Early-life stress can disrupt maturation of brain's reward circuits, promoting disorders NatureComms
Whole-cell patch-clamp recordings were obtained in the medial shell of the NAc . Data were collected with a Multiclamp 700B amplifier, Digidata 1550B , and using Clampex 11 . All recordings were acquired in voltage clamp at 34° Celsius and were digitized at 10 kHz and low pass filtered at 2 kHz. Patch pipette was filled with internal solution containing : 143 CsCl, 10 HEPES, 0.25 EGTA, 5 Phosphocreatine, 4 MgATP, 0.3 NaGTP and 1 mg/ml Neurobiotin .
. Briefly, after several washes with 0.01 M PBS containing 0.3% Triton X-100 , sections were treated for 30 min in 0.3% H-PBS-T, followed by blockade of nonspecific sites with 5% normal goat serum in PBS-T for 30 min. After rinsing with PBS three times, 5 min per wash, on a shaker at 95 rpm, sections were incubated for 48 h at 4 °C with rabbit anti-CRH primary antibody in the blocking solution . The sections were rinsed with PBS-T three times, 5 min per wash, followed by incubation in a 0.
. Sections were subjected to CRH IHC as described above. Fluorescein-conjugated tyramide was applied in the dark for 5–6 min on ice.Perfused brains of CRH-ires-Cre mice were post-fixed in 4% paraformaldehyde in 0.1 M PBS for 4–6 h, before cryoprotection in a 25% sucrose solution. Brains were frozen, then sectioned coronally into 20 µm thick slices using a Leica CM1900 cryostat .
For dual labeling, brain sections were first processed for Cre ISH as described above. Hybrid molecules were detected with anti-DIG mouse antibody , and cyanine 3-conjugated tyramide was applied. Sections were rinsed in PBS-T and then processed for CRH or ChR2-EYFP immunostaining. CRH signal was visualized with fluorescein-conjugated tyramide and ChR2-EYFP signal was visualized using anti-rabbit IgG conjugated to Alexa Fluor 488 .
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