and administration of orexin in VLPO arouses mice from sleep sug- gesting that orexin might be inhibiting the sleep-active VLPOGABA/Gal neurons. However ...
A. Basic Sleep Science
IV. Neural Circuits
GABAergic neurons of the thalamic reticular nucleus (TRN), most of which contain the calcium-binding protein, parvalbumin (PV), are involved in spindle generation. The expression of the GABA synthetic enzyme, glutamate decarboxylase 67 (GAD67), is decreased in cortical PV GABAergic neurons of Sz brains examined postmortem. Thus, one possible explanation for the spindle deficit in Sz is a reduction of GAD67 levels in TRN neurons. Thus, here we selectively deleted GAD67 in the TRN and tested the effect on sleep spindles. Methods: An adeno-associated virus constitutively expressing a Cre recombinase-Green fluorescence protein (GFP) fusion protein (AAVCre-GFP) was injected into TRN of homozygous GAD67 floxed mice. The time course of reduction in GAD67 after viral injection was evaluated by immunohistochemistry. Sleep-wake activity (EEG/EMG electrodes) was recorded and sleep-wakefulness states were analyzed according to standard procedures. A custom-designed script (Matlab) was used to detect individual spindles (10-15Hz) during non-REM (NREM) sleep. Results: 1 week after viral injection GAD67 expression in TRN was similar to control (83.3% of control, n=2). However, a pronounced reduction was observed two (11.5%, n=2) and three weeks (6.9%, n=2) after viral injection. In one animal with confirmed unilateral viral transduction in the TRN, NREM spindle density (Spindles/min NREM sleep) during the light period was decreased after 2 weeks of AAV injection (64.3% of control) when compared to a non-transduced control mouse (N=1) or to the same mouse recorded one 1 week after injection. Conclusion: Our results suggest we can successfully delete the GAD67 gene in TRN neurons. Our preliminary data further suggest that reduced GAD67 in TRN neurons leads to a decreased spindle density, similar to that observed in schizophrenia patients. Support (IfAny): This work was supported by Veterans Affairs Medical Research Service Awards 5I01BX001356 (RWM), I01BX002774 (RES) and I01BX001404 (RB), a VA Career Development Award 1IK2BX002130 (JMM) and by NIH: R21-NS079866 (RB), RO1-MH039683 (RWM), PO1-HL095491 (RWM), R21 NS093000 (REB). JTM received partial salary compensation and funding from Merck MISP, but has no conflict of interest with this work.
subarachnoid infusion of either vehicle (n=6) or one of two doses of CRF-R1 agonist, Stressin (STR; 0.3 μg; n=6 or 1μg; n=6; 0.2 μl/min over 3 hrs starting at ZT 2). Sections through the MnPO and VLPO were harvested and immunostained for c- Fos protein and glutamic acid decarboxylase (GAD). Results: Infusion of 6µg ANT decreased waking and increased NREM and REM sleep compared to vehicle and, and increased in percentage of GAD+ neurons expressing Fos in the MnPO (10.6 ± 1.1% versus 18.0 ± 1.9%) and the VLPO (11.2 ± 1.6% versus 22.3 ± 1.9%). Infusion of 1 µg STR increased waking and decreased NREM and REM sleep compared to vehicle. High dose STR infusion also decreased the percentage of GAD+ neurons expressing Fos-IR in MnPO (18.1 ± 9.9 versus 9.9 ± 1.5%) and VLPO (22.1 ± 1.3% versus 12.1 ± 1.8%). Conclusion: Vigilance state changes occurring in response to increased CRF-R1 signaling may be mediated by suppression of preoptic sleep-promoting neuronal systems as well as by activation of arousal promoting neurons. Support (If Any): Supported by the Department of Veterans Affairs (Grant BX00155605).
0132 EFFECTS OF FOOD RESTRICTION ON STABILITY AND FRAGMENTATION OF DAILY ACTIVITY RHYTHMS Li P1, Patxot M1, To T1, Escobar C2, Buijs RM3, Hu K1 1 Division of Sleep and Circadian Disorders, Brigham & Women’s Hospital/Harvard Medical School, Boston, MA, 2Departamento de Anatomía, Facultad de Medicina, Edificio “B” 4° Piso, Universidad Nacional Autónoma de México, México, MEXICO, 3Departamento de Biología Celulary Fisiología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México, MEXICO Introduction: Locomotor activity in rodents displays robust daily/ circadian rhythms in synchrony with light/dark cycles. Restricted feeding (FR) in a fixed and limited time period each day also affects daily activity rhythms, causing increased activity a few hours before the feeding period — food anticipatory activity (FAA). Here we tested whether FR during the dark phase affects the stability and fragmentation of daily activity rhythms, as mediated through the dorsomedial hypothalamic (DMH) nucleus — a neuronal node involved in the FAA. Methods: Locomotor activity was collected under 12h:12h light-dark cycles from18 Wistar rats in three groups. (Group 1) Six intact rats had ad libitum food access for >2 weeks. (Group 2) Six intact rats underwent a 16-day FR protocol in which food was only available between Zeitgeber time (ZT) 6–8 h. (Group 3) Six rats with the DMH lesion (DMHx) underwent an ad-lib protocol of >12 days followed by a 16-day FR protocol. Inter-daily stability (IS) and intra-daily variability (IV) were calculated using the last 7 days under each condition. To determine whether FR affects activity patterns outside the FAAfeeding period, IS and IV after excluding data between ZT3-8h were also obtained. Results: In the intact rats, FR caused a decrease of 15.7%±5.3%(SE) in IS (p=0.015) and an increase of 58.6%±15.5% in IV (p=0.0036). The FR effects remained after excluding the 5-hour FAA-feeding period. The DMH lesion did not affect IS and IV with ad-lib food access (both p >0.1). In the DMHx rats, FR did not affect IS (p>0.1) and caused an increase of 29.5% (±10%) in IV (p = 0.037). But the FR effect on IV disappeared when excluding the FAA-feeding period (change=8.5%±6.5%; p>0.1). Conclusion: Restricted feeding during the inactive phase leads to unstable and fragmented daily activity patterns across the 24 hours. Lesioning the DMH eliminates these FR effects.
