Abstracts

Rationale: Synaptic inhibition between neurons of the thalamic reticular nucleus (nRT) plays a major role in regulating thalamocortical circuit excitability and oscillations. We recently demonstrated that endogenous benzodiazepine (BZ) binding site ligands (endozepines) are expressed in nRT, but not the adjacent ventrobasal nucleus (VB). Endozepines increase intra-nRT GABAergic inhibition and suppress absence seizures. Outside-out patches pulled from VB neurons show a potentiated response to UV laser photostimulation of caged GABA when placed in nRT, indicating that VB sniffer patches can be used as endozepine biosensors. Endozepine actions may be mediated by peptides derived from the precursor diazepam binding inhibitor (DBI), such as octadecaneuropeptide or octapeptide. nm1054 (new mutation 1054) mutant mice lack a 400-kb part of chromosome 1 that includes the DBI gene. Here we investigated the effects of the nm1054 mutation on endozepine signaling in nRT.Methods: Acute horizontal brain slices were prepared from littermate wild-type (WT) and nm1054 knockout (KO) mice on postnatal days 21-30. Whole-cell voltage-clamp recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) were made from nRT neurons. Outside-out membrane patches were pulled from VB neurons and placed back in the slice in either VB or nRT, and the response to laser photolysis of caged GABA (100 ?M) was measured. Experiments were performed in the presence of ionotropic glutamate receptor antagonists to isolate GABAergic currents. In some cases slices were preincubated and experiments performed in the presence of the BZ binding site antagonist flumazenil (FLZ, 1 ?M).Results: nRT cells from KO mice (n=8) exhibited a decreased sIPSC duration (p<0.05) compared to cells from WT mice (n=11), suggesting the nm1054 mutation removes a source of positive allosteric modulation of GABAA receptors. In WT slices, patches moved to nRT (n=9) exhibited an increased uncaged IPSC duration compared to patches placed in VB (n=8) (p<0.001). In slices from nm1054 KO mice, patches placed in nRT (n=9) also showed an enhanced uncaging response compared to patches placed in VB (n=7) (p<0.05), but the degree of potentiation was significantly reduced compared to that observed in WT slices (p<0.05). Furthermore, the duration of uncaging responses for patches placed in nRT was diminished in KO slices compared to WT (p<0.01). The reduction in response potentiation in KO slices was similar to that induced in WT slices by FLZ treatment, indicating that this difference reflects altered endozepine signaling. There were no differences in uncaging responses between WT and KO when patches were placed in VB (p>0.4), signifying that these alterations are specific to nRT. Conclusions: Alterations in endozepine signaling in the nm1054 mouse indicate that DBI-derived peptides mediate at least some 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.