Supplemental Information. Direct Activation of Sleep-Promoting. VLPO Neurons by Volatile Anesthetics. Contributes to Anesthetic Hypnosis

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1 Current Biology, Volume 22 Supplemental Information Direct Activation of Sleep-Promoting VLPO Neurons by Volatile Anesthetics Contributes to Anesthetic Hypnosis Jason T. Moore, Jingqiu Chen, Bo Han, Qing Cheng Meng, Sigrid C. Veasey, Sheryl G. Beck, and Max B. Kelz Supplemental Inventory Supplemental Figures and Tables Figure S1, related to Figure 6 Table S1, related to Figure 4 Table S2, related to Figure 6 Supplemental Experimental Procedures Supplemental References

2 Figure S1. Targeted Ablation of VLPO Galaninergic Neurons, Related to Figure 6 4x DAPI (A and C) and 20x galanin in situ hybridization (B and D) images of a sample sham (A and B) and lesioned (C and D) animal. White boxes in (A) and (C) correspond to region imaged in (B) and (D). White boxes in (B) and (D) are 250 μm tall by 400 μm wide and correspond to the VLPO. In the control animal (B), there is a preservation of galanin-containing neurons throughout the preoptic hypothalamus, including the VLPO. While galanin-containing neurons remain medially, laterally and dorsally, they are dramatically reduced in VLPO (D). 2 of 7

3 Table S1. Basic Properties of Postsynaptic Currents Recorded from NA(-) VLPO Neurons, Related to Figure 4 EPSCs IPSCs Frequency (Hz) Amplitude (pa) Half-Width (ms) Frequency (Hz) Amplitude (pa) Half-Width (ms) Baseline 2.1 ± ± ± ± ± ± 0.2 (n=7) 240 μm Iso (n=7) 2.2 ± ± ± ± ± ± 1.4* Isoflurane (240 μm) only produced an increase in IPSC half-width (*p < 0.05). Table S2. Table of Best-Fit Parameters for Dose-Response Curves, Related to Figure 6 6 Days Post-Op 24 Days Post-Op Sham Lesion Sham Lesion ED 50, % ED 50 95% C.I., % Hill slope Hill slope 95% C.I The top and bottom constants were constrained to be 1 and 0, respectively.

4 Supplemental Experimental Procedures Animals All studies were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania. Wild-type male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) aged 3-6 months were used for all experiments. Prior to all experiments, animals were housed in a group environment (3-5 mice per cage), were provided access to food and water ad libitum, and were kept on a 12 hour on-12 hour off light-dark cycle with zeitgeber time 0 (ZT 0; lights on) at 7 pm. Anesthetic Exposure and Cell Counting Following two consecutive days of habituation with 100% oxygen for 90 minutes, mice (n=66) were placed in cylindrical loss of righting reflex chambers [1,2] and exposed to either isoflurane (1.2%, 0.6%, or 0.3% vaporized in 100% oxygen), halothane (1.0% vaporized in 100% oxygen), or 100% oxygen either in the 2 hours immediately following lights out (ZT 12-14, period of maximal activity and wakefulness) or during lights on (ZT 4-6, period of maximal sleep [3]). Each limb of the fresh gas flow passed through a resistor in order to ensure equal gas flow (200 ml/min) across all chambers. The chambers were heated by a 37 C water bath, which has been shown to prevent heat loss in mice exposed to an anesthetic in multi-hour trials [2]. Throughout all experiments, the concentrations of the volatile anesthetics were measured in triplicate by a Riken-FI21 refractometer (A.M. Bickford, Wales Center, NY), and oxygen and carbon dioxide levels were monitored by a Poet 8500Q (Criticare Systems, Waukesha, WI). After two hours of exposure, mice were immediately sacrificed by rapid cervical dislocation followed by brain harvest, postfixation in 4% paraformaldehyde, and paraffin embedding. Sections were cut at 10 μm and standard c-fos immunohistochemistry was performed as previously described [2]. Briefly: slide-mounted sections were deparaffined and rehydrated, followed by blocking in 5% goat serum and incubated in rabbit anti c-fos primary antibody (1:1000, Calbiochem, La Jolla, CA) overnight at 4 C. Unbound primary antibody was washed prior to incubation in biotinylated goat anti-rabbit secondary antibody (1:200, Invitrogen, Carlsbad, CA) for one hour at room temperature, followed by treatment with an ABC-peroxidase solution (Vector Labs, Burlingame, CA). Immunoreactivity was visualized by DAB chromagene. Counting was performed on sections spanning from mm (rostral) to mm (caudal) relative to bregma by two blinded experimenters using a standardized 400 x 250 μm grid positioned 300 μm lateral to midline as a marker for the location of VLPO. Hyperbaric Reversal C57BL/6J mice (n=12) were exposed to either 1.0% isoflurane vaporized in 100% oxygen (isoflurane group) or 100% oxygen alone (control group) for 30 minutes beginning at ZT5. Following equilibration, all mice were rapidly transferred into hyperbaric chambers containing either 1.0% isoflurane vaporized in 100% oxygen (isoflurane group) or 100% oxygen (control group). In the isoflurane group, the anesthetic was continued for an additional 30 minutes at 1 atmosphere to ensure that no mouse spontaneously regained its righting reflex during the rapid transfer into the hyperbaric chamber. The hyperbaric chambers were lined with sodalime to scrub exhaled carbon dioxide. The pressure within the chambers was increased to 70 atm with helium and held constant for 2 hours. Despite no change in the vapor pressure of isoflurane during the hyperbaric exposure, direct observation through a viewport revealed successful pressure reversal with all mice regaining their righting reflexes. Following a decompression lasting 15 minutes, the mice were rapidly sacrificed with their brains dissected, fixed, and stained as described above. 4 of 7

