Vu10010

All chemicals were purchased from VWR unless otherwise indicated. VU 10010 (M₄-selective positive allosteric modulator), SR 95531 hydrobromide (Gabazine, GABA_A antagonist), Baclofen (GABA_B antagonist), QX314 chloride (intracellular sodium channel blocker), and AF-DX 116 (selective M₂- muscarinic receptor antagonist) were obtained from Tocris Bioscience (Ellisville, Missouri) and 6, 7-Dinitroquinoxaline-2, 3-dione (DNQX, AMPA receptor antagonist), DL-2-Amino-5-phosphono pentanoic acid (APV, NMDA receptor antagonist) from Ascent Scientific (Bristol, U.K.). Biocytin (B-1592) was purchased from Life Technologies (Invitrogen).

To selectively stimulate hippocampal M4 mAChRs, 10 μM VU10010 (Tocris), a selective allosteric potentiator (Shirey et al., 2008 (link)), was infused into the hippocampus using a single cannula attached to a 7 day, 0.5 μl/h osmotic pump (Alzet Model 1007D). Vehicle (0.1% DMSO, 0.2% bovine serum albumin in saline) was infused in the control group of mice. The coordinates from Bregma were: anterior/posterior, −2.0 mm; medial/lateral, −1.3 mm; and dorsal/ventral, −2.1 mm. The osmotic pump was subcutaneously implanted caudal to the scapula. The mice were perfused 15 days after the end of the infusion period and their brains were harvested for immunohistochemical analysis. To evaluate the effects of the blood-brain barrier-permeable, potent and selective Chrm4 allosteric modulator VU0152100 (Brady et al., 2008 (link)) on NPCs, Nestin-GFP mice were treated with VU0152100 (0.5 mg/kg, Sigma-Aldrich) or vehicle via daily i.p. injections for 5 consecutive days and were perfused on day 6. To examine its effects on adult hippocampal neurogenesis, C57Bl/6J mice were treated with VU0152100 or vehicle via daily i.p. injections for 7 consecutive days. Mice were perfused 15 days after the end of the treatment and their brains were collected for histology. The VU0152100 was dissolved in 40% PEG800, 5% Tween20 and 10% DMSO in saline.