Pd 102807

8-9 week-old male C57Bl/6J mice were sacrificed by cervical dislocation, and their brains were removed and collected in ice-cold Hank's essential medium. The hippocampi were microdissected and minced and a single-cell suspension was prepared as previously described (Jhaveri et al., 2015 (link)). The resulting pellet was resuspended in 1 ml of complete neurosphere medium containing EGF (20 ng/ml; receptor grade, BD Biosciences) and bFGF (10 ng/ml; recombinant bovine, Roche). The cells were then plated in a 96-well plate in complete neurosphere medium containing DMEM/F-12, EGF and bFGF, with or without muscarine (10 μM, 50 μM), nicotine (10 μM, 50 μM), VU010010 (10 μM), VU0152100 (10 μM) or PD102807, a selective M4 mAChR antagonist (1 μM; Tocris Bioscience). Muscarine was dissolved in 100% DMSO, with the final concentration of DMSO being adjusted to 0.1% for each treatment, including vehicle. Norepinephrine (10 μM) and KCl (15 mM) were used to investigate the effects of VU010010 on subpopulations of quiescent NPCs. The plates were incubated at 37°C and the total number of neurospheres obtained in each treatment group was determined on day 14, with all conditions normalized to the control group for each experimental replicate and plotted as a percentage of the control.

PD 102807 (catalog 1671) and DNQX (6,7-dinitroquinoxaline-2,3-dione; catalog 0189) were purchased from Tocris. LY367385 (catalog L4420), sulpiride (catalog S8010), picrotoxin (catalog P1675), and other reagents were obtained from MilliporeSigma.

(−)-Bicuculline (BIC) chloride (20 µM, an A-type γ-aminobutyric acid [GABA] receptor antagonist), D-(−)-2-amino-5-phosphonopentanoic acid (AP5, 10 µM, a N-methyl-D-aspartate receptor antagonist), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM, AMPA/kainate receptor antagonist), tetrodotoxin citrate (TTX, 0.5 µM, a voltage-gated sodium channel blocker), acetylcholine (ACh, 100 µM, non-selective AChR agonist), scopolamine hydrobromide (10 nM, non-selective mAChR antagonist), mecamylamine hydrochloride (10 nM, non-selective nAChR antagonist), (−)-nicotine tartrate (100 µM, nAChR agonist), PNU282987 (10 µM, alpha7 nAChR agonist), TC2559 difumarate (10 µM, alpha4beta2 nAChR agonist), and CC4 (10 uM, alpha6beta2 nAChR agonist) were all purchased from Cayman Chemical (Ann Arbor, MI). Nifedipine (1 µM, L-type voltage-gated calcium channel blocker), (+)-muscarine chloride (Mus,10 µM, non-selective mAChR agonist), cesium chloride (Cs, 5 mM, broad-spectrum blocker of inwardly rectifying potassium (Kir) channels), methoctramine hydrate (50 nM, M2 mAChR antagonist) and phorbol 12-myristate 13-acetate (PMA, 50 nM, protein kinase C (PKC) activator) were purchased from Sigma-Aldrich. NS3861 (10 µM, alpha3beta2 and alpha3beta4 nAChR agonist) and PD102807 (400 nM, M4 mAChR antagonist) were purchased from Tocris (Bristol, United Kingdom).