Transgenic Archaerhodopsin-3 Expression in Hypocretin/Orexin Neurons Engenders Cellular Dysfunction and Features of Type 2 Narcolepsy.

Narcolepsy, characterized by excessive daytime sleepiness, is associated with dysfunction of the hypothalamic hypocretin/orexin (Hcrt) system, either due to extensive loss of Hcrt cells (Type 1, NT1) or hypothesized Hcrt signaling impairment (Type 2, NT2). Accordingly, efforts to recapitulate narcolepsy-like symptoms in mice have involved ablating these cells or interrupting Hcrt signaling. Here, we describe orexin/Arch mice, in which a modified archaerhodopsin-3 gene was inserted downstream of the prepro-orexin promoter, resulting in expression of the yellow light-sensitive Arch-3 proton pump specifically within Hcrt neurons. Histological examination along with ex vivo and in vivo electrophysiological recordings of male and female orexin/Arch mice demonstrated silencing of Hcrt neurons when these cells were photoilluminated. However, high expression of the Arch transgene affected cellular and physiological parameters independent of photoillumination. The excitability of Hcrt neurons was reduced, and both circadian and metabolic parameters were perturbed in a subset of orexin/Arch mice that exhibited high levels of Arch expression. Orexin/Arch mice also had increased REM sleep under baseline conditions but did not exhibit cataplexy, a sudden loss of muscle tone during wakefulness characteristic of NT1. These aberrations resembled some aspects of mouse models with Hcrt neuron ablation, yet the number of Hcrt neurons in orexin/Arch mice was not reduced. Thus, orexin/Arch mice may be useful to investigate Hcrt system dysfunction when these neurons are intact, as is thought to occur in narcolepsy without cataplexy (NT2). These results also demonstrate the utility of extended phenotypic screening of transgenic models when specific neural circuits have been manipulated.SIGNIFICANCE STATEMENT Optogenetics has become an invaluable tool for functional dissection of neural circuitry. While opsin expression is often achieved by viral injection, stably integrated transgenes offer some practical advantages. Here, we demonstrate successful transgenic expression of an inhibitory opsin in hypocretin/orexin neurons, which are thought to promote or maintain wakefulness. Both brief and prolonged illumination resulted in inhibition of these neurons and induced sleep. However, even in the absence of illumination, these cells exhibited altered electrical characteristics, particularly when transgene expression was high. These aberrant properties affected metabolism and sleep, resulting in a phenotype reminiscent of the narcolepsy Type 2, a sleep disorder for which no good animal model currently exists.

Excitation of Cortical nNOS/NK1R Neurons by Hypocretin 1 is Independent of Sleep Homeostasis.

We have proposed that cortical nNOS/NK1R interneurons have a role in sleep homeostasis. The hypocretins (orexins) are wake-promoting neuropeptides and hypocretin/orexin (Hcrt) neurons project to the cortex. Hcrt peptides affect deep layer cortical neurons, and Hcrt receptor 1 (Hcrtr1; Ox1r) mRNA is expressed in cortical nNOS/NK1R cells. Therefore, we investigated whether Hcrt neuron stimulation affects cingulate cortex nNOS/NK1R neurons. Bath application of HCRT1/orexin-A evoked an inward current and membrane depolarization in most nNOS/NK1R cells which persisted in tetrodotoxin; optogenetic stimulation of Hcrt terminals expressing channelrhodopsin-2 confirmed these results, and pharmacological studies determined that HCRTR1 mediated these responses. Single-cell RT-PCR found Hcrtr1 mRNA in 31% of nNOS/NK1R cells without any Hcrtr2 mRNA expression; immunohistochemical studies of Hcrtr1-EGFP mice confirmed that a minority of nNOS/NK1R cells express HCRTR1. When Hcrt neurons degenerated in orexin-tTA;TetO DTA mice, the increased EEG delta power during NREM sleep produced in response to 4 h sleep deprivation and c-FOS expression in cortical nNOS/NK1R cells during recovery sleep were indistinguishable from that of controls. We conclude that Hcrt excitatory input to these deep layer cells is mediated through HCRTR1 but is unlikely to be involved in the putative role of cortical nNOS/NK1R neurons in sleep homeostasis.

Cortical nNOS/NK1 Receptor Neurons are Regulated by Cholinergic Projections From the Basal Forebrain.

