FIGURE 3.

CHOP-mediated down-regulation of cell surface GABA_B receptors impairs GABA_B receptor-mediated downstream signaling. A, GABA_B receptor-induced phosphorylation of ERK1/2 was prevented by the GABA_B receptor antagonist CPG 56999A (CPG) and by thapsigargin-induced up-regulation of CHOP. Neurons were treated with thapsigargin (+TG) to up-regulate CHOP, whereas control cultures remained untreated. Subsequently, neurons were incubated for 10 min with 100 μm baclofen (Bac) in the presence or absence of 10 μm CPG 56999A (+CGP, GABA_B receptor antagonist). After fixation and permeabilization, neurons were stained for total ERK1/2 (ERK, green) and diphosphorylated ERK1/2 (pERK, red). Left panel, representative images. Scale bar = 5 μm. Right panel, quantification of pERK levels. Data are means ± S.E., 26 (Baclofen, Baclofen + CPG) and 28 (Baclofen + TG) neurons from three experiments. ***, p < 0.001, one-way analysis of variance, Bonferroni's multiple comparison test. B, up-regulation of CHOP diminished baclofen-induced inhibition of sPSCs. Left panel, representative current traces depicting spontaneous sPSCs recorded from untreated control neurons (n = 9) or from neurons treated with thapsigargin for up-regulation of CHOP (n = 5). Right panel, normalized amplitude and frequency values of the sPSCs. Mean amplitudes and frequency values were normalized to the control condition of the individual neuron. Con., control. Data are mean ± S.E. *, p < 0.05; Student's t test.