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Study Objectives As in various brain regions the activity of gamma-aminobutyric acid (GABA) neurons is largely unknown, we measured in vivo changes in calcium fluorescence in GABA neurons in the zona incerta (ZI) and the ventral lateral periaqueductal grey (vlPAG), two areas that have been implicated in regulating sleep. Methods vGAT-Cre mice were implanted with sleep electrodes, microinjected with rAAV-DIO-GCaMP6 into the ZI (n = 6) or vlPAG (n = 5) (isoflurane anesthesia) and a GRIN (Gradient-Index) lens inserted atop the injection site. Twenty-one days later, fluorescence in individual vGAT neurons was recorded over multiple REM cycles. Regions of interest corresponding to individual vGAT somata were automatically extracted with PCA–ICA analysis. Results In the ZI, 372 neurons were identified. Previously, we had recorded the activity of 310 vGAT neurons in the ZI and we combined the published dataset with the new dataset to create a comprehensive dataset of ZI vGAT neurons (total neurons = 682; mice = 11). In the vlPAG, 169 neurons (mice = 5) were identified. In both regions, most neurons were maximally active in REM sleep (R-Max; ZI = 51.0%, vlPAG = 60.9%). The second most abundant group was W-Max (ZI = 23.9%, vlPAG = 25.4%). In the ZI, but not in vlPAG, there were neurons that were NREMS-Max (11.7%). vlPAG had REMS-Off neurons (8.3%). In both areas, there were two minor classes: wake/REMS-Max and state indifferent. In the ZI, the NREMS-Max neurons fluoresced 30 s ahead of sleep onset. Conclusions These descriptive data show that the activity of GABA neurons is biased in favor of sleep in two brain regions implicated in sleep.

Highlights • Single amygdala GABA neuron projects to both brain stem and cortex. • Bifurcating amygdala neurons may regulate emotions and muscle tone., The amygdala regulates multiple behaviors and emotions by projecting to multiple brain regions. However, the topographical distribution of amygdala neurons projecting to specific brain areas is still unclear. In the present study, we focus on determining whether single amygdala neurons project to the brain stem ventrolateral periaqueductal grey (vlPAG) and to the medial prefrontal cortex (mPFC). The mPFC neurons are involved in detecting emotional content while the vlPAG neurons are involved in regulating muscle tone. In VGAT-Cre mice a cre-inducible retrograde AAV tracer expressing tdTomato was microinjected into the ventrolateral periaqueductal grey matter (vlPAG), while a second retrograde AAV tracer with generic expression of GFP was delivered into the medial prefrontal cortex (mPFC). The results identified a subgroup of bifurcating GABAergic neurons in the central nucleus (CeA) and basolateral amygdala (BLA) that projects to vlPAG and mPFC. Based on these projections we suggest that amygdala GABA neurons may be involved in triggering emotionally-induced cataplexy in the sleep disorder, narcolepsy.

It was in the influenza pandemic of 1918 that von Economo identified specific brain regions regulating sleep and wake. Since then researchers have used a variety of tools to determine how the brain shifts between states of consciousness. In every enterprise new tools have validated existing data, corrected errors and made new discoveries to advance science. The brain is a challenge but new tools can disentangle the brain network. We summarize the newest tool, a miniature microscope, that provides unprecedented view of activity of glia and neurons in freely behaving mice. With this tool we have observed that the activity of a majority of GABA and MCH neurons in the lateral hypothalamus is heavily biased toward sleep. We suggest that miniscope data identifies activity at the cellular level in normal versus diseased brains, and also in response to specific hypnotics. Shifts in activity in small networks across the brain will help identify point of criticality that switches the brain from wake to sleep.

Introduction Emerging data shows that GABA neurons in the zona incerta (ZI) play a prominent role in regulating sleep. Transfer of the orexin gene into ZI neurons blocks cataplexy in narcoleptic orexin-knockout mice (Liu et al., JNeurosci, 2011) and vGAT GABA neurons in the ZI anticipate onset of NREM (Blanco-Centurion et al., SLEEP, 2021). To identify the subtype of GABA neurons regulating sleep, we examined the activity of Lhx6 neurons. This study tests the hypothesis that Lhx6 neurons show peak calcium fluorescence during sleep, and that the fluorescence is further increased after prolonged waking. Methods In Lhx6-cre mice (mice=4; all females; 4-5 mo), rAAV-DIO-GCaMP6s was delivered stereotaxically to the ZI (isofluorane anesthesia) and a GRIN lens, along with EEG and EMG electrodes were implanted. 21d later a miniscope (INSCOPIX) was attached, and after 3d of adaptation, sleep and fluorescence in individual Lhx6 neurons were recorded for 4h (baseline). On another day, the mice were kept awake for 6h (gentle handling; 9a-3p) and fluorescence in Lhx6 neurons was recorded for 2h during recovery sleep. The imaged data from the two recording periods (baseline and recovery sleep) was combined into a single data file and the change in fluorescence was determined against the mean image frame (F0). Previously, we and others found that the GCamP calcium fluorescence is a direct measure of action potentials and serves as a marker of activity. Results 97 neurons were automatically extracted (PCA-ICA analysis; blinded without knowledge of sleep state). In 66 neurons (68%) the average fluorescence was significantly higher during REM, NREM or both, compared to waking (Mixed Model ANOVA; SPSS25; P Conclusion This is the first study to measure fluorescence in individual neurons after sleep loss. We find that the fluorescence in two-thirds of ZI Lhx6 neurons is tightly linked to sleep, and that the average fluorescence is further increased after prolonged waking. Microendoscopy is superior to indirect measures such as c-FOS or photometry in gauging sleep pressure in individual neurons. Support (If Any) VA BX000798, 1K6BX004216.

