Immunohistochemistry.

Animals were euthanized with an overdose of sodium pentobarbital. After ensuring the absence of a tail pinch response and pedal withdrawal reflex, animals were transcardially perfused with ice-cold phosphate-buffered saline (PBS) and then with 4% formaldehyde in PBS, as previously described (50). Brains were dissected out and immersed in 4% formaldehyde in PBS overnight at 4 °C. A Leica VT1000 S vibrating blade microtome was used to produce coronal brain sections (30 µm).

Tissue sections were first treated with blocking solution (10% normal donkey serum [NDS], supplemented with 0.3% Triton X-100 in PBS) for 1 h at room temperature (RT). After, sections were incubated with primary antibodies diluted in 10% NDS at RT overnight in a humidified chamber. The primary antibodies used for immunohistochemistry were the following: glutamine synthetase (1:2,000, rabbit, Invitrogen, PA1-46165), aldolase C (1:500, rabbit, Atlas Antibodies, HPA003282), aldehyde dehydrogenase 1 family member L1 (ALDH1L1; 1:500, rabbit, abcam, ab87117), S100b (1:500, guinea pig, Synaptic Systems, 287004), NeuN (1:500, mouse, Millipore, MAB377; 1:1,000, guinea pig, Millipore, ABN90), nNOS (1:300, goat, abcam, ab1376), substance P receptor (1:200, rabbit, Millipore, AB5060), somatostatin (1:500, rat, Millipore, MAB354), calmodulin (1:1,000, rabbit, Swant, 465), calbindin D28K (1:10,000, rabbit, Swant, CB38), calretinin (1:2,000, rabbit, Swant, 7699/4), parvalbumin (1:5,000, rabbit, Swant, PV25; 1:100, guinea pig, Synaptic Systems, 195004), ChAT (1:100, goat, Millipore, AB144P; 1:1,000, sheep, abcam, ab18736), DARPP-32 (1:500, rabbit, Millipore, AB1656), and NPY (1:2,000, rabbit, Immunostar, 22940). A Monoclonal Antibody Labeling kit (Life Technologies) was used to directly conjugate Alexa Fluor 647 to the NeuN antibody from Millipore (clone A60). After washing in PBS, the following species-specific, fluorophore-conjugated secondary antibodies were used to reveal antibody staining (1:500; Cy3, Alexa Fluor 488, Alexa Fluor 647, and DyLight 405 from Jackson Immunoresearch). Cell nuclei were marked with 4′6 ′-diamidino-2-phenylindole (DAPI) (1 μg/mL, Sigma-Aldrich, D9542). EdU was revealed using the Click-iT EdU Alexa Fluor 647 imaging kit (Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"C10340","term_id":"1535411","term_text":"C10340"}}C10340) following the manufacturer’s specifications. To control for nonspecific signals, tissue sections were incubated with secondary antibodies alone. Sections were coverslipped with Antifade Mounting Medium (VECTASHIELD, Vector Labs, H-1000).