SLEEP 2011 Abstract Supplement

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A. Basic Science II. Cell and Molecular Biology and Genetics 0030 OPTOGENETIC STIMULATION ENHANCES C-FOS AND INTERLEUKIN-1β LEVELS IN CULTURED NEURONS Jewett K 1 , Sengupta P 1 , Kirkpatrick R 2 , Clinton JM 1 , Krueger JM 1 1 WWAMI Medical Education Program, Program in Neuroscience, and Sleep and Performance Research Center, Washington State University, Spokane, WA, USA, 2 Veterinary Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA, USA Introduction: The pro-inflammatory cytokine interleukin-1β (IL-1) has a role in sleep regulation in health and disease states including the chronic inflammation associated sleep disorders. Yet a mechanistic connection between specific activity at the cellular level and IL-1 expression remains to be established. We have recently developed the methods for optogenetic stimulation of cultured networks of neurons and glial cells. Using that in vitro tool, here we test the hypothesis that enhanced level of induced activity in neurons elevates IL-1 production in cultured neurons. Methods: Cortices from newborn mouse (C57BL/6) brains were dissected in ice-cold Hibernate-E solution, digested in 2mg/mL papain, and then mechanically dissociated. The cells were transfected, using nucleofection technique, with YFP-tagged Syn-Channelrhodopsin-2 (SynChR2-YFP). The transfected cells were grown on poly-D-lysine coated coverslips in a 5% CO 2 incubator at 37°C in serum free NbActiv4 medium. Within about 4 days, cells form complex neuronal/glial networks. On day 8 in vitro (DIV8), these networks were stimulated using a train of light pulses (random, frequency 10 Hz) from light emitting diodes (470nm) under ambient growth conditions. After stimulation the cells were fixed and probed for c-Fos and IL-1 expression using immunofluorescence techniques, and imaged using a confocal microscope. Results: Stimulation for 90 minutes shows increase in c-Fos (in nuclei) and IL-1 (in cytoplasm) expression for most neurons. The extent of c- Fos and IL-1 expression depended on the duration of stimulation. Conclusion: Optogenetic stimulation increases the expression levels of c-Fos confirming cell activation. The elevated level of IL-1 in neurons indicates that the expression of IL-1 is dependent, at least in part, on neuronal activation. Neuron activation-induced IL1 expression may provide a mechanism linking cell activity to sleep regulation. Support (If Any): NIH grants R01NS025378 and R01NS031453 to James Krueger. SynChR2-YFP construct was a gift from Karl Deisseroth lab (Stanford University). 0031 EXPRESSION OF CHANNELRHODOPSINS IN PARVALBUMIN-POSITIVE BASAL FOREBRAIN NEURONS Kim T 1 , McKenna JT 1 , Brown RE 1 , Winston S 1 , Chen L 1 , Strecker RE 1 , Kocsis B 2 , Deisseroth K 3 , McCarley RW 1 , Basheer R 1 1 Psychiatry, Harvard Medical School-Boston VA Healthcare System, West Roxbury, MA, USA, 2 Psychiatry, Beth Israel Deaconess Medical Center, Boston, MA, USA, 3 Psychiatry and Behavioral Sciences, Stanford University, Stanford, MA, USA Introduction: The basal forebrain (BF) plays an important role in the modulation of cortical activity across sleep-wake cycles via corticopetal projections of cholinergic and non-cholinergic neurons. Among noncholinergic neurons, an important component consists of parvalbumin (PV)-containing, γ-aminobutyric acid (GABA)ergic neurons whose firing rates increase during electroencephalographic (EEG) low-voltage fast activity. However, their precise contribution to cortical activation and sleep-wake regulation is not well understood. Therefore, we sought to selectively incorporate channelrhodopsins (light-activated ion channels) into PV-positive neurons in BF in order to investigate the effect on sleep and wakefulness. Methods: Adeno-associated virus with double-floxed Channelrhodopsin2 (ChR2)-eYFP was injected stereotactically into BF of two types of transgenic mice (n=2/each). In both transgenic mice, Cre recombinase expression was under the control of the PV promoter, thus ChR2-eYFP should be expressed specifically in PV neurons. In homozygous PV-Cre mice, colocalization of ChR2-eYFP with PV protein was confirmed by immunohistochemistry. The second strain of transgenic mice (PV-Tomato) was generated by crossing PV-Cre mice with Cre-reporter Rosa/ Tomato mice, creating heterozygous PV-Cre mice with Cre-dependent expression of Tomato (red fluorescence), allowing confirmation that viral expression was Cre-dependent without the need for immunohistochemistry. Following viral injection, co-expression of ChR2-eYFP and Tomato was compared. Results: Preliminary data showed that >90% of ChR2-eYFP-positive BF neurons expressed PV and ~75% of the BF PV-positive neurons expressed ChR2-eYFP in PV-Cre mouse. In the second condition of PV-Tomato mice >90% of ChR2-eYFP-positive BF neurons expressed Tomato, and >90% of Tomato-positive BF neurons expressed ChR2- eYFP. Conclusion: Our