Study Objectives Sleep and wake are opposing behavioral states controlled by the activity of specific neurons that need to be located and mapped. To better understand how a waking brain falls asleep it is necessary to identify activity of individual phenotype-specific neurons, especially neurons that anticipate sleep onset. In freely behaving mice, we used microendoscopy to monitor calcium (Ca2+) fluorescence in individual hypothalamic neurons expressing the vesicular GABA transporter (vGAT), a validated marker of GABA neurons. Methods vGAT-Cre mice (male = 3; female = 2) transfected with rAAV-FLEX-GCaMP6M in the lateral hypothalamus were imaged 30 days later during multiple episodes of waking (W), non-rapid-eye movement sleep (NREMS) or REMS (REMS). Results 372 vGAT neurons were recorded in the zona incerta. 23.9% of the vGAT neurons showed maximal fluorescence during wake (classified as wake-max), 4% were NREM-max, 56.2% REM-max, 5.9% wake/REM max, while 9.9% were state-indifferent. In the NREM-max group, Ca2+ fluorescence began to increase before onset of NREM sleep, remained high throughout NREM sleep, and declined in REM sleep. Conclusions We found that 60.2% of the vGAT GABA neurons in the zona incerta had activity that was biased towards sleep (NREM and REMS). A subset of vGAT neurons (NREM-max) became active in advance of sleep onset and may induce sleep by inhibiting the activity of the arousal neurons. Abnormal activation of the NREM-max neurons may drive sleep attacks and hypersomnia.

Sleep and wake are opposing behavioral states controlled by the activity of specific neurons. The neurons responsible for sleep/wake control have not been fully identifed due to the lack of in-vivo high throughput technology. We use the deep-brain calcium (Ca2+) imaging method to identify activity of hypothalamic neurons expressing the vesicular GABA transporter (vGAT), a marker of GABAergic neurons. vGAT-cre mice (n=5) were microinjected with rAAV-FLEX-GCaMP6M into the lateral hypothalamus and 21d later the Ca2+ influx in vGAT neurons (n=372) was recorded in freely-behaving mice during waking (W), NREM and REM sleep. Post-mortem analysis revealed the lens tip located in the zona incerta/lateral hypothalamus (ZI-LH) and the change in fluorescence of neurons in the field of view was as follows: 54.9% of the vGAT neurons had peak fluorescence during REM sleep (REM-max), 17.2% were NREM-max, 22.8% were wake-max while 5.1% were both wake+REM max. Thus, three quarters of the recorded vGAT neurons in the ZI-LH were most active during sleep. In the NREM-max group Ca2+ fluorescence anticipated the initiation of NREM sleep onset and remained high throughout sleep (NREM and REM sleep). In the REM-max neurons Ca2+fluorescence increased before the onset of REM sleep and stayed elevated during the episode. Activation of the vGAT NREM-max neurons in the zona incerta and dorsal lateral hypothalamus would inhibit the arousal neurons to initiate and maintain sleep.

The abnormal or excessive fat accumulation that impairs health is one of the criteria that fulfills obesity. According to epidemiological data, obesity has become a worldwide public health problem that in turn would trigger additional pathologies such as cardiorespiratory dysfunctions, cancer, gastrointestinal disturbances, depression, sleep disorders, just to mention a few. Then, the search for a therapeutical intervention aimed to prevent and manage obesity has been the focus of study during the last years. As one can assume, the increased prevalence of obesity has translated to search of efficient pharmaceuticals designed to manage this health issue. However, to further complicate the scenario, scientific literature has described that obesity is the result of interaction between multiple events. Therefore, pharmacological approaches have faced a serious challenge for develop the adequate treatment. Here, we argue that a wide range of non-pharmacological/invasive techniques can be used to manage obesity, such as diets, cognitive behavioral interventions, exercise and transcranial direct current stimulation. Combining these techniques may allow improving quality of life of obese patients.

Narcolepsy was first identified almost 130 years ago, but it was only 15 years ago that it was identified as a neurodegenerative disease linked to a loss of orexin neurons in the brain. It is unclear what causes the orexin neurons to die, but our strategy has been to place the gene for orexin into surrogate neurons in the validated mouse models of narcolepsy, and test whether it can block narcolepsy symptoms, such as cataplexy. In both the orexin knockout and the orexin-ataxin-3 mouse models of narcolepsy we have found that cataplexy can be blocked if the surrogate neurons are part of the circuit responsible for cataplexy. We have also determined that the orexin gene can be inserted into surrogate neurons in the amygdala to block emotion-induced cataplexy. Through the use of optogenetics we anticipate that it will be possible to preemptively block cataplexy.