5 VLPO Lesions Mice received a 5-10 nl (0.5 mg/ml) bilateral injection of either unconjugated saporin (sham, n=7) or galanin-saporin (lesion, n=17) at stereotaxic coordinates corresponding to VLPO relative to bregma: 5.65 mm dorsal, ±0.65 mm lateral, and 0.05 mm rostral. The saporins were obtained from Advanced Targeting Systems (San Diego, CA). Injections were made via picospritzer over the course of 5 minutes. All animals were assayed in parallel for anesthetic sensitivity at 6 and 24 days post-surgery using previously described methods [1,2,4]. Following two days of habituation, mice were exposed to isoflurane anesthesia in cylindrical gas-tight, controlled-environment chambers arrayed in parallel. The righting reflex was assayed at isoflurane concentrations ranging from 0.52% to 1.17%, with an average step size of 0.04%. The isoflurane concentration was increased every 15 minutes until all mice had lost their righting reflex at two successive concentrations. A mouse was considered to have lost its righting reflex if it was not able to fully right itself on all four limbs within two minutes following the chambers being rotated 180º. Following behavioral testing on day 24, mice were sacrificed via cardiac perfusion. Sections were cut frozen at a thickness of 40 μm and stained for NeuN (1:1000, Millipore, Billerica, MA). Cell counts were performed as described above. Only animals showing 80% neuronal cell loss or greater bilaterally in VLPO were included in the lesion cohort (n=15). Galanin In Situ Hybridization Sense and antisense digoxigenin-labeled riboprobes were respectively synthesized from full length cdna clones of galanin (cdna clone MGC:54666 IMAGE: plasmid, Invitrogen) using SP6 and T7 RNA polymerases (Roche), respectively. Forty-micron paraformaldehyde fixed, free-floating coronal sections containing the VLPO were incubated with 0.1 M triethanolamine and 0.25% acetic anhydride for 10 min, and then pre-hybridized for 2 hours before being hybridized with DIG-labeled riboprobes (200 ng per 140 µl) for 16 hours overnight at 55ºC in a humidified chamber. The sections were washed with 4, 2, and 1X SSC each for 30 min at room temperature, and 0.1X SSC at 60ºC for 1 hour. The signal was detected using Tyramide Signal Amplification kit (TSA, Invitrogen), according to the manufacturer s protocol, using an anti-dig HRP (Roche) together with an Alexa 488 fluorescent dye to detect the DIGlabeled probe. Hypothalamic Slice Preparation and Electrophysiology Anesthetic-naïve mice were sacrificed by cervical dislocation followed by decapitation, and the head was placed in ice-cold pre-oxygenated sucrose solution consisting of 248 mm sucrose, 2.5 mm KCl, 1.25 mm NaH 2 PO 4, 2.0 mm MgSO 4, 2.5 mm CaCl 2, 10 mm dextrose, and 26 mm NaHCO 3. Following rapid removal of the brain, 200 μm thick coronal sections were cut through the hypothalamus using a Leica microslicer (Leica, Allendale, NJ) while the brain was continuously oxygenated in the ice-cold sucrose solution. Sections were transferred to a holding chamber containing artificial cerebrospinal fluid (acsf) continuously bubbled with 95% O 2 /5% CO 2. The composition of the acsf was 124 mm NaCl, 2.5 mm KCl, 1.25 mm NaH 2 PO 4, 2.0 mm MgSO 4, 2.5 mm CaCl 2, 10 mm dextrose, and 26 mm NaHCO 3. Slices were maintained at 37ºC for 1 hour, then kept at room temperature until recording. High Mg 2+ /low Ca 2+ acsf used for some experiments had 9.68 mm MgSO 4 and 160 μm CaCl 2. Whole cell voltage clamp and current clamp techniques were conducted as previously described [5-7]. During recording, slices were maintained in a recording chamber continuously perfused with oxygenated 32ºC acsf at a rate of 2 ml/min. The VLPO was localized via comparisons with a mouse stereotaxic atlas [8]; based on subsequent analyses of images of the electrode position and biocytin immunohistochemistry, cells that fell outside of the VLPO bounding box described above were excluded from analysis. After a cell was visualized using

