The initial rise was also

observed in HAL, but EPSCs declined soon below baseline in a dose-dependent fashion (Figures 7C and 7D). The progressive reduction of EPSCs during the train reflects the use dependence of the APD effect and is in line with the fluorescence measurements presented in Figure 7B. We used two strategies to substantiate our concept that the use-dependent inhibition of EPSC is causally linked to the blockade of voltage-gated sodium channels (Figure 6). First, we demonstrated that the effects of HAL on train-evoked EPSCs could be mimicked by a low concentration of the highly potent and selective sodium channel blocker TTX (25 nM, Figures 7C and 7D). Second, we functionally isolated axonal action potentials and recorded their extracellular see more equivalent, the so-called fiber volleys (FVs), in the absence

and presence of either a low concentration of TTX or several APDs. Under control conditions, FVs exhibited only a small decrement later in the train. In sharp contrast, TTX, which is known to block sodium channels in a use-dependent manner (Conti et al., 1996) as well as all of the three APDs tested (5 μM HAL, 30 μM CPZ, 30 μM RSP), produced a pronounced use-dependent inhibition of FVs (Figure 7E). The depressant effect of APDs on EPSCs during train stimulation is, therefore, sufficiently explained by their inhibitory action on axonal action potentials. Notably, the effect was not limited Birinapant clinical trial to the hippocampus but was also observed in the NAc, which is a major target region of dopaminergic Terminal deoxynucleotidyl transferase projections and contains mainly medium spiny neurons expressing D1 or D2 DA receptors. The behavior of EPSCs in NAc medium spiny interneurons during stimulus trains was remarkably different from that in hippocampal CA1 pyramidal cells because, even under control conditions, EPSCs displayed only a brief and weak initial enhancement before they progressively decayed (Figures 7F and 7G). As a consequence, the inhibitory effect of HAL (5 μM) was much more

pronounced when compared to the hippocampus (Figures 7F and 7G). Importantly, FVs in NAc proved to be approximately equally resistant to stimulus trains compared with hippocampal FVs, and HAL reduced FVs with similar efficacy (Figure 7H). These data indicate that, in the NAc with its dense dopaminergic innervation, transmitter release is especially sensitive to the use-dependent inhibition of axonal sodium channels by APDs. To determine whether the accumulation of APDs in synaptic vesicles is required for the efficient inhibition of exocytosis, we assessed the extent of the inhibition induced by 5 μM HAL in the presence of folimycin, which abolished the accumulation of HAL in synaptic vesicles (Figure 1). Exocytosis was measured with FM4-64 (Figure 8A). The fluorescence of the dye was unaffected by changes of the intravesicular pH value (Figure 2B) or by APD administration (Figure 2D).