Recent studies showed activation of the GABAergic neurons in the central nucleus of the amygdala (CeA) triggered cataplexy of sleep disorder narcolepsy. However, there is still no direct evidence on CeA GABAergic neurons’ real-time dynamic during cataplexy. We used a deep brain calcium imaging tool to image the intrinsic calcium transient as a marker of neuronal activity changes in the narcoleptic VGAT-Cre mice by expressing the calcium sensor GCaMP6 into genetically defined CeA GABAergic neurons. Two distinct GABAergic neuronal groups involved in cataplexy were identified: spontaneous cataplexy-ON and predator odor-induced cataplexy-ON neurons. Majority in the latter group were inactive during regular sleep/wake cycles but were specifically activated by predator odor and continued their intense activities into succeeding cataplexy bouts. Furthermore, we found that CeA GABAergic neurons became highly synchronized during predator odor-induced cataplexy. We suggest that the abnormal activation and synchronization of CeA GABAergic neurons may trigger emotion-induced cataplexy.

Recent studies showed activation of the GABAergic neurons in the central nucleus of the amygdala (CeA) triggered cataplexy of sleep disorder narcolepsy. However, there is still no direct evidence on CeA GABAergic neurons’ real-time dynamic during cataplexy. We used a deep brain calcium imaging tool to image the intrinsic calcium transient as a marker of neuronal activity changes in the narcoleptic VGAT-Cre mice by expressing the calcium sensor GCaMP6 into genetically defined CeA GABAergic neurons. Two distinct GABAergic neuronal groups involved in cataplexy were identified: spontaneous cataplexy-ON and predator odor-induced cataplexy-ON neurons. Majority in the latter group were inactive during regular sleep/wake cycles but were specifically activated by predator odor and continued their intense activities into succeeding cataplexy bouts. Furthermore, we found that CeA GABAergic neurons became highly synchronized during predator odor-induced cataplexy. We suggest that the abnormal activation and synchronization of CeA GABAergic neurons may trigger emotion-induced cataplexy.

Most brain neurons are active in waking, but hypothalamic neurons that synthesize the neuropeptide melanin-concentrating hormone (MCH) are claimed to be active only during sleep, particularly rapid eye movement (REM) sleep. Here we use deep-brain imaging to identify changes in fluorescence of the genetically encoded calcium (Ca(2+)) indicator GCaMP6 in individual hypothalamic neurons that contain MCH. An in vitro electrophysiology study determined a strong relationship between depolarization and Ca(2+) fluorescence in MCH neurons. In 10 freely behaving MCH-cre mice (male and female), the highest fluorescence occurred in all recorded neurons (n = 106) in REM sleep relative to quiet waking or non-REM sleep. Unexpectedly, 70% of the MCH neurons had strong fluorescence activity when the mice explored novel objects. Spatial and temporal mapping of the change in fluorescence between pairs of MCH neurons revealed dynamic activation of MCH neurons during REM sleep and activation of a subset of the same neurons during exploratory behavior. Functional network activity maps will facilitate comparisons of not only single-neuron activity, but also network responses in different conditions and disease. SIGNIFICANCE STATEMENT Functional activity maps identify brain circuits responding to specific behaviors, including rapid eye movement sleep (REM sleep), a sleep phase when the brain is as active as in waking. To provide the first activity map of individual neurons during REM sleep, we use deep-brain calcium imaging in unrestrained mice to map the activity of hypothalamic melanin-concentrating hormone (MCH) neurons. MCH neurons were found to be synchronously active during REM sleep, and also during the exploration of novel objects. Spatial mapping revealed dynamic network activation during REM sleep and activation of a subset of the neurons during exploratory behavior. Functional activity maps at the cellular level in specific behaviors, including sleep, are needed to establish a brain connectome.

Highlights • MCH neurons contain neither VGAT nor VGLUT2. • Majority of orexin neurons contain VGLUT2. • MCH neurons do not contain orexin., The neuropeptides orexin and melanin-concentrating hormone (MCH), as well as the neurotransmitters GABA (γ-Aminobutyric acid) and glutamate are chief modulators of the sleep-wake states in the posterior hypothalamus. To investigate co-expression of vesicular GABA transporter (VGAT, a marker of GABA neurons) and the vesicular glutamate transporter-2 (VGLUT2, a marker of glutamate neurons) in orexin and MCH neurons, we generated two transgenic mouse lines. One line selectively expressed the reporter gene EYFP in VGAT+ neurons, whereas the other line expressed reporter gene tdTomato in VGLUT2+ neurons. Co-localization between these genetic reporters and orexin or MCH immunofluorescent tags was determined using 3D computer reconstructions of Z stacks that were acquired using a multiphoton laser confocal microscope. Our results demonstrated that MCH neurons expressed neither VGAT nor VGLUT2, suggesting MCH neurons are a separate cluster of cells from VGAT+ GABAergic neurons and VGLUT2+ glutamatergic neurons. Moreover, most orexin neurons expressed VGLUT2, indicating these neurons are glutamatergic. Our data suggested that in the posterior hypothalamus there are four major distinct groups of neurons: VGAT+, orexin+/VGLUT2+, orexin-/VGLUT2+, and MCH neurons. This study facilitated our understanding of the role of these neurotransmitters and neuropeptides in relation to sleep/wake regulation.