results confirm that ChR2-eYFP expression was exclusively dependent on Cre recombinase under the control of the PV promoter. These results suggest that the Cre-dependent AAV expression system will be a useful tool to enable selective stimulation of BF PV neurons in order to examine their role in cortical arousal. Support (If Any): VA Merit Award (RB), NIMH Grant MH 39683 (RWM), HL095491 (BK) 0032 CD73 IN SLEEP REGULATION Zielinski MR, Taishi P, Krueger JM WWAMI Medical Education Program, Sleep and Performance Research Center, Washington State University, Spokane, WA, USA Introduction: Adenosine and extracellular ATP have multiple physiological actions including sleep and cerebral blood flow regulation. However, the exact mechanisms of adenosine-modulated sleep remain unknown. Extracellular ATP and ADP are converted to AMP by the enzyme CD39. Extracellular AMP is in turn converted to adenosine by the enzyme CD73. Here, we investigate the role of CD73 in sleep regulation. Methods: Male CD73KO mice (provided by Thompson LF, Oklahoma Medical Research Foundation, Oklahoma City, OK) and C57BL/6 control mice were implanted with cortical EEG and EMG electrodes. Spontaneous sleep and sleep responses to 6 h of sleep deprivation (SD) were determined by established criteria. Power spectral analyses were also performed. Adenosine-related molecules in the somatosensory cortex following SD were analyzed by real-time PCR. Further, CD73 expression was analyzed in male rats throughout the time-of-day and following 6 h of SD. Results: Spontaneous NREMS was enhanced in CD73KO mice compared to controls (P = 0.046). REMS did not differ between strains. NREMS following SD was enhanced in controls (P = 0.004). In contrast, SD failed to affect NREMS duration in CD73KO mice. EEG SWA during NREMS following SD was enhanced in both strains (P = 0.044). CD39, adenosine deaminase, adenosine kinase, and adenosine A2a receptor expression did not differ between strains or in their responses to SD. Adenosine A1 receptor expression was significantly lower in CD73 mice compared to controls (P = 0.004) but enhanced following SD only in CD73KOs (P = 0.003). In rats, CD73 expression was elevated following SD and with time-of-day dependent increases in sleep propensity (P = 0.012 and P < 0.001, respectively). Conclusion: These data indicate that CD73 is involved with sleep regulation. Further, these data suggest that mechanisms upstream of adenosine and CD73, such as extracellular ATP, are involved in regulating sleep. Support (If Any): NIH NS025378, NS031453 SLEEP, Volume 34, Abstract Supplement, 2011 A14
A. Basic Science II. Cell and Molecular Biology and Genetics 0033 NEUROTENSIN RECEPTOR 1 (NTSR1) IS INVOLVED IN BOTH AFFECT AND SLEEP Fitzpatrick K 1 , Hotz Vitaterna M 1 , Olker C 1 , Millstein J 3 , Gotter AL 2 , Winrow CJ 2 , Renger JJ 2 , Turek FW 1 1 Neurobiology & Physiology, Northwestern University, Evanston, IL, USA, 2 Neuroscience Department, Merck Research Laboratories, West Point, PA, USA, 3 Statistical Genetics, Sage Bionetworks, Seattle, WA, USA Introduction: Neurotensin, a neuropeptide that interacts with the dopaminergic system, has been previously associated with many psychiatric illnesses such as anxiety, addiction, and schizophrenia. Based on results from a large-scale sleep-wake phenotype and genotype analysis of 269 adult male mice from a [C57BL/6J X (BALB/cByJ X C57BL/6J F1)] N2 segregating cross, we identified neurotensin receptor 1 (NTSR1) as a candidate gene for affect and sleep through quantitative trait loci (QTL) and expression QTL (eQTL) analyses. Methods: 10 control C57BL/6 mice and 10 NTSR1 knockout mice were behaviorally tested in the open field, elevated plus maze, tail suspension, and forced swim tests. The same animals were used to record baseline sleep, sleep deprivation, and subsequent recovery, of which 8 knockout animals and 9 wild type controls successfully completed the protocol. Results: NTSR1 knockouts showed increased anxious behavior as compared to the wild types in the open field test, with significant differences in distance traveled, percent of time spent in the center, and percent of time spent in the corners. NTSR1 knockouts also showed an increase in despair behavior in the tail suspension test, with significant differences in bouts of immobility. NTSR1 knockouts also displayed a lower percentage of sleep time spent in REM sleep in the dark phase, and larger diurnal variation of REM minutes than the wild type controls in baseline conditions. In the recovery period following sleep deprivation, the NTSR1 knockout animals presented with more wake and less non-REM rebound sleep. Conclusion: Anxiety and depression have long been associated with alterations in sleep. Despite a wealth of evidence for a genetic component for depression and anxiety, the specific genes and gene networks associated with these affective disorders remain largely unknown. Here we present that a knockout of a