A2129

The quinolinium salt-based halide-sensitive fluorescence probe N-(ethoxycarbonylmethyl)-6-methoxyquinolium bromide (MQAE; ab145418, Abcam) was used as a measure of chloride ion influx activity13 (link). Following cell culture, the PRNCs were incubated with 5 mM MQAE for 2 h at RT in the dark, and subsequently washed twice with NbActive 4 (BrainBit). Cells were then treated with 50 μM GABA (A2129, Sigma-Aldrich) or 10 nM GABA analog THIP, δ-GABA_AR specific agonist8 (link) (T101, Sigma-Aldrich), then fluorescence intensity was consequently measured at 0, 5, 10, and 20 min at RT. For inhibition assay, the cells were pretreated with 1 μM flumazenil (F6300, Sigma-Aldrich) or 1 μM picrotoxin (P1675, Sigma-Aldrich) or PBS (control) for 45 min at RT, and then stimulated with 50 μM GABA for 10 min at RT. Intercellular MQAE is quenched by 10 μM tributyltin chloride (T50202, Sigma-Aldrich) and 10 μM nigericin sodium salt (N7143, Sigma-Aldrich). The fluorescence intensity was measured by the Gemini EX florescence plate reader (Ex/Em = 360/460; Molecular Devices). The kinetic analysis was performed by using GraphPad Prism 6^® software.

Animals were treated with 5-bromo-2ʹ-deoxyuridine (BrdU; Sigma-Aldrich, B5002) at a concentration of 0.7% in fish water for 2 h. BrdU is a nonradioactive analog of thymidine incorporated into proliferating cells’ DNA during the S phase of mitosis. Fish were then allowed to survive for another 22 h (short-term survival) or 2 weeks (long-term survival) before being processed for BrdU immunodetection. For the acute treatment, animals were treated with BrdU at a concentration of 0.7% in fish water for 1 h before analysis. In acute experiments described in Fig. 5a, animals were injected intraperitoneally (volume: 2 μl) with either saline, nicotine (300 μΜ; Sigma-Aldrich, SML1236), GABA (500 μΜ; Sigma-Aldrich, A2129), or gabazine (100 μΜ; Sigma-Aldrich, SR95531). Immediately after injection, the animals were treated with BrdU for 1 h, as described above.

For the experiments conducted to evaluate the impact of pharmacological agents on proliferation and neurogenesis in vivo, animals were anesthetized using 0.03% tricaine methane sulfonate (MS-222; Sigma-Aldrich, E10521) in fish water and injected intraperitoneally (volume: 2 μl) with saline, ACh (2 mM; Sigma-Aldrich, A6625), muscarine (50 μΜ; Sigma-Aldrich, M104), nicotine (300 μΜ; Sigma-Aldrich, SML1236), GABA (500 μΜ; Sigma-Aldrich, A2129), or gabazine (100 μΜ; Sigma-Aldrich, SR95531). Immediately after injection, the animals were treated with BrdU as described above (see “BrdU treatment” section). For ex vivo evaluation of NSPC receptor activation’s contribution to proliferation, animals were anesthetized and dissected as for the electrophysiological recordings. Isolated intact spinal cords were then transferred to a continuously aired chamber containing the pharmacological agents, and BrdU diluted in the extracellular solution used for electrophysiological recording. In some experiments the extracellular solution contained TTX (1 μM) to abolish the synaptic transmission in the spinal cord networks. After the pharmacological treatments, the animals and tissues were processed for immunodetection of the incorporated BrdU.