Since the discovery of the neuropeptide melanin-concentrating hormone (MCH) more than half a century ago, MCH neurons have spawned hundreds of scientific studies. MCH neurons are a phylogenetically well-conserved group of hypothalamic neurons. The MCH neurons project diffusely throughout the CNS but only reside within a restricted area encompassing the incerto-lateral and perifornical hypothalamus. Here we review optogenetic studies focused on understanding the functions of the MCH neurons, particularly their role as sleep modulators. We attempted to put optogenetic findings in context with other studies focused on neuronal/behavioral modulation by MCH or its receptors. We also laid a theoretical framework to understand better the data on MCH neuronal activity in relation to the activity of the orexin neurons. For readers not familiar with optogenetics, we also went over major developments in this new field.

Neurons containing melanin-concentrating hormone (MCH) are located in the hypothalamus. In mice, optogenetic activation of the MCH neurons induces both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep at night, the normal wake-active period for nocturnal rodents [R. R. Konadhode et al. (2013) J. Neurosci., 33, 10257-10263]. Here we selectively activate these neurons in rats to test the validity of the sleep network hypothesis in another species. Channelrhodopsin-2 (ChR2) driven by the MCH promoter was selectively expressed by MCH neurons after injection of rAAV-MCHp-ChR2-EYFP into the hypothalamus of Long-Evans rats. An in vitro study confirmed that the optogenetic activation of MCH neurons faithfully triggered action potentials. In the second study, in Long-Evans rats, rAAV-MCH-ChR2, or the control vector, rAAV-MCH-EYFP, were delivered into the hypothalamus. Three weeks later, baseline sleep was recorded for 48 h without optogenetic stimulation (0 Hz). Subsequently, at the start of the lights-off cycle, the MCH neurons were stimulated at 5, 10, or 30 Hz (1 mW at tip; 1 min on - 4 min off) for 24 h. Sleep was recorded during the 24-h stimulation period. Optogenetic activation of MCH neurons increased both REM and NREM sleep at night, whereas during the day cycle, only REM sleep was increased. Delta power, an indicator of sleep intensity, was also increased. In control rats without ChR2, optogenetic stimulation did not increase sleep or delta power. These results lend further support to the view that sleep-active MCH neurons contribute to drive sleep in mammals.

Narcolepsy is a chronic sleep disorder linked to the loss of orexin-producing neurons in the hypothalamus. Cataplexy, a sudden loss of muscle tone during waking, is an important distinguishing symptom of narcolepsy and it is often triggered by strong emotions. The neural circuit underlying cataplexy attacks is not known, but is likely to involve the amygdala, a region implicated in regulating emotions. In mice models of narcolepsy, transfer of the orexin gene into surrogate neurons has been successful in ameliorating narcoleptic symptoms. However, it is not known whether this method also blocks cataplexy triggered by strong emotions. To examine this possibility, the gene encoding mouse prepro-orexin was transferred into amygdala neurons of orexin-knockout (KO) mice (rAAV-orexin; n = 8). Orexin-KO mice that did not receive gene transfer (no-rAAV; n = 7) or received only the reporter gene (rAAV-GFP; n = 7) served as controls. Three weeks later, the animal's sleep and behaviour were recorded at night (no-odour control night), followed by another recording at night in the presence of predator odour (odour night). Orexin-KO mice given the orexin gene transfer into surrogate amygdala neurons had significantly less spontaneous bouts of cataplexy, and predator odour did not induce cataplexy compared with control mice. Moreover, the mice with orexin gene transfer were awake more during the odour night. These results demonstrate that orexin gene transfer into amygdala neurons can suppress both spontaneous and emotion-induced cataplexy attacks in narcoleptic mice. It suggests that manipulating amygdala pathways is a potential strategy for treating cataplexy in narcolepsy.

Cataplexy, a sudden unexpected muscle paralysis, is a debilitating symptom of the neurodegenerative sleep disorder, narcolepsy. During these attacks, the person is paralyzed, but fully conscious and aware of their surroundings. To identify potential neurons that might serve as surrogate orexin neurons to suppress such attacks, the gene for orexin (hypocretin), a peptide lost in most human narcoleptics, was delivered into the brains of the orexin-ataxin-3 transgenic mouse model of human narcolepsy. Three weeks after the recombinant adenoassociated virus (rAAV)-mediated orexin gene transfer, sleep–wake behavior was assessed. rAAV-orexin gene delivery into neurons of the zona incerta (ZI), or the lateral hypothalamus (LH) blocked cataplexy. Orexin gene transfer into the striatum or in the melanin-concentrating hormone neurons in the ZI or LH had no such effect, indicating site specificity. In transgenic mice lacking orexin neurons but given rAAV-orexin, detectable levels of orexin-A were evident in the CSF, indicating release of the peptide from the surrogate neurons. Retrograde tracer studies showed that the amygdala innervates the ZI consistent with evidence that strong emotions trigger cataplexy. In turn, the ZI projects to the locus ceruleus, indicating that the ZI is part of a circuit that stabilizes motor tone. Our results indicate that these neurons might also be recruited to block the muscle paralysis in narcolepsy.

