Supplementary Information s7

Supplementary Information

Supplementary Figure Legend

Supplementary Figure 1. The REM flip-flop switch is part of a cascading pair of switches that generate forebrain vs. brainstem-spinal manifestations of REM sleep. The VLPO and the ascending arousal systems, particularly the monoaminergic pathways (TMN, LC, Raphe), form a mutually inhibitory flip-flop switch that controls transitions into sleep. When the extended VLPO (VLPOex) is active, it further suppresses the brainstem monoaminergic nuclei, which removes a crucial excitatory input to the REM-off population and allows the REM-on population to gain ascendancy. The REM-on neurons in turn activate an array of brainstem and spinal pathways that produce the phenomena of REM sleep. The orexin or hypocretin neurons (ORX) play the role of a “finger” that, when active presses the wake-sleep flip-flop switch into the wake position by reinforcing the activity of the arousal neurons. Similarly, the orexin inputs to the vlPAG-LPT normally prevent the emergence of REM phenomena except during sleep (when the orexin neurons are nearly silent). Absence of the orexin influence not only permits unwanted switching of the wake-sleep state, but also allows REM phenomena (atonia during cataplexy, dreams during hypnagogic hallucinations) to emerge during wakefulness (i.e., without tripping the wake-sleep flip-flop switch). The neurons that contain melanin-concentrating hormone (MCH) have the same targets as the orexin neurons, but the opposite influence (mainly inhibitory) and opposite activity pattern (mainly REM active). Hence, their net effect is to reinforce the influence of the orexin neurons.

Acknowledgement of grant support

This work was supported by grants NS051609, MH55772, AG09975 and HL60292 from the United States Public Health Service.

1 Supplementary Methods Animals. Pathogen-free, adult, male Sprague-Dawley rats (275- 300 grams, Harlan) were individually housed and had free access to food and water. All animals were housed under controlled conditions (12 hr light starting at 07:00 am, 100 lux) in an isolated ventilated chamber maintained at 20-22 oC. All protocols were approved by the Institutional Animal Care and Use Committees of Beth Israel Deaconess Medical Center and Harvard Medical School. EEG/EMG lead implantation and sleep analysis. After animals were anesthetized with chloral hydrate (350mg/kg), the skull was exposed. Four EEG screw electrodes were implanted into the skull, in the frontal (2) and parietal bones (2) of each side, and two flexible EMG wire electrodes were placed into the neck muscles. The free ends of the leads were soldered into a socket that was attached to the skull with dental cement, and the incision was then closed by wound clips. One week after surgery, the sockets were connected via flexible recording cables and a commutator to a Grass polygraph and computer and signals were digitized by an Apple Macintosh computer running ICELUS (G Systems Inc). EEG/EMG was recorded at the end of third week after surgery for 24 hours. Tracer injections and lesions. Under chloral hydrate anesthesia (7% in saline, 350 mg/kg), a fine glass pipette was positioned at pre-calculated targets and injections made of tracers: CTB (1.0 %, 1-10 nl cholera toxin subunit B, CTB, List Biological), FG (5%, 3-20 nl Fluorogold, Molecular Probes), AAV-GFP (adeno-associated viral vector containing the gene for green fluorescent protein, 50-100 nl, Harvard Gene Therapy Initiative Research Vector Core); or toxins: ibotenic acid (10% of ibotenic acid, 20-30 nl, Sigma), orexin-saporin (20-100 nl, Advanced Targeting Systems), 5,7-DHT (25%, 50 nl 5-7-dihydroxytryptamine (5,7-DHT, Sigma), or 6- hydroxydopamine (10%, 100 nl injection into the fourth ventricle). After 2 additional min, the pipette was slowly withdrawn and the incision was closed with wound clips. Animals survived for 7 days. The coordinates were based on Paxinos and Watson’s rat brain atlas for tracer and neurotoxin injections, as listed below.

Table 2. Coordinates (millimeters) from bregma for tracer or neurotoxin injections. vlPA LPT PPT LDT vSL SLD PC MPB DR MnR PRF VMM M G D N S AP -8.04 - - -8.76 -9.24 -9.24 -9.24 -9.24 -7.8 -7.8 -7.8 11.4 0 8.04 8.28 ML 1.0 1.4 2.0 1.0 1.0 1.0 1.4 2.0 0 0 1.0 1.0 0 DV 5.5 6.5 6.4 6 7.2 6.6 6.4 6.8 6.0 8.0 8.0 8.0 5.0 VMM = ventromedial medulla; MS = medial septum; PC = precoeruleus region; PRF = pontine reticular formation. Perfusion. Animals were deeply anesthesized by chloral hydrate (500 mg/kg), then perfused with 50 ml saline followed by 500 ml 10% formalin through the heart. The brains were removed, post-fixed for 4 hr in 10% formalin, and then equilibrated in 20% sucrose in PBS overnight. Immunohistochemistry. The brains were sectioned on a freezing microtome at 40 µm into four series. Sections were washed in 0.1 M phosphate-buffered saline, pH 7.4 (PBS, 2 changes) and then incubated in the primary antiserum (rabbit polycolonal antibody agonist c-Fos, 1: 150,000, AB-5, residues 4-17 from human cFos, Oncogene; polycolonal goat antibody against ChAT, AB144, 1:2000, immunogen: human placental enzyme, Chemicon; goat polycolonal antibody against 5-HT, 1:10,000, AB125, immunogen = 5-Hydroxytryptamine-glutaraldehyde-Poly- lysine, Chemicon; mouse monocolonal antibody against TH, 1:30,000, Lot # 22941, isolated and purified from rat PC12 cells, DiaSorin; goat anti- CTB, 1:100,000, catalog # 114, verified by a single band when examined by SDS-polyacrylamide gels electrophoresis, List Biological; rabbit anti-GFP 1:20,000, raised against jellyfish GFP, 970-1001, Molecular Probes; monoclonal

