Vigilance no 23, 1

Description Introduction: Several studies support an implication of synaptic proteins in sleep regulation. Nonetheless, little is known about the role of the protein synthesis machinery in sleep. 4E-BP1 and 4E-BP2 are translational repressors, which once phosphorylated by mTORC1, release inhibition of protein synthesis. 4E-BP1 is highly expressed in the suprachiasmatic nucleus and has been implicated in circadian rhythms. 4E-BP2 that is widely expressed in the brain is critical for memory and plasticity. In addition, 4E-BP2 knockout (KO) mice exhibit autistic behaviors. However, there is no data on their implication in sleep regulation. Hence, the aim of this study is to verify the contribution of 4E-BP1 and 4E-BP2 in sleep regulation. Methods: Wild-type (WT), 4E-BP1 and 4E-BP2 KO mice were implanted with electroencephalography (EEG) and electromyography (EMG) electrodes and recorded continuously for 48 h including a 24-h baseline followed by a 6-h sleep deprivation (SD) starting at the beginning of the second day. Behavioral states were assessed based on EEG/EMG traces. Changes in mRNA expression after SD in the cerebral cortex were assessed by qPCR in the same three genotypes. Results: 4E-BP1 KO mice exhibit less wakefulness during the night without changes in sleep fragmentation. Changes in EEG activity were observed for wakefulness and NREM sleep, including less overall delta activity during baseline. However, after SD, the sleep of 4E-BP1 KO mice is similar to WT mice. Analyses for 4E-BP2 KO mice and of the gene expression response to SD are underway. Because 4E-BP2 is more expressed in the brain, a stronger phenotype is expected. Conclusion: Our findings support a role for the protein synthesis machinery in the regulation of sleep and wakefulness. More precisely, our results indicate that the translational repressor 4E-BP1 regulates vigilance state duration and EEG activity. P2 GSK3 responds to sleep loss and is involved in sleep regulation O'Callaghan, Emma; KHLGHATYAN, Jivan ; Lu, Emilie ; BEAULIEU, Jean Martin ; Mongrain, Valerie 1,2 Center for Advanced Research in Sleep Medicine and Research Center, Hôpital du Sacré-Coeur de Montréal, Department of Neuroscience, Université de Montréal, Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec and Université Laval Abstract Description Introduction: Sleep is required for normal brain function, and sleep loss detrimentally impacts cognitive functions and brain health. Moreover, most psychiatric conditions are characterized by sleep disturbances. However, molecular mechanisms underlying sleep regulation and disease-associated sleep disturbances are poorly understood. Glycogen Synthase Kinase 3 beta (GSK3) plays roles in the neuronal plasticity thought to underlie sleep, and is associated with cognitive functions and mood. We investigated how sleep loss impacts GSK3 level in the brain and assessed the contribution of GSK3 to sleep regulation. Methods: 1) Protein levels of GSK3 and phosphorylated-GSK3 (pGSK3) were examined in the murine cortex by Western Blot following a 6-h sleep deprivation (SD) or undisturbed baseline sleep. 2) Knockout (KO) of GSK3 was performed using the CRISPR/Cas9 system delivered by AAV viral vectors (one encoding Cas9 nuclease and one guide RNA targeting Gsk3 gene), in two cerebral cortex locations above which electrodes for electroencephalography (EEG) were implanted in adult male mice. Littermates were injected in parallel with a Cas9 coding AAV virus and a control AAV. EEG recordings were performed followed by vigilance state identification and spectral analysis to quantify vigilance state duration and quality. 3) The role of GSK3 in the EEG response to SD was simultaneously assessed in CRISPR/Cas9 KO and control animals. Results: 1) A 6-hDescription Introduction: Sleep is required for normal brain function, and sleep loss detrimentally impacts cognitive functions and brain health. Moreover, most psychiatric conditions are characterized by sleep disturbances. However, molecular mechanisms underlying sleep regulation and disease-associated sleep disturbances are poorly understood. Glycogen Synthase Kinase 3 beta (GSK3) plays roles in the neuronal plasticity thought to underlie sleep, and is associated with cognitive functions and mood. We investigated how sleep loss impacts GSK3 level in the brain and assessed the contribution of GSK3 to sleep regulation. Methods: 1) Protein levels of GSK3 and phosphorylated-GSK3 (pGSK3) were examined in the murine cortex by Western Blot following a 6-h sleep deprivation (SD) or undisturbed baseline sleep. 