Sleep deprivation (SD) produces numerous deleterious changes in brain cells, including apoptosis. It has been demonstrated that growth hormone (GH) stimulates cell growth and counteracts apoptosis, although this anti-apoptotic effect has not been tested against SD. To determine the protective effect of GH administration on cell proliferation and survival in the dentate gyrus (DG) of the hippocampus after sleep deprivation; we injected Wistar adult rats with a low dose of recombinant human GH (rhGH 5 ng/kg) per seven days and then we gently sleep deprived the animals for 48 consecutive hours. 5-Bromodeoxiuridine (BrdU) was administered to assess cell proliferation after the GH treatment and NeuN was used as marker of cell fate. Our results indicate that GH produced a three fold increase in the number of BrdU positive cells within the DG [Control = 1044 ± 106.38 cells, rhGH = 2952 ± 99.84 cells, P

Gene transfer has proven to be an effective neurobiological tool in a number of neurodegenerative diseases, but it is not known if it can correct a sleep disorder. Narcolepsy is a neurodegenerative sleep disorder linked to the loss of neurons containing the neuropeptide orexin, also known as hypocretin. Here, a replication-defective herpes simplex virus-1 amplicon-based vector was constructed to transfer the gene for mouse prepro-orexin into mice with a genetic deletion of the orexin gene. After in vitro tests confirmed successful gene transfer into cells, the gene vector was delivered to the lateral hypothalamus of orexin knockout (KO) mice where the orexin peptide was robustly expressed in the somata and processes of numerous neurons, and the peptide product was detected in the cerebrospinal fluid. During the 4-day life-span of the vector the incidence of cataplexy declined by 60%, and the levels of rapid eye movement sleep during the second half of the night were similar to levels in wild-type mice, indicating that narcoleptic sleep–wake behavior in orexin KO mice can be improved by targeted gene transfer.

Ablation of the SCN, an established circadian clock, does not abolish food entrainment, suggesting that the food-entrainable oscillator (FEO) must lie outside the SCN. Typically, animals show anticipatory locomotor activity and rise in core body temperature under the influence of the FEO. Signals from the FEO would, therefore, converge onto arousal neurons so that the animal might forage for food. In the present study, we investigate whether the neuropeptide orexin, which has been linked to arousal, might transduce the arousal signal. Orexin-knockout (orexin-KO) and wildtype (WT) mice (both C57BL/6J derived) were implanted with MiniMitter transmitters that recorded core body temperature and activity (12 h LD cycle). After a week of ad-libitum feeding, the mice were given access to food for 4 h (ZT 4-8) for nine days followed by 2-days of fasting. When orexin-KO mice were placed in a restricted feeding schedule, both core body temperature and activity entrained to the feeding schedule. In these mice gross locomotor activity was severely blunted during the nine day period of restricted feeding (-79.4+/-6.3%) from the WT, but they showed an increase in core body temperature in anticipation to the meal time similar to the WT mice. There was no difference in the amount of food intake between the genotypes. We conclude that orexin is not required for entrainment of activity and temperature to a restricted feeding schedule, but is required for the robust expression of gross locomotor activity in anticipation of the scheduled feeding.

Narcolepsy is linked to a widespread loss of neurons containing the neuropeptide hypocretin (HCRT), also named orexin. A transgenic (TG) rat model has been developed to mimic the neuronal loss found in narcoleptic humans. In these rats, HCRT neurons gradually die as a result of the expression of a poly-glutamine repeat under the control of the HCRT promoter. To better characterize the changes in HCRT-1 levels in response to the gradual HCRT neuronal loss cerebrospinal fluid (CSF) HCRT-1 levels were measured in various age groups (2–82 weeks) of wild-type (WT) and TG Sprague–Dawley rats. TG rats showed a sharp decline in CSF HCRT-1 level at week 4 with levels remaining consistently low (26%±9%, mean±S.D.) thereafter compared with WT rats. In TG rats, HCRT-1 levels were dramatically lower in target regions such as the cortex and brainstem (100-fold), indicating decreased HCRT-1 levels at terminals. In TG rats, CSF HCRT-1 levels significantly increased in response to 6 h of prolonged waking, indicating that the remaining HCRT neurons can be stimulated to release more neuropeptide. Rapid eye movement (REM) sleep in TG rats ( n =5) was consistent with a HCRT deficiency. In TG rats HCRT immunoreactive (HCRT-ir) neurons were present in the lateral hypothalamus (LH), even in old rats (24 months) but some HCRT-ir somata were in various stages of disintegration. The low output of these neurons is consistent with a widespread dysfunction of these neurons, and establishes this model as a tool to investigate the consequences of partial hypocretin deficiency.

With aging there is a significant decline in the normal architecture of sleep and a reduction in the diurnal amplitude of core body temperature. Regular moderate exercise has been shown to have a positive impact in the elderly and here we investigate whether sleep-wake patterning can also be improved. Young (3 months) and old (22 months) F344 rats were exercised once a day for 50min at night onset over an 8-week period. Thereafter, polysomnographic recordings were obtained immediately after exercise. To determine the lasting consequences of exercise, sleep was also recorded 2 days and 2 weeks after exercise had ended. Old rats that were exercised had a significant weight loss, were awake more during the last third of their active period, had less sleep fragmentation and the amplitude of the diurnal rhythm of core body temperature was significantly increased. Old exercised rats also had an overall increase in the amplitude of EEG power (0.5-16Hz) during wake and theta EEG power during REM sleep. In young rats regular exercise increased EEG delta power (0.5-4Hz) during NREM sleep. Our data indicate regular exercise in old rats improves sleep architecture, EEG power and diurnal rhythm of temperature.

