult tg/tg mice and their coisogenic C57BL/6J (+/+) controls with the use of the single-electrode switch-clamp technique. The principal aim of this study was to investigate the mechanisms responsible for the tg gene-linked prolongation (mean 60%) of a giant synaptic response, the potassium-induced paroxysmal depolarizing shift (PDS) at depolarized membrane potentials (V(m) -47 to -54 mV) during synchronous network bursting induced by 10 mM potassium ([K⁺](o)). 2. To examine the role of intrinsic voltage-dependent conductances underlying the mutant PDS prolongation, neurons were voltage clamped by the use of microelectrodes filled with 100 mM QX-314 or QX-222 chloride (voltage-gated sodium channel blockers) and 2 M cesium sulphate (potassium channel blocker). The whole-cell currents active during the PDS showed a significantly prolonged duration (mean 34%) at depolarized V(m)s in tg/tg compared with +/+ cells, indicating that a defect in voltage-dependent conductances is unlikely to completely account for the mutant phenotype. 3. Bath application of 40 μM (DL)-2-aminophosphonovalerate (DL-APV) produced a 30% reduction in PDS duration in both genotypes but failed to significantly alter the tg gene- linked prolongation compared with the wild type. These data indicate that the mutant PDS abnormality does not result from a selective increase of the N- methyl-D-aspartate (NMDA) receptor-mediated excitatory synaptic component. 4. Blockade of γ-aminobutyric acid-A (GABA(A)) transmission with picrotoxin (50 μM) or bicuculline (1-5 μM) completely eliminated the difference in PDS duration between the genotypes. Furthermore, although both GABA(A) receptor antagonists increased the mean PDS duration in +/+ neurons, they did not significantly alter it in tg/tg neurons. These findings are consistent with a reduction in GABA(A) receptor-mediated synaptic inhibition during bursting in the tg CA3 hippocampal network. 5. To test this hypothesis, bursting CA3 pyramidal neurons were loaded intracellularly with chloride by the use of KCl-filled microelectrodes to examine the effect of reversing the hyperpolarizing chloride-dependent GABA(A) receptor-mediated inhibitory postsynaptic component of the PDS. Chloride loading prolonged PDS duration in both genotypes, but the increase was greater in +/+ than in tg/tg neurons, indicating that a smaller GABA(A) inhibitory postsynaptic potential (IPSP) component was reversed in the mutant. 6. To estimate the relative contributions of fast excitatory and inhibitory inputs to the PDS prolongation, a temporal analysis of the synaptic conductances and reversal potential changes underlying the PDS was performed in +/+ and tg/tg neurons under single-electrode voltage clamp. With the use of paroxysmal current measurements at different holding potentials, comparisons of current-voltage plots made at 1-ms intervals revealed 1) a significant slowing of the rise and decay of the synaptic conductance and 2) a negative and positive shift in the reversal potential at time periods corresponding to the peak and decay, respectively, of the PDS in mutant compared with wild-type neurons. 7. Because the voltage-clamp data were obtained under conditions where NMDA receptors were blocked by bath-applied 20 μM D-APV, and GABA(B) receptor- coupled K⁺ channels by cesium loading, the remaining conductances during the PDS were due primarily to non-NMDA and GABA(A) receptor-mediated PSPs. Conductance time course plots constructed to assess the contributions of these two PSP components to the total conductance change during the PDS revealed 1) an enhancement of the GABA(A) synaptic conductance change during the repolarizing phase and 2) a reduced amplitude and slower decay of the fast non-NMDA excitatory synaptic conductance change in the mutant compared with the wild type. 8. We propose that a gene-linked alteration in synaptic inhibition, characterized by a net reduction in the hyperpolarizing IPSP and a positive shift of the GABA(A)-dependent synaptic equilibrium potential, may contribute to the prolongation of the K⁺-induced PDS during synchronous network bursting in CA3 pyramidal neurons of the epileptic tg mutant.