2) Knockout (KO) of GSK3 was performed using the CRISPR/Cas9 system delivered by AAV viral vectors (one encoding Cas9 nuclease and one guide RNA targeting Gsk3 gene), in two cerebral cortex locations above which electrodes for electroencephalography (EEG) were implanted in adult male mice. Littermates were injected in parallel with a Cas9 coding AAV virus and a control AAV. EEG recordings were performed followed by vigilance state identification and spectral analysis to quantify vigilance state duration and quality. 3) The role of GSK3 in the EEG response to SD was simultaneously assessed in CRISPR/Cas9 KO and control animals. Results: 1) A 6-h Vigilance Volume 25, number/numéro 1 17 SD increased levels of GSK3 and pGSK3 in the mouse cerebral cortex. Additionally, SD tended to decrease the pGSK3/GSK3 ratio suggesting increased GSK3 activity. 2) Preliminary EEG analysis suggests that KO of GSK3 increases the duration of wakefulness and decreases the duration of non-rapid eye movement sleep, in addition to increasing sleep intensity. 3) The effect of GSK3 KO on recovery following SD is being analyzed. Conclusion: These data suggest that GSK3 responds to wakefulness and sleep history, and participates in the regulation of sleep. P3 Working memory but not decision making deteriorates after cumulative sleep restriction Santisteban, Jose Arturo; Brown, Thomas ; Neibert, Mariana ; Gruber, Reut 1,2 Department of Psychiatry, McGill University, Douglas Mental Health University Institute, Douglas Mental Health University Instittue Abstract Description Introduction: Eighteen percent of young adults are sleep deprived. Sleep deprivation leads to significant neurobehavioral impairments and compromises job and academic performance and driving safety. Data is scarce and contradictory regarding the impact of cumulative sleep deprivation on neurobehavioural functoning (NBF). The objective of this study was to assess the impact of cumulative sleep restriction on the NBF of young adults. Methods: Participants Sixty-five healthy participants (ages 18-34). DesignA double-blind, placebo controlled, randomized trial. Participants were randomized into experimental and placebo conditions. Each participant completed a period of baseline protocol and an experimental period. The experimental period lasted 6 nights and had 2 conditions: 1) sleep restriction condition participants were asked to restrict their sleep by one hour per night 2) placebo conditionparticipants were asked to use a lamp which had no clinical effects for 30 minutes during day time. NBF was assessed at baseline (Day 1) and following sleep manipulation (Day 12). Measures Sleep duration was assessed using actigraphy, an accelerometer that measures sleep objectively based on movement. NBF was measured using the Cambridge Neuropsychological Test Automated Battery Spatial Span task and the Cambridge Gambling Task for working memory and decisionmaking, respectively. Results: Poorer performance on spatial span task was found in the experimental condition compared to the placebo condition, controlling for baseline spatial span length, sex, age, sleep efficiency during experimental week, baseline sleep duration, and chronotype, (F(1,49)=5.18, p < 0.05).No significant effects for sleep restriction were found on decision making measures when comparing the experimental condition to the placebo condition. Conclusion: Young adults performance on spatial working memory task deteriorated following cumulative sleep deprivation whereas their performance on decision making task was not affected. The present study indicates that sleep deprivation has a differential impact on the NBF of young adults.Description Introduction: