Regulation of REM and Non-REM Sleep by Periaqueductal GABAergic Neurons.

Animals

All experimental procedures were approved by the Animal Care and Use Committee at the University of California, Berkeley. Optogenetic manipulation experiments were performed in male or female GAD2 -Cre (Jackson Laboratory stock 010702) and VGLUT2-Cre (016963) mice. Optrode recording, calcium imaging, lesion, and rabies-mediated trans-synaptic tracing experiments were all performed in male or female GAD2 -Cre, VGLUT2 - Cre , TH - Cre (008601), and ChAT - Cre (006410) mice. Animals were housed on a 12-h dark/12-h light cycle (light on between 0700 and 1900 hours). Animals with implants for EEG/EMG recordings, optogenetic stimulation, optrode recordings, or calcium imaging were housed individually. Mice injected with viruses for trans-synaptic tracing were housed in groups of up to six animals. Surgical procedures Adult (6–12-week-old) mice were anesthetized with isoflurane (3% induction, 1.5% maintenance) and placed on a stereotaxic frame. Body temperature was kept stable throughout the procedure by using a heating pad. After asepsis, the skin was incised to expose the skull, and the overlying connective tissue was removed. For access to the vlPAG, a craniotomy (1 mm diameter) was made on top of the right superior colliculus (4.6–4.8 mm posterior to bregma, 0.6–0.7 mm lateral). AAV2-EF1a-FLEX-ChR2–eYFP, AAV2-EF1a-FLEX–eYFP (produced by University of North Carolina Vector Core), AAV1-Syn-Flex-GCaMP6f (University of Pennsylvania Vector Core), or AAV-DJ-EF1a-FLEX-ChR2–eYFP (University of Stanford Gene Vector and Virus Core) was loaded into a sharp micropipette mounted on a Nanoject II or Nanoject III and injected slowly at a depth of 2.5 mm from the brain surface (300–500 nl, unilateral). An optical fiber (0.2 mm diameter) was inserted with the tip 0.2 mm above the virus injection site. To stimulate axon projections of vlPAG GABAergic neurons in the dorsolateral pons, the optical fiber was implanted within the pons (5.2–5.3 mm posterior to bregma, 0.9 mm lateral, 3.0 mm depth). For cell-type-specific lesion experiments, AAV2-Flex-taCasp3-Tevp (University of North Carolina Vector Core) was injected bilaterally into the vlPAG (200–300 nl). For optogenetic experiments, data from animals where the tip of the optical fiber was not within the aimed location were excluded. For lesion experiments, data from animals where virus expression was not restricted to the vlPAG were excluded. For EEG and EMG recordings, a reference screw was inserted into the skull on top of the left cerebellum. EEG recordings were made from two screws, one on top of hippocampus (about 2 mm posterior to bregma) and one on top of the prefrontal cortex (about 1 mm anterior to bregma). Two EMG electrodes were inserted into the left and right neck muscles. For EEG/EMG recordings in differential mode, four screws were fixed on the skull, two posterior and two anterior of bregma. All screws, electrodes, microdrives, connectors, and GRIN lenses required for EEG/EMG recordings, optrode recordings, and calcium imaging were secured to the skull using dental cement. Rabies virus tracing For retrograde tracing, AAV-CAG-FLEx loxP -TC 66T (1.0 × 10 12 gc/mL) and AAV8-CAG-FLEx loxP -RG (1.8 × 10 12 gc/mL) was mixed at a 1:1 ratio and stereotaxically injected. For tracing from glutamatergic neurons in the dorsolateral pons, 100 nl was injected into VGLUT2-Cre mice at −5.3 mm AP, 0.8 mm ML, 3.3 mm DV, unilaterally. For tracing from the LDT, 300–500 nl was injected in ChAT - Cre mice at −5.2 mm AP, 0.7 mm ML, 3.2 mm DV, bilaterally. For tracing from the noradrenergic neurons in the LC, 200–300 nl was injected into TH-Cre mice at −5.34 mm AP, 0.8 mm ML, 3.2 mm DV, unilaterally. Two weeks after the AAV injection, 300–400 nl of RV dG -eGFP+EnvA (1.0 × 10 9 cfu/ml) was injected into the same location. The injection pipette for AAV was tilted at 20 degrees from vertical while the injection pipette for RV was inserted vertically to limit virus coinfection along a common injection tract. We excluded animals with inefficient trans-synaptic labeling indicated by 20) and L -ratio (