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. 2005 Feb 2;25(5):1159–1168. doi: 10.1523/JNEUROSCI.3953-04.2005

Figure 8.

Figure 8.

Effect of trafficking on nicotine-induced long-term pCREB. A, The protocol started with a 1 min incubation of culture medium only (No-Stim; B), 10 μm nicotine (Nic; C), 10 μm nicotine plus 50 nm MLA added 1 min earlier to block α7-nAChRs (Nic/MLA; D), or KCl (E). After rinsing, the cells were given a first recovery period of 20 min to allow trafficking. Then, to induce pCREB, the cells received a 5 min second incubation in culture medium with cadmium (to block VGCCs) and no additives (control), nicotine (nic), or nicotine with αBgt applied 10 min earlier (nic/αBgt) to block α7-nAChRs, followed by rinsing and a second 20 min recovery period before immunostaining for nuclear pCREB. Cells were pretreated either with medium alone (Med) or with botulinum toxin C1 (BotC1) to block SNARE-dependent trafficking. Values represent the mean ± SEM of results compiled from three experiments. Blockade of SNARE-dependent trafficking by botulinum toxin specifically inhibited the ability of α7-nAChRS to drive CREB activation during the second incubation, if and only if the receptors had been exposed to nicotine in the first incubation (*p < 0.001 comparing BotC1 and Med for Nic-induced pCREB in C). Neither the blockade of trafficking nor the first incubation with nicotine or KCl had any other effect in the pCREB assay.