Eighteen percent of young adults are sleep deprived. Sleep deprivation leads to significant neurobehavioral impairments and compromises job and academic performance and driving safety. Data is scarce and contradictory regarding the impact of cumulative sleep deprivation on neurobehavioural functoning (NBF). The objective of this study was to assess the impact of cumulative sleep restriction on the NBF of young adults. Methods: Participants Sixty-five healthy participants (ages 18-34). DesignA double-blind, placebo controlled, randomized trial. Participants were randomized into experimental and placebo conditions. Each participant completed a period of baseline protocol and an experimental period. The experimental period lasted 6 nights and had 2 conditions: 1) sleep restriction condition participants were asked to restrict their sleep by one hour per night 2) placebo conditionparticipants were asked to use a lamp which had no clinical effects for 30 minutes during day time. NBF was assessed at baseline (Day 1) and following sleep manipulation (Day 12). Measures Sleep duration was assessed using actigraphy, an accelerometer that measures sleep objectively based on movement. NBF was measured using the Cambridge Neuropsychological Test Automated Battery Spatial Span task and the Cambridge Gambling Task for working memory and decisionmaking, respectively. Results: Poorer performance on spatial span task was found in the experimental condition compared to the placebo condition, controlling for baseline spatial span length, sex, age, sleep efficiency during experimental week, baseline sleep duration, and chronotype, (F(1,49)=5.18, p < 0.05).No significant effects for sleep restriction were found on decision making measures when comparing the experimental condition to the placebo condition. Conclusion: Young adults performance on spatial working memory task deteriorated following cumulative sleep deprivation whereas their performance on decision making task was not affected. The present study indicates that sleep deprivation has a differential impact on the NBF of young adults. P4 A dedicated brainstem circuit controls REM sleep Fraigne, Jimmy ; Torontali, Zoltan ; Thomasian, Aren ; Li, Daniel ; Peever, John 1 University of Toronto, Dept. Cell & System Biology Abstract Description It remains unclear which neural circuit triggers REM sleep and REM sleep atonia, but glutamate neurons in the subcoeruleus (SubCGLUT) are hypothesized to control REM sleep as well as REM sleep atonia by activating GABA neurons in the ventral medulla (vMGABA). Here, we aimed to determine how optogenetic activation and inhibition of the SubCGLUT-vMGABA circuit impact REM sleep and REM sleep atonia. To control the neuronal activity of the glutamatergic SubC neurons, we bilaterally infused 200nL of an adeno-associated viral vector (AAV) containing either a light-sensitive excitatory opsin (AAV-EF1-DIO-ChETAeYFP) or a light-sensitive inhibitory opsin (AAVEF1-DIO-ARCH-eYFP) or an inert control protein (AAVEF1DIO-eYFP) into the SubC of 33 Vglut2-cre mice. Animals were instrumented for EEG and EMG recordings. SubCGLUT neurons were activated or silenced specifically during REM sleep. In another set of animals, the SubCGLUT-vMGABA circuit was inhibited continuously during REM sleep at the level of the vM. Only animals that had histological verification of eYFP expression in the SubC region and projection fibers in the vM were used for analysis. We used Vglut2 fluorescent in situ hybridization and/or Vglut2-tdTomato expressing mice to confirm the specificity of our virally-mediated opsin expression. We found that activation of SubCGLUT neurons increased the length of REM sleep episodes by 773% (n=5, p<0.01), and further decreased motor activity during REM sleep (n=5, p<0.01). In contrast, inhibition of SubC cells shortened the duration of REM sleep episodesDescription It remains unclear which neural circuit triggers REM sleep and REM sleep atonia, but glutamate neurons in the subcoeruleus (SubCGLUT) are hypothesized to control REM sleep as well as REM sleep atonia by activating GABA neurons in the ventral medulla (vMGABA). Here, we aimed