Abstracts

Rationale: Synaptic inhibition between neurons of the thalamic reticular nucleus (nRT) plays a major role in regulating thalamocortical circuit excitability, oscillations, and absence seizures. We recently demonstrated that endogenous benzodiazepine (BZ) binding site ligands (endozepines) are expressed in nRT, but not the adjacent ventrobasal nucleus. Furthermore, we showed that nm1054 mice, which harbor a 400-kb chromosomal deletion that includes the Diazepam binding inhibitor (Dbi) gene, exhibit deficient endozepine signaling in nRT and altered absence seizures. nm1054 mice, however, lack at least 6 genes and exhibit morphological abnormalities such as hydrocephalus and domed crania. Therefore, we re-examined this question in mice in which exon 2 of the Dbi gene is specifically deleted and replaced with a single loxP site (DBI KO).Methods: For in vivo EEG recordings, mice were bilaterally implanted with subdural wires positioned over the parietal cortex on postnatal day 35 and older. EEG recordings were performed at least one week post-surgery and analyzed for 3.75-6 Hz spike-and-wave discharge (SWD) activity. For in vitro electrophysiology, acute horizontal brain slices were prepared from littermate wild-type (WT) and DBI knockout (KO) mice on postnatal days 33-44. Whole-cell voltage-clamp recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) were made from nRT neurons using a near-isotonic chloride pipette solution. Experiments were performed in the presence of ionotropic glutamate receptor antagonists to isolate GABAergic currents. Data were compared before and during 10 min bath application of the BZ binding site antagonist flumazenil (FLZ, 1 M). Current-clamp recordings were performed using a K-gluconate-based pipette solution in the absence of receptor blockers.Results: DBI KO mice do not exhibit the gross brain and cranial abnormalities observed in nm1054 mice. Immunoreactivity for DBI protein in the thalamus was confirmed to be essentially absent in the KO mice compared to WT. Preliminary EEG findings showed an approximately 3-fold higher incidence of spontaneous SWD activity in KO mice (n=3) compared to WT (n=4). nRT cells from KO mice (n=12) exhibited a decreased sIPSC duration (p<0.001) compared to cells from WT mice (n=12), suggesting that KO mice lack a source of positive allosteric modulation of GABAA receptors. In addition, whereas FLZ decreased sIPSC duration in WT cells (n=8, p<0.001), FLZ had no effect on sIPSC duration in KO cells (n=8, p>0.2), indicating that Dbi gene products are acting through BZ binding sites. Current-clamp recordings showed no significant differences in intrinsic membrane or firing properties between WT (n=6) and KO nRT neurons (n=7) (p>0.6).Conclusions: Alterations in endozepine signaling and seizure activity in DBI KO mice support the hypothesis that DBI-derived peptides mediate antiepileptic endozepine actions in nRT. Future studies will investigate the role of endogenous DBI-derived peptides in the regulation of thalamocortical circuitry in both normal and seizure states.