6 an infrared filter and differential interference contrast, a gigaohm seal was made with a recording electrode and then the membrane was ruptured to obtain a whole-cell recording. The electrodes had a resistance of 5-8 M when filled with an intracellular solution of 130 mm K- gluconate, 5 mm NaCl, 10 mm phosphocreatine disodium salt, 1mM MgCl 2, 10 mm HEPES, 0.02 mm EGTA, 0.5 mm Na 2 GTP, 2 mm MgATP, and 0.1% biocytin (ph 7.3, mosm); some voltage-clamp recordings were performed with an intracellular solution of 70 mm K- gluconate, 70 mm KCl, 2 mm NaCl, 10 mm phosphocreatine disodium salt, 10 mm HEPES, 4 mm EGTA, 0.3 mm Na 2 GTP, 4mM MgATP, and 0.1% biocytin (ph 7.3, mosm). Series and membrane resistances were monitored regularly throughout the experiments, and recordings were discarded if these parameters changed significantly from baseline. Cells were initially characterized in current-clamp mode, then baseline and drugresponses were recorded by alternating between current- and voltage-clamp modes using an Multiclamp 700 amplifier, a Digidata 1320 A/D converter and pclamp 10 software (Axon Instruments, Foster City, CA). VLPO neurons were classified according to whether they showed a depolarizing (NA(+); n=15) or hyperpolarizing (NA(-); n=34) response to the administration of 100 μm norepinephrine (NA) [9,10]. Non-volatile drugs were diluted from stock in fresh oxygenated acsf immediately prior to use. Isoflurane was sonicated into solution in preoxygenated acsf, then stored in a gas-tight syringe. The isoflurane diluted in acsf was delivered via a syringe pump to prevent evaporation; anesthetic concentrations of samples taken from the recording chamber were verified by mass spectrometry according to published protocols [2,4]. Traces were analyzed using Clampfit 10.2 (Axon Instruments, Foster City, CA), MiniAnalysis (Synaptosoft, Fort Lee, NJ), and custom code written in MATLAB (The MathWorks Inc, Natick, MA). Statistics Unless otherwise stated, all data are reported as means ± standard error. One- and two-way ANOVAs were used to test for statistical significance, followed where appropriate by post-hoc t- tests corrected for multiple comparisons; a p-value 0.05 was considered significant. For the behavioral dose-response data in the lesion experiment, a sigmoid dose-response function (Prism 5.0, GraphPad Software, San Diego, CA) was used to find the best fit curves for the population data. The bottom and top parameters were held constant at 0 and 1, respectively, while the ED 50 and Hill slope parameters were unconstrained. 6 of 7

7 Supplemental References 1. Sun Y, Chen J, Pruckmayr G, Baumgardner JE, Eckmann DM, et al. (2006) High throughput modular chambers for rapid evaluation of anesthetic sensitivity. BMC Anesthesiol 6: 13. doi: / Kelz MB, Sun Y, Chen J, Cheng Meng Q, Moore JT, et al. (2008) An essential role for orexins in emergence from general anesthesia. Proc Natl Acad Sci USA 105: doi: /pnas Pack AI, Galante RJ, Maislin G, Cater J, Metaxas D, et al. (2007) Novel method for highthroughput phenotyping of sleep in mice. Physiol Genomics 28: doi: /physiolgenomics Friedman EB, Sun Y, Moore JT, Hung H-T, Meng QC, et al. (2010) A conserved behavioral state barrier impedes transitions between anesthetic-induced unconsciousness and wakefulness: evidence for neural inertia. PLoS ONE 5: e doi: /journal.pone Beck SG, Pan Y-Z, Akanwa AC, Kirby LG (2004) Median and dorsal raphe neurons are not electrophysiologically identical. J Neurophysiol 91: doi: /jn Crawford LK, Craige CP, Beck SG (2010) Increased intrinsic excitability of lateral wing serotonin neurons of the dorsal raphe: a mechanism for selective activation in stress circuits. J Neurophysiol 103: doi: /jn Calizo LH, Ma X, Pan Y-Z, Lemos J, Craige C, et al. (2011) Raphe serotonin neurons are not homogenous: Electrophysiological, morphological and neurochemical evidence. Neuropharmacology. doi: /j.neuropharm Franklin KBJ, Paxinos G (2007) The Mouse Brain in Stereotaxic Coordinates. 3rd ed. New York: Academic Press. 9. Gallopin T, Fort P, Eggermann E, Cauli B, Luppi P-H, et al. (2000) Identification of sleeppromoting neurons in vitro. Nature 404: doi: / Liu Y-W, Li J, Ye J-H (2010) Histamine regulates activities of neurons in the ventrolateral preoptic nucleus. J Physiol (Lond) 588: doi: /jphysiol