Hypocretin-2 Saporin Lesions of the Ventrolateral Periaquaductal Gray (vlPAG) Increase REM Sleep in Hypocretin Knockout Mice

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

Neurons Containing Orexin or Melanin Concentrating Hormone Reciprocally Regulate Wake and Sleep

There is considerable amount of data on arousal neurons whereas there is a paucity of knowledge regarding neurons that make us fall asleep. Indeed, current network models of sleep-wake regulation list many arousal neuronal populations compared to only one sleep group located in the preoptic area. There are neurons outside the preoptic area that are active during sleep, but they have never been selectively manipulated. Indeed, none of the sleep-active neurons have been selectively stimulated. To close this knowledge gap we used optogenetics to selectively manipulate neurons containing melanin concentrating hormone (MCH). The MCH neurons are located in the posterior hypothalamus intermingled with the orexin arousal neurons. Our data indicated that optogenetic stimulation of MCH neurons in wildtype mice (J Neuroscience, 2013) robustly increased both non-REM and REM sleep. MCH neuron stimulation increased sleep during the animal’s normal active period, which is compelling evidence that stimulation of MCH neurons has a powerful effect in counteracting the strong arousal signal from all of the arousal neurons. The MCH neurons represent the only group of sleep-active neurons that when selectively stimulated induce sleep. From a translational perspective this is potentially useful in sleep disorders, such as insomnia, where sleep needs to be triggered against a strong arousal drive. Our studies indicate that the MCH neurons belong within an overall model of sleep-wake regulation

Sleep rhythmicity and homeostasis in mice with targeted disruption of mPeriod genes

In mammals, sleep is regulated by circadian and homeostatic mechanisms. The circadian component, residing in the suprachiasmatic nucleus (SCN), regulates the timing of sleep, whereas homeostatic factors determine the amount of sleep. It is believed that these two processes regulating sleep are independent because sleep amount is unchanged after SCN lesions. However, because such lesions necessarily damage neuronal connectivity, it is preferable to investigate this question in a genetic model that overcomes the confounding influence of circadian rhythmicity. Mice with disruption of both mouse Period genes (mPer)1 and mPer2 have a robust diurnal sleep-wake rhythm in an entrained light-dark cycle but lose rhythmicity in a free-run condition. Here, we examine the role of the mPer genes on the rhythmic and homeostatic regulation of sleep. In entrained conditions, when averaged over the 24-h period, there were no significant differences in waking, slow-wave sleep (SWS), or rapid eye movement (REM) sleep between mPer1, mPer2, mPer3, mPer1-mPer2 double-mutant, and wild-type mice. The mice were then kept awake for 6 h (light period 6-12), and the mPer mutants exhibited increased sleep drive, indicating an intact sleep homeostatic response in the absence of the mPer genes. In free-run conditions (constant darkness), the mPer1-mPer2 double mutants became arrhythmic, but they continued to maintain their sleep levels even after 36 days in free-running conditions. Although mPer1 and mPer2 represent key elements of the molecular clock in the SCN, they are not required for homeostatic regulation of the daily amounts of waking, SWS, or REM sleep

Anandamide Enhances Extracellular Levels of Adenosine and Induces Sleep: An In Vivo Microdialysis Study

STUDY OBJECTIVES:The principal component of marijuana, delta-9-tetrahydrocannabinol increases sleep in humans. Endogenous cannabinoids, such as N-arachidonoylethanolamine (anandamide), also increase sleep. However, the mechanism by which these molecules promote sleep is not known but might involve a sleep-inducing molecule such as adenosine. Microdialysis samples were collected from the basal forebrain in order to detect levels of adenosine before and after injection of anandamide. DESIGN:Rats were implanted for sleep studies, and a cannula was placed in the basal forebrain to collect microdialysis samples. Samples were analyzed using high-performance liquid chromatography. SETTINGS:Basic neuroscience research laboratory. PARTICIPANTS AND INTERVENTIONS:Three-month-old male F344 rats. At the start of the lights-on period, animals received systemic injections of dimethyl sulfoxide (vehicle), anandamide, SR141716A (cannabinoid receptor 1 [CB1] antagonist), or SR141716A and anandamide. One hour after injections, microdialysis samples were collected (5 microL) from the basal forebrain every hour over a 20-minute period for 5 hours. The samples were immediately analyzed via high-performance liquid chromatography for adenosine levels. Sleep was also recorded continuously over the same period. MEASUREMENTS AND RESULTS:Anandamide increased adenosine levels compared to vehicle controls with the peak levels being reached during the third hour after drug injection. There was a significant increase in slow-wave sleep during the third hour. The induction in sleep and the rise in adenosine were blocked by the CB1-receptor antagonist, SR141716A. CONCLUSIONS:Anandamide increased adenosine levels in the basal forebrain and also increased sleep. The soporific effects of anandamide were mediated by the CB1 receptor, since the effects were blocked by the CB1-receptor antagonist. These findings identify a potential therapeutic use of endocannabinoids to induce sleep in conditions where sleep may be severely attenuated

Anandamide enhances extracellular levels of adenosine and induces sleep: an in vivo microdialysis study.

STUDY OBJECTIVES:The principal component of marijuana, delta-9-tetrahydrocannabinol increases sleep in humans. Endogenous cannabinoids, such as N-arachidonoylethanolamine (anandamide), also increase sleep. However, the mechanism by which these molecules promote sleep is not known but might involve a sleep-inducing molecule such as adenosine. Microdialysis samples were collected from the basal forebrain in order to detect levels of adenosine before and after injection of anandamide. DESIGN:Rats were implanted for sleep studies, and a cannula was placed in the basal forebrain to collect microdialysis samples. Samples were analyzed using high-performance liquid chromatography. SETTINGS:Basic neuroscience research laboratory. PARTICIPANTS AND INTERVENTIONS:Three-month-old male F344 rats. At the start of the lights-on period, animals received systemic injections of dimethyl sulfoxide (vehicle), anandamide, SR141716A (cannabinoid receptor 1 [CB1] antagonist), or SR141716A and anandamide. One hour after injections, microdialysis samples were collected (5 microL) from the basal forebrain every hour over a 20-minute period for 5 hours. The samples were immediately analyzed via high-performance liquid chromatography for adenosine levels. Sleep was also recorded continuously over the same period. MEASUREMENTS AND RESULTS:Anandamide increased adenosine levels compared to vehicle controls with the peak levels being reached during the third hour after drug injection. There was a significant increase in slow-wave sleep during the third hour. The induction in sleep and the rise in adenosine were blocked by the CB1-receptor antagonist, SR141716A. CONCLUSIONS:Anandamide increased adenosine levels in the basal forebrain and also increased sleep. The soporific effects of anandamide were mediated by the CB1 receptor, since the effects were blocked by the CB1-receptor antagonist. These findings identify a potential therapeutic use of endocannabinoids to induce sleep in conditions where sleep may be severely attenuated

VGAT and VGLUT2 expression in MCH and orexin neurons in double transgenic reporter mice

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. Keywords: GABA, Glutamate, Sleep, Arousa

Endocannabinoid modulation of cortical up-states and NREM sleep.

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