Cholinergic (ACh) basal forebrain (BF) neurons are active during wakefulness and rapid eye movement (REM) sleep and are involved in sleep homeostasis. We have previously shown in adult animals that cortical neurons that express neuronal nitric oxide synthase (nNOS) and the receptor for Substance P (NK1R) are activated during non-REM (NREM) sleep in proportion to homeostatic sleep drive. Here, we show that BF neurons modulate cortical nNOS/NK1R cells. In vitro optogenetic stimulation of BF terminals both activated and inhibited nNOS/NK1R neurons. Pharmacological studies revealed cholinergic responses mediated by postsynaptic activation of muscarinic receptors (mAChRs; M3R > M2/4R > M1R) and that presynaptic M3R and M2R activation reduced glutamatergic input onto nNOS/NK1R neurons whereas nicotinic receptor (nAChR)-mediated responses of nNOS/NK1R neurons were mixed. Cholinergic responses of nNOS/NK1R neurons were largely unaffected by prolonged wakefulness. ACh release, including from BF cells, appears to largely excite cortical nNOS/NK1R cells while reducing glutamatergic inputs onto these neurons. We propose that cholinergic signaling onto cortical nNOS/NK1R neurons may contribute to the regulation of cortical activity across arousal states, but that this response is likely independent of the role of these neurons in sleep homeostasis.

Sleep/Wake Physiology and Quantitative Electroencephalogram Analysis of the Neuroligin-3 Knockout Rat Model of Autism Spectrum Disorder.

Study Objectives: Neuroligin-3 (NLGN3) is one of the many genes associated with autism spectrum disorder (ASD). Sleep dysfunction is highly prevalent in ASD, but has not been rigorously examined in ASD models. Here, we evaluated sleep/wake physiology and behavioral phenotypes of rats with genetic ablation of Nlgn3. Methods: Male Nlgn3 knockout (KO) and wild-type (WT) rats were assessed using a test battery for ASD-related behaviors and also implanted with telemeters to record the electroencephalogram (EEG), electromyogram, body temperature, and locomotor activity. 24-h EEG recordings were analyzed for sleep/wake states and spectral composition. Results: Nlgn3 KO rats were hyperactive, exhibited excessive chewing behavior, and had impaired prepulse inhibition to an auditory startle stimulus. KO rats also spent less time in non-rapid eye movement (NREM) sleep, more time in rapid eye movement (REM) sleep, exhibited elevated theta power (4-9 Hz) during wakefulness and REM, and elevated delta power (0.5-4 Hz) during NREM. Beta (12-30 Hz) power and gamma (30-50 Hz) power were suppressed across all vigilance states. Conclusions: The sleep disruptions in Nlgn3 KO rats are consistent with observations of sleep disturbances in ASD patients. The EEG provides objective measures of brain function to complement rodent behavioral analyses and therefore may be a useful tool to study ASD.

Cntnap2 Knockout Rats and Mice Exhibit Epileptiform Activity and Abnormal Sleep-Wake Physiology.

Study Objectives: Although recent innovations have enabled modification of the rat genome, it is unclear whether enhanced utility of rodents as human disease models will result. We compared electroencephalogram (EEG) and behavioral phenotypes of rats and mice with homozygous deletion of Cntnap2, a gene associated with cortical dysplasia-focal epilepsy (CDFE) and autism spectrum disorders (ASD). Methods: Male contactin-associated protein-like 2 (Cntnap2) knockout (KO) and wild-type (WT) rats and male Cntnap2 KO and WT mice were implanted with telemeters to record EEG, electromyogram, body temperature, and locomotor activity. Animals were subjected to a test battery for ASD-related behaviors, followed by 24-hr EEG recordings that were analyzed for sleep-wake parameters and subjected to spectral analysis. Results: Cntnap2 KO rats exhibited severe motor seizures, hyperactivity, and increased consolidation of wakefulness and REM sleep. By contrast, Cntnap2 KO mice demonstrated absence seizure-like events, hypoactivity, and wake fragmentation. Although seizures observed in Cntnap2 KO rats were more similar to those in CDFE patients than in KO mice, neither model fully recapitulated the full spectrum of disease symptoms. However, KOs in both species had reduced spectral power in the alpha (9-12 Hz) range during wake, suggesting a conserved EEG biomarker. Conclusions: Deletion of Cntnap2 impacts similar behaviors and EEG measures in rats and mice, but with profound differences in nature and phenotypic severity. These observations highlight the importance of cross-species comparisons to understand conserved gene functions and the limitations of single- species models to provide translational insights relevant to human diseases.

Locus Coeruleus and Tuberomammillary Nuclei Ablations Attenuate Hypocretin/Orexin Antagonist-Mediated REM Sleep.