to determine how optogenetic activation and inhibition of the SubCGLUT-vMGABA circuit impact REM sleep and REM sleep atonia. To control the neuronal activity of the glutamatergic SubC neurons, we bilaterally infused 200nL of an adeno-associated viral vector (AAV) containing either a light-sensitive excitatory opsin (AAV-EF1-DIO-ChETAeYFP) or a light-sensitive inhibitory opsin (AAVEF1-DIO-ARCH-eYFP) or an inert control protein (AAVEF1DIO-eYFP) into the SubC of 33 Vglut2-cre mice. Animals were instrumented for EEG and EMG recordings. SubCGLUT neurons were activated or silenced specifically during REM sleep. In another set of animals, the SubCGLUT-vMGABA circuit was inhibited continuously during REM sleep at the level of the vM. Only animals that had histological verification of eYFP expression in the SubC region and projection fibers in the vM were used for analysis. We used Vglut2 fluorescent in situ hybridization and/or Vglut2-tdTomato expressing mice to confirm the specificity of our virally-mediated opsin expression. We found that activation of SubCGLUT neurons increased the length of REM sleep episodes by 773% (n=5, p<0.01), and further decreased motor activity during REM sleep (n=5, p<0.01). In contrast, inhibition of SubC cells shortened the duration of REM sleep episodes Vigilance Volume 25, number/numéro 1 18 (n=6, p<0.01), and increased overall motor activity by 26% (n=5, p<0.01). Importantly, silencing SubCGLUT transmission at the vM (SubCGLUT-vMGABA) increased overall motor activity during REM sleep (n=3, p<0.05) without affecting REM sleep amounts (n=3, p=0.639). These results support the hypothesis that neurons in the SubCGLUT-vMGABA circuit control both REM sleep and REM sleep atonia. P5 Cataplexy produces muscle paralysis by recruiting the REM sleep circuit Torontali, Zoltan ; Fraigne, Jimmy ; Peever, John 3 University of Toronto, Department of Cell and Systems Biology, University of Toronto Department Cell and Systems Biology, University of Toronto Department of Cell and Systems Biology Abstract Description Cataplexy is the uncontrollable onset of skeletal muscle paralysis during wakefulness and is a debilitating symptom of the sleep disorder narcolepsy. Cataplexy is thought to result from the intrusion of REM sleep paralysis into wakefulness. The subcoeruleus nucleus (SubC) is hypothesized to be the core circuit that generates REM sleep paralysis. Here, we found that cataplexy is exacerbated by activating the SubC in narcoleptic mice and can produce cataplexy-like attacks in wild-type mice. We used chemogenetics to either activate or inactivate the SubC in both wildtype and narcoleptic mice with recording both encephalogram and electromyogram activity to define behavioural states. We found that SubC activation triggered cataplexy-like attacks in wild type mice and that SubC activation promoted cataplexy in narcoleptic mice, whereas its inhibition decreased these attacks. Our results support our working hypothesis that cataplexy is triggered by recruiting the brainstem circuitry that underlies REM sleep paralysis. Support: This research was funded by the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada (NSERC), and the Ontario Graduate Scholarship (OGS).Description Cataplexy is the uncontrollable onset of skeletal muscle paralysis during wakefulness and is a debilitating symptom of the sleep disorder narcolepsy. Cataplexy is thought to result from the intrusion of REM sleep paralysis into wakefulness. The subcoeruleus nucleus (SubC) is hypothesized to be the core circuit that generates REM sleep paralysis. Here, we found that cataplexy is exacerbated by activating the SubC in narcoleptic mice and can produce cataplexy-like attacks in wild-type mice. We used chemogenetics to either activate or inactivate the SubC in both wildtype and narcoleptic mice with recording both encephalogram and electromyogram activity to define behavioural states. We found that SubC activation triggered cataplexy-like attacks in wild type mice and that SubC activation promoted cataplexy in narcoleptic mice, whereas its inhibition decreased these attacks. Our results support our working hypothesis