It is currently hypothesized that the drive to sleep is determined by the activity of the basal forebrain (BF) cholinergic neurons, which release adenosine (AD), perhaps because of increased metabolic activity associated with the neuronal discharge during waking, and the accumulating AD begins to inhibit these neurons so that sleep-active neurons can become active. This hypothesis grew from the observation that AD induces sleep and AD levels increase with wake in the basal forebrain, but surprisingly it still remains untested. Here we directly test whether the basal forebrain cholinergic neurons are central to the AD regulation of sleep drive by administering 192–IgG–saporin to lesion the BF cholinergic neurons and then measuring AD levels in the BF. In rats with 95% lesion of the BF cholinergic neurons, AD levels in the BF did not increase with 6 h of prolonged waking. However, the lesioned rats had intact sleep drive after 6 and 12 h of prolonged waking, indicating that the AD accumulation in the BF is not necessary for sleep drive. Next we determined that, in the absence of the BF cholinergic neurons, the selective adenosine A1receptor agonist N6-cyclohexyladenosine, administered to the BF, continued to be effective in inducing sleep, indicating that the BF cholinergic neurons are not essential to sleep induction. Thus, neither the activity of the BF cholinergic neurons nor the accumulation of AD in the BF during wake is necessary for sleep drive.

The neuropeptide hypocretin, also known as orexin, has been implicated in waking since its deletion leads to the sleep disorder narcolepsy. Hypocretin neurons project to major arousal areas, and in an effort to determine which region is responsible for the changes in sleep-wake architecture we have developed the neurotoxin hypocretin2-saporin, which lesions hypocretin receptor bearing neurons. Here, in rats, we investigate the effects of hypocretin2-saporin lesions of the substantia nigra and ventral tegmental area in the regulation of sleep and wakefulness. Bilateral injection of hypocretin2-sap into both the ventral tegmental area and substantia nigra (92 and 184 ng/microl, 0.25 microl in the ventral tegmental area and 0.5 microl in the substantia nigra) or into the substantia nigra alone (184 ng/microl, 0.5 microl) produced insomnia. The insomnia seemed to be associated with a large increase in locomotion on days 4 and 6 postinjection, as hyperactivity and stereotypic movements were consistently observed on the video recordings in all lesioned rats. In these rats, a nearly complete loss of both tyrosine hydroxylase and neuron-specific nuclear protein (neuronal nuclei) immunoreactive cells in the substantia nigra as well as diminution of tyrosine hydroxylase-immunoreactive fibers in the caudate putamen was found. Following bilateral injection of hypocretin2-sap at a lower concentration (46 ng/microl, 0.25 microl in the ventral tegmental area and 0.5 microl in the substantia nigra), very little reduction in the number of tyrosine hydroxylase- and neuronal nuclei-immunoreactive neurons and only a temporary increase in wakefulness (17.4% increase during light-off period on day 6 postinjection) were observed. Ventral tegmental area lesions (184 ng/mul of hypocretin2-sap, 0.25 microl, bilateral injections) did not produce significant changes in sleep, although most of the tyrosine hydroxylase- and neuronal nuclei-immunoreactive neurons in the ventral tegmental area were destroyed. Insomnia following hypocretin2-sap lesions of the substantia nigra could be secondary to increased motor activity resulting from reduction of tonic inhibitory control by the substantia nigra.

The hypocretin neurons have been implicated in regulating sleep-wake states as they are lost in patients with the sleep disorder narcolepsy. Hypocretin (HCRT) neurons are located only in the perifornical region of the posterior hypothalamus and heavily innervate pontine brainstem neurons, such as the locus coeruleus (LC), which have traditionally been implicated in promoting arousal. It is not known how the hypocretin innervation of the pons regulates sleep-wake states as pontine lesions have never been shown to increase sleep. It is likely that in previous studies specific neurons were not lesioned. Therefore, in this study, we applied saporin-based neurotoxins to the dorsolateral pons and monitored sleep in rats. Anti-dopamine-beta-hydroxylase-saporin killed the LC neurons but sleep was affected only during a two hour light-dark transition period. Application of hypocretin2-saporin killed fewer LC neurons relative to other adjacent neurons. This occurred because the LC neurons possess the hypocretin receptor 1 but the ligand hypocretin 2 binds to this receptor with less affinity relative to the hypocretin receptor 2. The hypocretin2-saporin lesioned rats compared to controls had increased sleep during the dark period and displayed increased limb movements during REM sleep. None of the lesioned rats had sleep onset REM sleep periods or cataplexy. We conclude that the hypocretin innervation to the pons functions to awaken the animal when the lights turn off (via its innervation of the LC), sustains arousal and represses sleep during the rest of the night (via a wider innervation of other pontine neurons), and modulates muscle tone.