candidate gene in the region of a mouse QTL for sleep characteristics, known to have comorbidity in humans with affective disorders, shows similar behavioral characteristics associated with affective disorders in humans. Support (If Any): Merck & Co., Inc. 0034 TOLL-LIKE RECEPTOR 4 IS A REGULATOR OF MONOCYTE AND ELECTROENCEPHALOGRAPHIC RESPONSES TO SLEEP LOSS Wisor J, Clegern WC, Schmidt MA Washington State University, Spokane, WA, USA Introduction: Sleep loss triggers changes in inflammatory signaling pathways in the brain and periphery. The mechanisms that underlie these changes are ill-defined. The Toll-like receptor 4 (TLR4) activates inflammatory signaling cascades in response to endogenous and pathogen-associated ligands known to be elevated in association with sleep loss. TLR4 is therefore a possible mediator of some of the inflammation-related effects of sleep loss. Here we describe the baseline electroencephalographic sleep phenotype and the biochemical and electroencephalographic responses to sleep loss in TLR4-deficient mice. Methods: : TLR4-deficient mice and wild type controls were subjected to electroencephalographic and electromyographic recordings during spontaneous sleep/wake cycles and during and after sleep deprivation sessions of 3, 6 and 24-hr duration. Results: Relative to wild type control mice, TLR4-deficient mice exhibited an increase in the duration of the primary daily waking bout oc- curring at dark onset in a light/dark cycle. The amount of time spent in non-rapid eye movement sleep by TLR4-deficient mice was reduced in proportion to increased wakefulness in the hours immediately after dark onset. Subsequent to sleep deprivation, EEG measures of increased sleep drive were attenuated in TLR4-deficient mice relative to wild type mice. TLR4 was enriched 10-fold in brain cells positive for the cell surface marker CD11b (cells of the monocyte lineage) relative to CD11b-negative cells in wild type mouse brains. To assess whether this population was affected selectively by TLR4 knockout, flow cytometry was used to count F4/80- and CD45-positive cells in the brains of sleepdeprived and time of day control mice. While wild type mice exhibited a significant reduction in the number of CD11b-positive cells in the brain after 24-hr sleep deprivation, TLR4-deficient mice did not. Conclusion: These data demonstrate that innate immune signaling pathways active in the monocyte lineage, including presumably microglia, detect and mediate in part the cerebral reaction to sleep loss. Support (If Any): These experiments were supported by a Washington State University, Spokane Faculty Seed grant and a Washington State University New Faculty Seed grant. 0035 GENE EXPRESSION IN SLEEP-DEPRIVED PURINE TYPE 2X7 RECEPTOR KNOCKOUT MICE Honn KA, Davis CJ, Bohnet SG, Krueger JM WWAMI Medical Education Program, Sleep and Performance Research Center, Washington State University, Spokane, WA, USA Introduction: The purine type 2X7 receptor (P2X7R) is involved in cytokine release and sleep regulation. ATP is released during neurotransmission. Further, P2X7R expression changes with sleep loss and time of day. Mice lacking the P2X7R have reduced duration of NREMS and electroencephalography (EEG) slow wave activity (SWA) during NREMS following sleep deprivation (SD) as compared with wildtype mice (WT). We posit that the attenuated sleep responses to sleep loss observed in the P2X7R knockout (KO) mice is accompanied by changes in mRNAs previously linked to sleep regulation. Methods: Levels of mRNAs previously linked to sleep were determined using reverse transcriptase polymerase chain reaction (PCR). Two strains of mice were used, control C57BL6 and P2X7RKO mice. For each strain, one group of mice (N=8) was deprived of sleep by gentle handling during the last 6h of daylight and the control groups (N=8 each) were allowed to sleep undisturbed. All mice were sacrificed at dark onset. The hypothalamus and the somatosensory cortex were harvested, RNA extracted, cDNA prepared and PCR performed by standard methods. Results: SD enhanced brain-derived neurotrophic factor (BDNF), the P2X4 receptor (P2X4R), and adenosine deaminase (Ada) mRNAs in the cortices of both strains. In the hypothalamus of WT mice, BDNF, P2X4R, and Ada mRNAs also increased, but in this tissue these mRNAs failed to change in the P2X7RKO mice. SD enhanced tumor necrosis factor alpha (TNF) mRNA in WT cortices but not in P2X7RKO cortices. In contrast, in the hypothalamus, SD failed to alter TNF mRNA expression in WT mice but decreased it in the P2X7RKO mice. Conclusion: Differential brain expression in WT and P2X7RKO mice of BDNF, P2X4R, Ada and TNF mRNAs may be involved in the biochemical causal pathways that lead to the differential sleep responses to sleep loss exhibited by these two strains of mice. Support (If Any): NIH NS031453, NS025378 A15 SLEEP, Volume 34, Abstract Supplement, 2011
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