that cataplexy is triggered by recruiting the brainstem circuitry that underlies REM sleep paralysis. Support: This research was funded by the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada (NSERC), and the Ontario Graduate Scholarship (OGS). P6 Spontaneous Activity Networks Present In Sleep Houldin, Evan ; Ray, Laura ; Owen, Adrian ; Fogel, Stuart 2 Western University, University of Ottawa Abstract Description Although there is an extensive literature on spontaneous activity networks (SANs) present in the waking state, with select studies examining the impact of sleep on a subset of wake SANs (e.g. Horovitz, 2009, PNAS), no studies to date have fully characterized the repertoire of SANs present in sleep, with an aim to identify potentially new SANs. Although the function of SANs is currently undetermined, they are thought to support cognitive processing during wake (Smith, 2009, PNAS). Past studies have examined the impact of sedation (Boveroux, 2010, Anesthesiology) and disorders of consciousness on SANs (Boly, 2009, HBM), indicating the persistence of SANs such as the default mode network. Unlike these conditions however, sleep is a healthy alternate functional mode of the brain, one that may be supported by correspondingly unique SANs. This study examined whether sleep is accompanied by the presence of SANs different from the canonical waking set.Data from a combined EEG/fMRI sleep study was examined in each of wake, N2, N3 and REM states. 35 healthy subjects (20 female, mean age 23.7, without sleep disorders) were recruited for an evening study and asked to sleep naturally in the scanner for approximately 90 minutes, with no prior sleep deprivation. A separate wake resting state scan was also collected immediately prior to the sleep scan. Sleep scoring of the EEG data was used to classify the fMRI data according to each sleep stage. Independent component analysis was then performed on the datasets for each sleep stage and the resulting independent components were compared with spatial templates derived from a separate wake SAN study. No new SANs were discovered in any sleep stage, suggesting that the repertoire of SANs present in wake comprises the full set. Further, this suggests that sleep functions are subserved by unique interactions between the canonical SANs.Description Although there is an extensive literature on spontaneous activity networks (SANs) present in the waking state, with select studies examining the impact of sleep on a subset of wake SANs (e.g. Horovitz, 2009, PNAS), no studies to date have fully characterized the repertoire of SANs present in sleep, with an aim to identify potentially new SANs. Although the function of SANs is currently undetermined, they are thought to support cognitive processing during wake (Smith, 2009, PNAS). Past studies have examined the impact of sedation (Boveroux, 2010, Anesthesiology) and disorders of consciousness on SANs (Boly, 2009, HBM), indicating the persistence of SANs such as the default mode network. Unlike these conditions however, sleep is a healthy alternate functional mode of the brain, one that may be supported by correspondingly unique SANs. This study examined whether sleep is accompanied by the presence of SANs different from the canonical waking set.Data from a combined EEG/fMRI sleep study was examined in each of wake, N2, N3 and REM states. 35 healthy subjects (20 female, mean age 23.7, without sleep disorders) were recruited for an evening study and asked to sleep naturally in the scanner for approximately 90 minutes, with no prior sleep deprivation. A separate wake resting state scan was also collected immediately prior to the sleep scan. Sleep scoring of the EEG data was used to classify the fMRI data according to each sleep stage. Independent component analysis was then performed on the datasets for each sleep stage and the resulting independent components were compared with spatial templates derived from a separate wake SAN study. No new SANs were discovered in any sleep stage, suggesting that the repertoire of SANs present in wake comprises the full set. Further, this suggests that sleep functions are