Tug905

Postnatal day 1 pups were euthanized, their brains immediately removed, and the hypothalamus microdissected. Tissue fragments were successively dissociated with a Pasteur pipette in PBS with 5.5 mM glucose, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were suspended in 5 mL of proliferation media: Dulbecco’s modified Eagle’s medium (DMEM)-F12/Glutamax (Gibco) supplemented with growth factors (10 ng/mL bFGF and 10 ng/mL EGF, 100 U/mL penicillin, 100 mg/mL streptomycin, and 1% B27 supplement). The floating neurospheres were allowed to grow in uncoated 25 cm² flasks incubated in a humidified incubator with a 5% CO₂ atmosphere. On culture day 7, neurospheres were collected by centrifugation, dissociated, and plated, in fresh proliferation medium, onto Poly-D-Lysine (PDL; Sigma P1024)-coated 12 well culture plates for RNA extraction or glass coverslips for immunocytochemistry. After 5 days, or once the monolayer reached confluence, cell differentiation was induced by switching proliferation medium for media without growth factors for 7 or 18 days^{33 (link)}. Cells were differentiated in the presence of 10 μM TUG905, BDNF (10 ng/mL; Sigma), IL-6 (2 ng/mL; Sigma), or SB 203,580 (0.1 μM Sigma) individually or in combination.

For central administration of GPR40 exogenous ligands, 7-week old mice were submitted to cannula implantation in the right lateral ventricle under xylazine (10 mg/kg, ip) and ketamine (100 mg/kg, ip) anesthesia. The coordinates were as follows: anteroposterior, 0.34 mm; lateral, 1.0 mm; and depth, 2.2 mm. The efficiency of cannula placement and viability was confirmed by icv administration of angiotensin II and measurement of the drinking response. Ventricular-cannulated mice were treated daily for 7 days with 2.0 μL of vehicle [1:1:3; Ethanol/ DMSO/ artificial cerebrospinal fluid (Tocris)], GW9508 (2.0 mM; Tocris Bioscience) or TUG905 (2.0 mM, synthesized as previously described^{50 (link)}.
BrdU (Sigma) was used to evaluate cell proliferation and survival. BrdU is a thymidine analogue that is incorporated into the DNA double-helix during the S-phase of the cell cycle, and thus marks actively proliferating cells^{70 (link)}. All animals received BrdU (0.1 M phosphate-buffered saline [PBS], pH = 7.2; 10 μg/day icv and 50 mg 1 × day ip) and were euthanized 1 or 28 days later (Fig. 1A) to assay proliferation or survival of new cells, respectively.
For some experiments, mice were treated either with an immunoneutralizing rabbit anti-BDNF antibody (0.8 μg/100 μL, sc-546, Santa Cruz Biotechnology) or a pre-immune rabbit serum (R9133, Sigma), by ip injection every 3 days for 2 weeks.