0131 EFFECTS OF CRF RECEPTOR-1 AGONIST AND ANTAGONIST ON SLEEP AND NEURONAL C-FOS EXPRESSION IN THE PREOPTIC HYPOTHALAMUS Kumar S, Gvilia I, Hsieh K, McGinty D, Szymusiak R V.A. Greater Los Angeles Healthcare System, Los Angeles, CA Introduction: Corticotropin releasing factor (CRF) neurons in the hypothalamic paraventricular nucleus and extended amygdala regulate endocrine and behavioral responses to stress. Sleep suppression frequently accompanies the stress response. Wake-promoting effects of CRF are mediated, in part, by excitation of hypocretin neurons in the lateral hypothalamus and noradrenergic neurons in the locus coeruleus. The extent to which CRF acts on sleep-promoting neuronal systems is not clear. We examined the effects of subarachnoid infusion of a CRF agonist and antagonist on sleep and on GABAergic neuronal activity in sleep regulatory nuclei in the rat preoptic hypothalamus, the median preoptic nucleus (MnPO) and ventrolateral preoptic area (VLPO). Methods: Adult Sprague Dawley rats were chronically implanted for electrographic sleep-wake state recording and a guide cannula targeting the ventral subarachnoid space rostral to the optic chiasm. In experiment 1, groups of rats were administered either vehicle (n=7) or one of two doses of CRF-receptor1 (R1) antagonist, Antalarmin (ANT; 2 μg; n=7 or 6 μg; n=7) by subarachnoid infusion (0.2 μl/min over 3 hrs starting at ZT 8). In experiment 2, groups of rats were administered A49
SLEEP, Volume 40, Abstract Supplement, 2017
A. Basic Sleep Science
IV. Neural Circuits
Support (If Any): This work was supported by NIH grants R00HL102241, R01AG048108 and P01AG009975 (to K.H.). P.L. was supported by the International Postdoctoral Exchange Fellowship 20150042 from the China Postdoctoral Council.
in promoting wakefulness and regulating sleep. Furthermore, patients with PWS, have fewer oxytocin neurons which we hypothesize reduces orexin signaling, resulting in daytime sleepiness, abnormal REM sleep and cataplexy. Methods: Using optogenetics and EEG, EMG, with video recordings, we have examined sleep/wake behavior in wild type, orexin null and MAGEL2 null, a model of PWS, mice. We have selectively expressed a light sensitive channel, ChR2, in oxytocin neurons of the PVH and have targeted optical fibers to illuminate the oxytocin fibers of the lateral hypothalamus. Results: Our results indicate activation of oxytocin positive fibers wake mice from sleep and increase the amount of wake during the day. Conclusion: Enhancing the activity level of the oxytocin system may help people with PWS to maintain wakefulness throughout the day. Further research with these mice should provide helpful insights into the daytime sleepiness people with PWS experience. Support (If Any): Foundation for Prader-Willi Research.