2 mouse anti-parvalbumin 1:20,000, produced by hybridization of mouse myeloma cells with spleen cells from mice immunized with paravalbumin purified from carp muscles, lot#235, Swant) for one day at room temperature. Sections were then washed in PBS and incubated in biotinylated secondary antiserum (against appropriate species IgG, 1:1,000 in PBS) for one hour, washed in PBS and incubated in avidin-biotin-horseradish peroxidase conjugate (Vector) for 1 hour. Sections were then washed again and incubated in a 0.06% solution of 3,3- diaminobenzidine tetrahydrochloride (DAB, Sigma) plus 0.02% H2O2. The sections were stained brown with DAB only or black by adding 0.05% cobalt chloride and 0.01% nickel ammonium sulfate to the DAB solutions. The antibodies against Fos, TH, orexin, ChAT, C-Fos and serotonin all have been used extensively in our laboratory and their specificity has been established in rat brain in prior control studies 37. The parvalbumin antiserum stained a pattern of cellular morphology and distribution identical with previous studies. The GFP and CTB antisera did not stain control brains. In situ hybridization of GAD mRNA andVGLUT2 mRNA. Sections with immunostainning (CTB, or Fos, FG) were acetylated and hybridized overnight (55 oC) with a 35S-labeled cRNA probe synthesized from a plasmid containing the sequence of GAD67 (3.2 kb, generous gift from N. Tillakaratne, University of California, Los Angeles) or VGLUT2 (1615–1979 of GenBank entry AF271235). After a succession of one hour washes (2xSSC/1mM DTT, 50 oC; 0.2xSSC/1mMDTT, 55 oC; 0.2xSSC/1mM DTT, 60 oC), the tissue was treated with RNase-A (Boehringer-Mannheim, Indianapolis) and washed under conditions of increasing stringency, including a 30 min wash at 60°C in 0.1× SSC. The tissue was then dehydrated in alcohols and air-dried. Slides were developed in Kodak D-19, fixed, and then dehydrated and coverslipped. OX-2 mRNA in situ hybridization was described previously37. Cell counting. A one in four series of sections was used for cell counting analysis. We counted cells with a clearly defined nucleus in the vlPAG within a 400x400 m box and LPT within a 400x600 m box (see Figure 1) from three sections through the center of the region. To count cells in the ventral SLD, we constructed a 400x400 m box with the dorsal edge placed against the ventral border of Barrington’s nucleus from two adjacent sections (see Figure 3). A 400x300 m box was used to count cells in the dorsal SLD from the same sections. The precoeruleus region box was 200x200 m with its lateral edge along the mesencephalic trigeminal tract and its ventral edge along the ventral margin of the central gray matter. The MPB box was 200X500 m bounded by the ventral edge of the superior cerebellar peduncle dorsally and the mesencephalic trigeminal tract medially in three sections at the same level as the SLD. The PPT and LDT lesions were assessed by loss of ChAT-ir cells within the LDT and the PPT regions. Locus coeruleus (LC) lesions were assessed by counting TH-ir cells in the LC from 4-5 sections from the caudal edge of the precoeruleus region through the body of the LC. MnR and DRN lesions were determined by counting remaining 5-HT-ir neurons in 3 sections from the level of the vlPAG and LPT. Because immunostaining does not penetrate the full section thickness, stereological methods could not be used. Cell counts were corrected with Abercrombie’s formula based upon the mean diameter of nuclei. Because it is difficulty to control the lesion size with ibotenic acid, our lesions sometimes included nearby areas. Hence, we also placed control lesions adjacent to target areas. For instance, in some cases with lesions in the SLD or PPT, but also including the MPB, we found increased total sleep time. We controlled for this by cases with lesions involving the MPB but not the SLD or PPT, which showed similar degrees of sleep increase, thus suggesting that the change in sleep behavior was due to damage to the MPB. Statistical methods. Statistical differences in cell counts and sleep behavior were assessed with an ANOVA followed by post-hoc Student’s t-test.