GoKawiilAnimals
Animal care and surgical procedures were approved and overseen by the Harvard University Institutional Animal Care and Use Committee and the Harvard Center for Comparative Medicine or the Institutional Animal Care and Use Committee at Boston Children’s Hospital. The following mouse lines were used: C57BL/6 (Jackson Laboratory, 000664), GRfl/fl (Jackson Laboratory, 021021), Vglut1cre (Jackson Laboratory, 023527), Vgatcre (Jackson Laboratory, 028862), Olig2cre (Jackson Laboratory, 025567), Sun1-GFPfl/fl (Jackson Laboratory, 021039), and PvalbFlpO (Jackson Laboratory, 022730). Mice were maintained in a reverse 12 h light:12 h dark cycle (22:00–10:00) in a temperature- and humidity-controlled environment and provided food and water ad libitum. For DR conditions, mice were maintained in chronic darkness until collection. To control for circadian rhythm and to prevent light stimulation of DR mice, mice were collected under red light between 10:00–12:00 (Zeitgeiber time 12–14 h) for all genomic, imaging, ELISA and LC–MS experiments. Male and female mice were randomly assigned to experimental groups and used in similar proportions. For the developmental genomic experiments, mice were collected between P7 and P35. Details of animal age and sex are indicated in each protocol section. For imaging experiments, mice were collected at P21, P28, P35 and 8–12-weeks of age (adult). For ELISA and LC–MS experiments, serum samples were collected at P10, P14, P21 and P28. For electrophysiology experiments, mice were patched between P33 and P35. For CORT treatment experiments, DR C57BL/6 mice received either 10 mg kg−1 CORT or corn oil (vehicle) at P14 via intraperitoneal injection under red light.
Cell lines
NIH/3T3 cells (ATCC, CRL-1658) and HEK293T cells (ATCC, CRL-3216) were maintained in standard DMEM supplemented with 10% CCS, sodium pyruvate, MEM non-essential amino acids, and penicillin/streptomycin. Before GR CUT&RUN, 3T3 cells were grown in phenol-red-free DMEM containing 10% charcoal-stripped FBS for 48 h and then treated with 200 nM dexamethasone or vehicle (0.01% ethanol) for 2 h. Before Flag immunoprecipitations, HEK293T cells were grown in phenol-red-free DMEM containing 10% charcoal-stripped FBS for 24 h and then treated with 200 nM dexamethasone for 2 h.
Serum collection
Mice were anaesthetized with 10 mg ml−1 ketamine and 1 mg ml−1 xylazine in PBS by intraperitoneal injection for terminal blood draws. Blood was collected from the left atrium of the heart with an insulin syringe. To isolate serum, blood was incubated for 30 min at room temperature and then centrifuged at 1,300g for 10 min at 4 °C. The supernatant (serum) was isolated and stored at −80 °C prior to corticosterone ELISA and LC–MS.
Neonatal stereotactic surgery
P0–P1 wild-type, GRfl/fl or PVFlp/Flp;GRfl/fl mice were anaesthetized on ice and positioned within a neonatal adapter (Stoelting 51625) on a stereotaxic frame (Kopf) in which the temperature of the mouse was maintained around 4 °C using an ethanol bath containing dry ice. A small patch of skin above the V1 injection site (medial–lateral: ±1.8 mm; anterior–posterior: +0.5 mm from lambda; dorsal–ventral: −0.1 mm) was cut, and a small opening into the skull was drilled with a 30G needle. AAV (500 nl) was injected at approximately 200 nl/min, and the pipette was left in place for 5 min after infusion to allow for viral spreading. For genomic and electrophysiology assays required broad infection, mice were injected bilaterally, whereas for histology experiments, mice were injected unilaterally. Following injection, pups were recovered at 37 °C before being returned to their home cage.
Adult stereotactic surgery
For the adult PNN staining and ODP experiments, GRfl/fl mice were anaesthetized by isoflurane inhalation (3–5% induction, 1% maintenance) and positioned within a stereotaxic frame (Kopf). Animal temperature was maintained at 37 °C with a heat pad. Fur around the scalp area was removed with a shaver and sterilized with three alternating washes with betadine and 70% ethanol. A burr hole was drilled through the skull above V1 (medial–lateral: ±2.5 mm; anterior–posterior: −3.5 mm from bregma; dorsal–ventral: −0.5 mm and −0.25 mm). To broadly target superficial and deep cortical layers, 500 nl AAV was injected with a glass pipette at a depth of −0.5 mm (~200 nl/min), and an additional 500 nl was injected at a depth of −0.25 mm. After each injection, the pipette was left in place for 5 min to allow for viral spreading. All mice received postoperative analgesic (buprenorphine slow-release formulation, 1 mg kg−1).
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