Abstract
During the consolidation of fear memory, it has been shown that GABA(A) receptors (GABA(A)R) are rapidly downregulated in amygdala. This rapid decrease in GABA(A)R functioning may permit transient hyperexcitablity, contributing to cellular mechanisms of memory consolidation. Memory consolidation also requires brain-derived neurotrophic factor (BDNF) activation of tyrosine receptor kinase B (TrkB) receptors in the amygdala and hippocampus. We hypothesized that rapid internalization of GABA(A)Rα1 is mediated via TrkB activation of PKA and PKC-dependent processes. Primary neuronal cell cultures, from postnatal day 14-21 mouse amygdala and hippocampus, were analyzed with immunofluorescence using cell-surface, whole-cell permeabilization, and antibody internalization techniques, as well as with (3)H-muscimol binding assays. In both hippocampal and amygdala cultures, we found a >60% reduction in surface GABA(A)Rα1 within 5 min of BDNF treatment. Notably, the rapid decrease in surface GABA(A)Rα1 was confirmed biochemically using surface biotinylation assays followed by western blotting. This rapid effect was accompanied by TrkB phosphorylation and increased internal GABA(A)Rα1 immunofluorescence, and was blocked by k252a, a broad-spectrum tyrosine kinase antagonist. To further demonstrate TrkB specificity, we used previously characterized TrkB(F616A) mice, in which the highly selective TrkB-mutant specific antagonist, 1NMPP1, prevented the BDNF-dependent GABA(A)Rα1 internalization. In hippocampus, we found both PKA and PKC inhibition, using Rp-8-Br-cAMP and Calphostin C, respectively, blocked GABA(A)Rα1 internalization, whereas inhibition of MAPK (U0126) and PI3K (LY294002) did not prevent rapid internalization. By contrast in amygdala cultures, Rp-8-Br-cAMP had no effect. Together, these data suggest that rapid GABA(A)R internalization during memory consolidation is BDNF-TrkB dependent. Further, it appears that hippocampal GABA(A)R internalization is PKA and PKC dependent, while it may be primarily PKC dependent in amygdala, implying differential roles for TrkB-dependent kinase activation in BDNF-dependent memory formation.