Rapid eye movement (REM) sleep is hypothesized to result from the activity of REM sleep-generating and REM sleep-inhibiting neurons. The serotoninergic (5-HT) neurons of the dorsal raphe nucleus (DRN) represents one such population of REM-sleep inhibiting neurons since they are silent during REM sleep. Consistent with the decrease in activity of 5-HT neurons, the brain extracellular levels of 5-HT are lower during REM sleep compared to wakefulness. It is not known whether serotonin release is also reduced as a consequence of REM sleep rebound. Using microdialysis sampling coupled to HPLC-ECD, we measured the extracellular levels of 5-HT and its metabolite (5-HIAA) in the medial medullary reticular formation (mMRF) of freely behaving rats during normal sleep, REM sleep deprivation as well as during REM sleep rebound. We found that the levels 5-HT and 5-HIAA were significantly decreased by REM sleep deprivation. The reduction of 5-HT release was maintained during REM sleep rebound but the extracellular level of its main metabolite was increased. In addition, even during REM sleep rebound, 5-HT release during sleep was low compared to wakefulness. Taken together these data support the permissive role of 5-HT neurotransmission for REM sleep expression.

Up-/down-state transitions are a form of network activity observed when sensory input into the cortex is diminished such as during non-REM sleep. Up-states emerge from coordinated signaling between glutamatergic and GABAergic synapses and are modulated by systems that affect the balance between inhibition and excitation. We hypothesized that the endocannabinoid (EC) system, a neuromodulatory system intrinsic to the cortical microcircuitry, is an important regulator of up-states and sleep. To test this hypothesis, up-states were recorded from layer V/VI pyramidal neurons in organotypic cultures of wild-type or CB1R knockout (KO) mouse prefrontal cortex. Activation of the cannabinoid 1 receptor (CB1) with exogenous agonists or by blocking metabolism of endocannabinoids, anandamide or 2-arachidonoyl glycerol, increased up-state amplitude and facilitated action potential discharge during up-states. The CB1 agonist also produced a layer II/III-selective reduction in synaptic GABAergic signaling that may underlie its effects on up-state amplitude and spiking. Application of CB1 antagonists revealed that an endogenous EC tone regulates up-state duration. Paradoxically, the duration of up-states in CB1 KO cultures was increased suggesting that chronic absence of EC signaling alters cortical activity. Consistent with increased cortical excitability, CB1 KO mice exhibited increased wakefulness as a result of reduced NREM sleep and NREM bout duration. Under baseline conditions, NREM delta (0.5-4 Hz) power was not different in CB1 KO mice, but during recovery from forced sleep deprivation, KO mice had reduced NREM delta power and increased sleep fragmentation. Overall, these findings demonstrate that the EC system actively regulates cortical up-states and important features of NREM sleep such as its duration and low frequency cortical oscillations.

Melanin concentrating hormone (MCH) is a cyclic neuropeptide present in the hypothalamus of all vertebrates. MCH is implicated in a number of behaviors but direct evidence is lacking. To selectively stimulate the MCH neurons the gene for the light-sensitive cation channel, channelrhodopsin-2, was inserted into the MCH neurons of wild-type mice. Three weeks later MCH neurons were stimulated for 1 min every 5 min for 24 h. A 10 Hz stimulation at the start of the night hastened sleep onset, reduced length of wake bouts by 50%, increased total time in non-REM and REM sleep at night, and increased sleep intensity during the day cycle. Sleep induction at a circadian time when all of the arousal neurons are active indicates that MCH stimulation can powerfully counteract the combined wake-promoting signal of the arousal neurons. This could be potentially useful in treatment of insomnia.

STUDY OBJECTIVES Narcolepsy is a sleep disorder characterized by loss of orexin neurons. Previously, our group demonstrated that transfer of the orexin gene into surrogate neurons in the lateral hypothalamus and the zona incerta significantly reduced cataplexy bouts in the orexin-ataxin-3 mice model of narcolepsy. The current study determined the effects of orexin gene transfer into the dorsolateral pontine neurons in the orexin knockout (KO) mice model of narcolepsy. The dorsolateral pons was chosen because it plays a critical role in regulating muscle tone and thus it is conceivable to be involved in cataplexy as well. Cataplexy is the pathognomonic symptom in narcolepsy. DESIGN Independent groups of orexin KO mice were given bilateral microinjections (0.75 μL each side) of either recombinant adenoassociated virus-orexin (rAAV-orexin; n = 7), or rAAV-green fluorescent protein (rAAV-GFP; n = 7) into the dorsolateral pons. A group of orexin KO mice that did not receive rAAV (n = 7) and a group of wild-type mice (C57BL/J6; n = 5) were used as controls. Three weeks after rAAV-mediated gene transfer narcolepsy symptoms were examined using sleep and behavioral recordings. Number, location of the orexin-immunoreactive neurons, and relative density of orexin immunoreactive fibers were determined. MEASUREMENTS AND RESULTS Orexin gene transfer into the dorsolateral pons significantly decreased cataplexy and modestly improved wake maintenance compared to the orexin KO mice that did not receive rAAV. In contrast, GFP gene transfer worsened narcoleptic symptoms compared to the no-rAAV orexin KO group. CONCLUSION Orexin gene transfer into the dorsolateral pontine neurons can control cataplexy attacks and modestly improve wake maintenance.