subserved by unique interactions between the canonical SANs. Vigilance Volume 25, number/numéro 1 19 P7 Frequency analysis and modeling of Evoked Response Potentials for wake-like and sleep-like states in cultured neural networks Xue, Mengran ; Taishi, Ping ; Roy, Sandip ; Krueger, James M. 2,1 Washington State UniversityPullman, Washington State UniversitySpokane Abstract Description Introduction: Evoked response potentials (ERP) reflect the functional state of brain networks. In vivo, ERP frequency content during sleep as compared to waking, has an amplified low-frequency content. Preliminary data indicate that sleep regulatory substance interleukin-1 beta (IL1) induces a similar amplification of low-frequency content in vitro. We develop generative mathematical model for the in vitro ERP signal. Methods: Co-cultures of neurons/glia derived from 1-day old C57BL6 mice were grown on multi-electrode arrays as described by Jewett et al Eurp. J. Neurosci. 2015. Recordings were taken after the addition of 0.0 or 0.1 ng IL1 s on day 15. Electrical-induced ERPs were calculated by signaling averaging all responses greater than 2 standard deviations above baseline noise levels. Frequency content of the averaged ERP was calculated by Fast Fourier Transform (FFT). Models were fit to the averaged ERP data to minimize the integrated-squared error between the model prediction and the data. Results: The ERP with IL1 had ~20dB higher delta-wave amplitudes compared to samples without IL1 added. The accentuation of the low-frequency band reflects the more common occurrence of larger-amplitude biphasic evoked responses when IL1 is added. The frequencyand timedomain characteristics of the ERPs together suggest a third-order linear transfer model for the evoked response with and without IL1. The average ERPs with and without IL1 exhibited a common set of modes (time constants), but nevertheless reflect an altered input-output response. Specifically, a linear transfer function with three poles is employed to match the trajectory of one ERP signal. The best fit models are as follows, for the with and without IL1 cases, respectively: H(s)=(-700s-30)/(s^3+6s^2+12s+8) e^(-0.1s) and H(s)=(50s+330)/(s^3+6s^2+12s+8) e^(-0.3s). The poles of both transfer functions (the denominators) are the same while the zeros (the numerators) are distinct from each other. Support: NIH/NINDS NS025378.Description Introduction: Evoked response potentials (ERP) reflect the functional state of brain networks. In vivo, ERP frequency content during sleep as compared to waking, has an amplified low-frequency content. Preliminary data indicate that sleep regulatory substance interleukin-1 beta (IL1) induces a similar amplification of low-frequency content in vitro. We develop generative mathematical model for the in vitro ERP signal. Methods: Co-cultures of neurons/glia derived from 1-day old C57BL6 mice were grown on multi-electrode arrays as described by Jewett et al Eurp. J. Neurosci. 2015. Recordings were taken after the addition of 0.0 or 0.1 ng IL1 s on day 15. Electrical-induced ERPs were calculated by signaling averaging all responses greater than 2 standard deviations above baseline noise levels. Frequency content of the averaged ERP was calculated by Fast Fourier Transform (FFT). Models were fit to the averaged ERP data to minimize the integrated-squared error between the model prediction and the data. Results: The ERP with IL1 had ~20dB higher delta-wave amplitudes compared to samples without IL1 added. The accentuation of the low-frequency band reflects the more common occurrence of larger-amplitude biphasic evoked responses when IL1 is added. The frequencyand timedomain characteristics of the ERPs together suggest a third-order linear transfer model for the evoked response with and without IL1. The average ERPs with and without IL1 exhibited a common set of modes (time constants), but nevertheless reflect an altered input-output response. Specifically, a linear transfer function with three poles is employed to match the trajectory of one ERP signal. The best fit models are as follows, for the with and without IL1 cases, respectively: H(s)=(-700s-30)/(s^3+6s^2+12s+8) e^(-0.1s) and H(s)=(50s+330)/(s^3+6s^2+12s+8) e^(-0.3s). The poles of both transfer functions (the denominators) are the same while the zeros (the numerators) are distinct from each other. Support: NIH/NINDS NS025378. P8 Evoked Response Potentials are attenuated in mice lacking the interleukin-1 receptor accessory protein. Sahabandu, Dinuka ; Taishi, Ping ; Roy, Sandip ; Krueger, James M. 1,2 Washington State UniversityPullman, Washington State UniversitySpokane Abstract Description Introduction: Interleukin-1 (IL1) is a well characterized sleep regulatory substance. IL1 signals in part via a neuron-specific IL1 receptor accessory protein (AcPb). Mice lacking AcPb have attenuated sleep homeostasis after sleep deprivation. Sleepand wake-like states occur in neuronal/glial co-cultures grown on multi-electrode arrays (Jewett et al Eurp. J. Neurosci. 2015). We now characterize evoked response potentials (ERPs) in cultured cells from wild type (WT) and AcPb knockout (KO) mice. In animals, sleep ERPs are larger than during waking. Methods: Co-cultures of neurons/glia derived from 1-day old AcPbKO and WT mice were grown on multi-electrode arrays as described by Jewett. Recordings were taken on culture days 5, 10 and 14 and after the addition of 0.0, 0.01 or 0.1 ng IL1 to culture wells on day 15. To elicit ERPs, electrical stimuli began at the time of IL1 treatment. ERPs for individual electrodes were calculated by signal averaging all responses greater than 2 standard deviations above baseline noise levels. Results: ERP peak to peak voltages and energy increase 200-500% in cells from WT mice on days 10 and 14 compared to day 5. ERP peak to peak voltages and energy increase 125-250% in cells derived from AcPbKO mice on days 10 and 14 compared to day 5. On day 14 absolute ERP values were greater in WT cells than cells from AcPbKO mice. With the addition of IL1, ERP values in WT cells increased in response to 0.1 ngIL1 whereas they decreased in AcPbKO cells. Conclusions: ERPs in mature WT cells are of larger magnitude and are more responsive to electrical stimulation with or without the presence of IL1 than are cells lacking AcPb. AcPb plays a role in the expression of ERPs that parallel the attenuated sleep homeostasis in AcPbKO mice. Support: NIH/NINDS (USA) grants NS025378 and NS096250Description Introduction: Interleukin-1 (IL1) is a well characterized sleep regulatory substance. IL1 signals in part via a neuron-specific IL1 receptor accessory protein (AcPb). Mice lacking AcPb have attenuated sleep homeostasis after sleep deprivation. Sleepand wake-like states occur in neuronal/glial co-cultures grown on multi-electrode arrays (Jewett et al Eurp. J. Neurosci. 2015). We now characterize evoked response potentials (ERPs) in cultured cells from wild type (WT) and AcPb knockout (KO) mice. In animals, sleep ERPs are larger than during waking. Methods: Co-cultures of neurons/glia derived from 1-day old AcPbKO and WT mice were grown on multi-electrode arrays as described by Jewett. Recordings were taken on culture days 5, 10 and 14 and after the addition of 0.0, 0.01 or 0.1 ng IL1 to culture wells on day 15. To elicit ERPs, electrical stimuli began at the time of IL1 treatment. ERPs for individual electrodes were calculated by signal averaging all responses greater than 2 standard deviations above baseline noise levels. Results: ERP peak to peak voltages and energy increase 200-500% in cells from WT mice on days 10 and 14 compared to day 5. ERP peak to peak voltages and energy increase 125-250% in cells derived from AcPbKO mice on days 10 and 14 compared to day 5. On day 14 absolute ERP values were greater in WT cells than cells from AcPbKO mice. With the addition of IL1, ERP values in WT cells increased in response to 0.1 ngIL1 whereas they decreased in AcPbKO cells. Conclusions: ERPs in mature WT cells are of larger magnitude and are more responsive to electrical stimulation with or without the presence of IL1 than are cells lacking AcPb. AcPb plays a role in the expression of ERPs that parallel the attenuated sleep homeostasis in AcPbKO mice. Support: NIH/NINDS (USA) grants