Radioimmunoassay

All procedures followed the Institute of Laboratory Animal Research guidelines and were approved by the Animal Care and Use Committee of the National Institute of Mental Health. Transgenic mice expressing HSV-TK under the GFAP promoter were generated from a previously-generated plasmid^{28 (link)} using standard techniques and bred on a mixed C57Bl/6:CD-1 background. Male v-WT and v-TK mice were treated with valganciclovir for 8 weeks (dexamethasone experiment), 10-19 weeks (endocrine), 12 weeks (behavior) or 4 weeks (histology; histology after 12 weeks in Supplementary Fig. 1), beginning at 8 weeks of age. Male C57Bl/6 mice were irradiated under pentobarbital anesthesia, as described previously^{29 (link)}, and tested 9 weeks later. For immunohistochemical analyses, mice were given BrdU 6 weeks (for PVN analysis) or 24 hours prior to sacrifice, brain sections were immunostained as previously described^{29 (link)}, and labeled cells were counted stereologically.
Serum corticosterone was measured by radioimmunoassay (MP Biomedicals) from submandibular blood samples obtained directly from the home cage condition or after exploration of a novel box, restraint, or isoflurane exposure. For the dexamethasone suppression test, dexamethasone (Sigma; 50 μg/kg in propylene glycol) or vehicle were injected 90 min prior to restraint, and blood was sampled immediately following 10 min restraint.
Behavioral tests were performed following 30 min of restraint or directly from the home cage. Different cohorts of mice were tested in the NSF test, elevated plus maze, forced swim test and sucrose preference test as previously described.^{12 (link), 18 (link), 21 , 30 (link)} Statistical analyses were performed by t-test, log-rank test, or ANOVA with Fisher's LSD test for post hoc comparisons. Significance was set at P<0.05.

C57BL/6J male mice (2–5 months of age) were housed individually on a 12 hr/12 hr light/dark schedule with lights on at 7 A.M. (ZT0) and handled for 6 days. Mice were sleep-deprived (SD) in their home cages for 5 hours by gentle handling beginning at ZT5 or left undisturbed (non-sleep-deprived mice, NSD). For contextual fear conditioning experiments, animals were placed in a novel chamber for 3 minutes, and received a 2-second, 1.5 mA footshock after 2.5 minutes. Half of the mice were deprived of sleep for 5 hours post-training. Mice received intra-peritoneal injections of rolipram (ROL; 1 mg/kg) or vehicle (2% DMSO in 0.9% saline) immediately and 2.5 hours post-training. Testing of contextual memory was performed 24 hours after training in the trained context and 48 hours after training in a novel chamber.
Electrophysiological recordings were carried out as previously reported28 (link). 1-train LTP was induced by a single 100 Hz, 1-second duration train of stimuli. 4-train LTP consisted of 4 trains applied with a 5-minute inter-train interval; for massed 4-train LTP a 5-second inter-train interval was used. Theta-burst stimulation (TBS) consisted of 40-ms duration, 100 Hz bursts delivered at 5 Hz for 3 seconds (15 bursts of 4 pulses per burst, for a total of 60 pulses). Chemical LTP was induced by treatment of slices for 15 minutes with 5µM forskolin (FSK) in 0.1% ethanol, or a combination of 50µM forskolin and 30µM 3-isobutyl-1-methylxanthine (IBMX, in water). Rolipram (0.1µM in 0.1% DMSO) was applied for 60 minutes, beginning 30 minutes before tetanization.
cAMP assays on CA1 regions of hippocampal slices 10 minutes after treatment for 15 minutes with forskolin (50µM), forskolin + IBMX (30µM), or vehicle (0.1% EtOH) were performed by radioimmunoassay according to kit instructions. cAMP-specific PDE activity assays29 (link) and Western blots for PDE4A530 (link) were performed as previously described.
Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.