Ten years ago the sleep disorder narcolepsy was linked to the neuropeptide hypocretin (HCRT), also known as orexin. This disorder is characterized by excessive day time sleepiness, inappropriate triggering of rapid-eye movement (REM) sleep and cataplexy, which is a sudden loss of muscle tone during waking. It is still not known how HCRT regulates REM sleep or muscle tone since HCRT neurons are localized only in the lateral hypothalamus while REM sleep and muscle atonia are generated from the brainstem. To identify a potential neuronal circuit, the neurotoxin hypocretin-2-saporin (HCRT2-SAP) was used to lesion neurons in the ventral lateral periaquaductal gray (vlPAG). The first experiment utilized hypocretin knock-out (HCRT-ko) mice with the expectation that deletion of both HCRT and its target neurons would exacerbate narcoleptic symptoms. Indeed, HCRT-ko mice (n = 8) given the neurotoxin HCRT2-SAP (16.5 ng/23nl/sec each side) in the vlPAG had levels of REM sleep and sleep fragmentation that were considerably higher compared to HCRT-ko given saline (+39%; n = 7) or wildtype mice (+177%; n = 9). However, cataplexy attacks did not increase, nor were levels of wake or non-REM sleep changed. Experiment 2 determined the effects in mice where HCRT was present but the downstream target neurons in the vlPAG were deleted by the neurotoxin. This experiment utilized an FVB-transgenic strain of mice where eGFP identifies GABA neurons. We verified this and also determined that eGFP neurons were immunopositive for the HCRT-2 receptor. vlPAG lesions in these mice increased REM sleep (+79% versus saline controls) and it was significantly correlated (r = 0.89) with loss of eGFP neurons. These results identify the vlPAG as one site that loses its inhibitory control over REM sleep, but does not cause cataplexy, as a result of hypocretin deficiency.

A distributed network of neurons is hypothesized to be responsible for wake, non-REM (rapid eye movement) and REM sleep. First-order circuit models have emerged, and in the last few years these models have been empirically tested. We review the evidence and advance the hypothesis that cholinergic, histamine, and noradrenergic neurons trigger arousal in the event of an alarm (either internal or external) and maintain vigilance. In post-traumatic stress disorder (PTSD), the hyperarousal and frequent awakenings during sleep might be related to overactivation of these neurons.

Neurons containing the neuropeptide hypocretin (HCRT, orexin) are localized only in the lateral hypothalamus, from where they innervate multiple regions implicated in arousal, including the basal forebrain. HCRT activation of downstream arousal neurons is likely to stimulate release of endogenous factors. One such factor is adenosine, which in the basal forebrain increases in level with wakefulness and decreases with sleep, and is hypothesized to regulate the waxing and waning of sleep drive. Does loss of HCRT neurons affect adenosine levels in the basal forebrain? Is the increased sleep that accompanies HCRT loss a consequence of higher adenosine levels in the basal forebrain? In the present study, we investigated these questions by lesioning the HCRT neurons with HCRT-2-saporin (HCRT-2-SAP) and measuring sleep and extracellular levels of adenosine in the basal forebrain. In separate groups of rats, the neurotoxin HCRT-2-SAP or saline was administered locally to the lateral hypothalamus, and 80 days later adenosine and sleep were assessed. Rats given the neurotoxin had a 94% loss of HCRT neurons. These rats woke less at night, and had more rapid eye movement sleep, which is consistent with HCRT hypofunction. These rats also had more sleep after brief periods of sleep deprivation. However, in the lesioned rats, adenosine levels did not increase with 6 h of sleep deprivation, whereas an increase in adenosine levels occurred in rats without lesion of the HCRT neurons. These findings indicate that adenosine levels do not increase with wakefulness in rats with a HCRT lesion, and that the increased sleep in these rats occurs independently of adenosine levels in the basal forebrain.

The hypocretin (HCRT) neurons are located only in the perifornical area of the lateral hypothalamus and heavily innervate the cholinergic neurons in the basal forebrain (BF), histamine neurons in the tuberomammillary nucleus (TMN), and the noradrenergic locus ceruleus (LC) neurons, three neuronal populations that have traditionally been implicated in regulating arousal. Based on the innervation, HCRT neurons may regulate arousal by driving these downstream arousal neurons. Here, we directly test this hypothesis by a simultaneous triple lesion of these neurons using three saporin-conjugated neurotoxins. Three weeks after lesion, the daily levels of wake were not changed in rats with double or triple lesions, although rats with triple lesions were asleep more during the light-to-dark transition period. The double- and triple-lesioned rats also had more stable sleep architecture compared with nonlesioned saline rats. These results suggest that the cholinergic BF, TMN, and LC neurons jointly modulate arousal at a specific circadian time, but they are not essential links in the circuitry responsible for daily levels of wake, as traditionally hypothesized.

Virtually all organisms manifest regular periods of behavioral quiescence and activity. In mammals and birds, these periods have evolved into regular episodes of wakefulness and sleep. The sleep period itself has further differentiated into two distinct states, slow wave sleep (SWS) and rapid eye movement (REM) sleep. In humans, SWS has further differentiated into four distinct stages, each identified by a specific pattern on the electroencephalogram (EEG). REM sleep is very similar to wakefulness in many ways; the main difference is that there is behavioral quiescence during REM sleep. For this reason, REM sleep is often referred to as paradoxical sleep.