NS025378 and NS096250 Vigilance Volume 25, number/numéro 1 20 P9 Brain transmembrane TNF signals upon soluble TNF receptor stimulation to inhibit evoked response potential amplitudes Chen, Liangyu ; Sahabandu, Dinuka ; Taishi, Ping ; Roy, Sandip ; Li, Shuyi ; Gibbons, Cody M. ; Krueger, James M. 3,2 Shengjing Hospital, China Medical University, Shenyang, China, Washington State University, Spokane, Washington State University, Pullman Abstract Description Introduction:Tumor necrosis factor alpha (TNF) can induce whole organism sleep, local unilateral intense sleep, sleep-like states within cortical columns (Churchill et al Neurosci 2008), and a deep sleep like state in neuronal/glial co-cultures in vitro (Jewett et al 2015 Eurp. J. Neurosci.). In brain, the dominate form of TNF is the 26kD transmembrane TNF (tmTNF). In sympathetic axons, the soluble TNF receptor (sTNFR) reverse signals via tmTNF. We determined whether the sTNFR, and if the absence of TNFRs, affected electrically-induced evoked response potentials (ERP) in neuronal/glial co-cultures using cells from wild type (WT) and double TNFR knockout (KO) mice. During sleep in vivo cortical ERPs are of greater magnitude than during waking. In vivo and in vitro neuronal/glial co-cultures, ERPs are enhanced by the soluble 17kD form of TNF which signals via the transmembrane TNFRs. Methods:Neuronal/glial co-cultures of cells from WT and double TNFR KO mice were prepared per Jewett et al and ERPs were determined on development days 5, 10, and 14, and on day 14 with and without the addition of 2 different doses of the sTNFR. Results: ERP peak to peak amplitudes were about 20% lower in the TNFR KO mice than in WT mice on culture days 10 and 14; on day 5 values were similar in cells from both strains. After addition of sTNFR to the cultures on day 14, ERP amplitudes and ERP energy were less in the cells derived from the TNFR KO mice than in those derived from WT cells. Conclusion:We conclude; the sTNFR, either by its absence or by the addition of exogenous sTNFR, affects the emergent network property, ERPs. Support: NIH/NINDS (USA) NS096250 and NS025378.Description Introduction:Tumor necrosis factor alpha (TNF) can induce whole organism sleep, local unilateral intense sleep, sleep-like states within cortical columns (Churchill et al Neurosci 2008), and a deep sleep like state in neuronal/glial co-cultures in vitro (Jewett et al 2015 Eurp. J. Neurosci.). In brain, the dominate form of TNF is the 26kD transmembrane TNF (tmTNF). In sympathetic axons, the soluble TNF receptor (sTNFR) reverse signals via tmTNF. We determined whether the sTNFR, and if the absence of TNFRs, affected electrically-induced evoked response potentials (ERP) in neuronal/glial co-cultures using cells from wild type (WT) and double TNFR knockout (KO) mice. During sleep in vivo cortical ERPs are of greater magnitude than during waking. In vivo and in vitro neuronal/glial co-cultures, ERPs are enhanced by the soluble 17kD form of TNF which signals via the transmembrane TNFRs. Methods:Neuronal/glial co-cultures of cells from WT and double TNFR KO mice were prepared per Jewett et al and ERPs were determined on development days 5, 10, and 14, and on day 14 with and without the addition of 2 different doses of the sTNFR. Results: ERP peak to peak amplitudes were about 20% lower in the TNFR KO mice than in WT mice on culture days 10 and 14; on day 5 values were similar in cells from both strains. After addition of sTNFR to the cultures on day 14, ERP amplitudes and ERP energy were less in the cells derived from the TNFR KO mice than in those derived from WT cells. Conclusion:We conclude; the sTNFR, either by its absence or by the addition of exogenous sTNFR, affects the emergent network property, ERPs. Support: NIH/NINDS (USA) NS096250 and NS025378. P10 Synchronization and delta power are emergent network properties tailored by interleukin-1 receptor accessory proteins Nguyen, Joseph T. ; Taishi, Ping ; Sahabandu, Dinuka ; Jewett, Kathryn A. ; Roy, Sandip ; Krueger, James M. 3,2 Washington State University, Spokane, Washington State UniversityPullman, Washington State University-