0133 OREXIN MEDIATES FEED-FORWARD INHIBITION OF VLPO SLEEP-ACTIVE NEURONS - A MECHANISM FOR CONTROLLING AROUSAL De Luca R, Park D, Bandaru S, Arrigoni E BIDMC/Harvard Medical School, Boston, MA Introduction: The ventrolateral preoptic area (VLPO) plays an essential role in the initiation and maintenance of sleep. It contains a cluster of sleep-active neurons that are GABAergic and co-expresses galanin (VLPOGABA/Gal). VLPO is innervated by wake-promoting neurons and the VLPOGABA/Gal neurons are strongly inhibited by noradrenaline, carbachol and serotonin. VLPO also receives input from orexin neurons and administration of orexin in VLPO arouses mice from sleep suggesting that orexin might be inhibiting the sleep-active VLPOGABA/Gal neurons. However, orexin is an excitatory peptide, thus orexin’s effect in the VLPO remains unclear. In this study we investigate the effect of orexin on VLPO neurons in brain slices. Methods: We recorded VLPO neurons in brain slices from WT and Vgat-IRES-cre mice. We filled the recorded cells with biocytin for anatomical localization. We recorded from VLPO GFP-labelled GABAergic neurons in Vgat-IRES-cre mice that were injected in VLPO with an AAV-flex-GFP. We identify VLPO-sleep active neurons based on the inhibitory responses to noradrenaline or carbachol and/ or by the presence of VGAT and galanine mRNAs using single cell RT-PCR. Results: We found a dual response to orexin in VLPO. About 60% of VLPO neurons were excited by orexin but 40% were inhibited through release of GABA. The VLPO neurons that were inhibited by orexin were also inhibited by carbachol or noradrenaline suggesting that they could be the VLPOGABA/Gal sleep-active neurons. We tested this hypothesis by recording VLPO GABAergic neurons (GFP-labelled). Orexin increased the frequency of spontaneous IPSCs in 50% of the VLPO GABAergic neurons and these neurons expressed galanin mRNA. Conclusion: Orexin inhibits VLPOGABA/Gal sleep-active neuron by increasing GABAergic afferent input. This GABAergic input could originate from the local neurons that are directly activated by orexin. We propose that during wakefulness VLPOGABA/Gal sleep-active neurons are strongly and directly inhibited by wake-promoting signals, such as noradrenaline, carbachol and serotonin. Whereas, orexin could act by activating local GABAergic neurons that in turn produce feed-forward inhibition of VLPOGABA/Gal sleep-promoting neurons. Support (If Any): R01NS091126 and P01HL095491.
0135 GABAERGIC NEURONS IN THE PREOPTIC HYPOTHALAMUS PROJECT TO MIDBRAIN STRUCTURES INVOLVED IN REM SLEEP CONTROL Kumar S, Hsieh K, Chase M, Szymusiak R V.A. Greater Los Angeles Healthcare System, Los Angeles, CA Introduction: GABAergic, sleep-active neurons in the preoptic hypothalamus are key components of hypothalamic-brainstem circuits that regulate sleep and arousal. Many sleep-active neurons recorded in the median preoptic nucleus (MnPO) and the ventrolateral preoptic area (VLPO) exhibit elevated discharge rates during both nonREM and REM sleep compared to waking. A population of neurons in the dorsal lateral preoptic area (DLPO) exhibit REM-active discharge. Preoptic nonREM/REM-active neurons may participate in control of the nonREM-REM cycle. To evaluate this, we examined projections of GABAergic neurons in the preoptic area to midbrain nuclei implicated in REM sleep control, the ventrolateral periaqueductal gray (vlPAG) and the dorsal raphe nucleus (DRN). Methods: Adult Sprague-Dawley rats received unilateral injections of the retrograde anatomical tracer, cholera toxin subunit-b Alexa Fluor 594 conjugate (CTb), targeting the vlPAG or DRN. After a 14-day survival period to allow for retrograde transport of tracer, rats were euthanatized and tissue sections through the preoptic hypothalamus were processed for visualization of CTb and of glutamic acid decarboxylase (GAD), a marker of GABAergic neurons. Results: CTb injections that successfully targeted the vlPAG resulted in a moderate density of CTb-labeled cell bodies in the VLPO with co-localization of GAD occurring in ~30% CTb+ neurons. The density of CTb labeling in the MnPO and DLPO was somewhat lower, with co-localization of GAD occurring in ~15–20% of CTB+ neurons. CTb injections in the DRN yielded moderate to high density of retrogradely labeled neurons in the VLPO, with co-localization of GAD in ~40–50% of CTb+ neurons. Compared to VLPO, co-localization of CTb and GAD in the MnPO and DLPO was low. Conclusion: This study confirmed anatomical connections between GABAergic neurons in sleep-regulatory regions of the preoptic area to REM sleep-regulatory regions in the midbrain, with the highest density of GABAergic projection neurons originating in the VLPO. These connections may functionally integrate neuronal systems that control sleep onset and nonREM sleep to brainstem REM sleep generating circuits. Support (If Any): Supported by the Department of Veterans Affairs (Grant BX00155605) and the National Institutes of Health (Grant R01-NS092383).
0134 OXYTOCIN FIBERS IN THE LATERAL HYPOTHALAMUS PROMOTE AROUSAL IN A MOUSE MODEL OF PWS Mahoney CE1, Kroeger D1, Grinevich V2, Scammell TE1 1 Harvard Medical School/BIDMC, Boston, MA, 2German Cancer Research Center, Heidleberg, GERMANY Introduction: Daytime sleepiness, disrupted sleep, and cataplexy-like falling episodes are common in Prader-Willi Syndrome (PWS), but the cause of these symptoms is unknown. The effects of oxytocin on sleep/ wake behavior are not well understood, but orexins activate the oxytocin neurons, and oxytocin activates the orexin neurons. We hypothesize that this positive feedback loop normally plays an essential role SLEEP, Volume 40, Abstract Supplement, 2017
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