Hypocretin 1 and 2 (Hcrts; also known as orexin A and B), excitatory neuropeptides synthesized in cells located in the tuberal hypothalamus, play a central role in the control of arousal. Hcrt inputs to the locus coeruleus norepinephrine (LC NE) system and the posterior hypothalamic histaminergic tuberomammillary nuclei (TMN HA) are important efferent pathways for Hcrt-induced wakefulness. The LC expresses Hcrt receptor 1 (HcrtR1), whereas HcrtR2 is found in the TMN. Although the dual Hcrt/orexin receptor antagonist almorexant (ALM) decreases wakefulness and increases NREM and REM sleep time, the neural circuitry that mediates these effects is currently unknown. To test the hypothesis that ALM induces sleep by selectively disfacilitating subcortical wake-promoting populations, we ablated LC NE neurons (LCx) or TMN HA neurons (TMNx) in rats using cell-type-specific saporin conjugates and evaluated sleep/wake following treatment with ALM and the GABAA receptor modulator zolpidem (ZOL). Both LCx and TMNx attenuated the promotion of REM sleep by ALM without affecting ALM-mediated increases in NREM sleep. Thus, eliminating either HcrtR1 signaling in the LC or HcrtR2 signaling in the TMN yields similar effects on ALM-induced REM sleep without affecting NREM sleep time. In contrast, neither lesion altered ZOL efficacy on any measure of sleep-wake regulation. These results contrast with those of a previous study in which ablation of basal forebrain cholinergic neurons attenuated ALM-induced increases in NREM sleep time without affecting REM sleep, indicating that Hcrt neurotransmission influences distinct aspects of NREM and REM sleep at different locations in the sleep-wake regulatory network.

Quantitative Electroencephalographic Analysis Provides an Early-Stage Indicator of Disease Onset and Progression in the zQ175 Knock-In Mouse Model of Huntington's Disease.

STUDY OBJECTIVES: Patients with Huntington's disease (HD) show a high prevalence of sleep disorders that typically occur prior to the onset of motoric symptoms and neurodegeneration. Our understanding of the pathophysiological alterations in premanifest HD is limited, hindering the ability to measure disease modification in response to treatment. We used a full-length knock-in HD model to determine early changes in the electroencephalogram (EEG) and sleep that may predict the onset and progression of the disease. METHODS: A 10-month longitudinal study was designed to determine the effect of the HD mutation on the EEG and sleep/wake changes in heterozygous (HET) and homozygous (HOM) zQ175 mice and wild-type (WT) littermates from 8 to 48 w of age. Mice were instrumented with tethered headmounts to record EEG/electromyography signals. Telemeters were implanted to continuously measure locomotor activity (LMA) and body temperature (Tb). Sleep deprivation (SDep) was performed at 8, 12, 16, 24, 32, and 48 w of age. RESULTS: The HD mutation disrupted the EEG field potential from 8-12 w in an age- and mutant huntington dose-dependent manner, prior to changes in sleep/wake states, LMA, and Tb. Prominent effects of the HD mutation on the EEG included a progressive reduction in low frequency power, a slowing of rapid eye movement peak theta frequency, and the emergence of state-dependent beta/gamma oscillations. There was no effect of genotype on the relative increase in nonrapid eye movement delta power or sleep time in response to SDep. CONCLUSIONS: The expression of the Huntington's disease (HD) mutation results in complex EEG alterations that occur prior to deficits in behavioral measures and are one of the earliest phenotypes uncovered in this mouse model. Despite these EEG changes, homeostatic responses to sleep loss were preserved in HET and HOM zQ175 mice. Greater insight into the localization and response of these EEG alterations to novel therapies may enable early intervention and improve outcomes for patients with HD.

Group I metabotropic glutamate receptor antagonists alter select behaviors in a mouse model for fragile X syndrome.

RATIONALE: Studies in the Fmr1 knockout (KO) mouse, a model of fragile X syndrome (FXS), suggest that excessive signaling through group I metabotropic glutamate receptors (mGluRs), comprised of subtypes mGluR1 and mGluR5, may play a role in the pathogenesis of FXS. Currently, no studies have assessed the effect of mGluR1 modulation on Fmr1 KO behavior, and there has not been an extensive behavioral analysis of mGluR5 manipulation in Fmr1 KO mice. OBJECTIVES: The goals for this study were to determine if pharmacologic blockade of mGluR1 may affect Fmr1 KO behavior as well as to expand on the current literature regarding pharmacologic blockade of mGluR5 on Fmr1 KO behavior. METHODS: Reduction of mGluR1 or mGluR5 activity was evaluated on a variety of behavioral assays in wild-type (WT) and Fmr1 KO mice through the use of antagonists: JNJ16259685 (JNJ, mGluR1 antagonist) and MPEP (mGluR5 antagonist). RESULTS: JNJ and MPEP decreased marble burying in both WT and Fmr1 KO mice without reductions in activity. Neither JNJ nor MPEP affected the prepulse inhibition in either WT or Fmr1 KO mice. JNJ did not affect Fmr1 KO motor coordination but did impair WT performance. MPEP improved a measure of motor learning in Fmr1 KO but not WT mice. While both JNJ and MPEP decreased the audiogenic seizures in the Fmr1 KO, MPEP completely abolished the manifestation of seizures. CONCLUSION: These data illustrate that, while the manipulation of either mGluR1 or mGluR5 can affect select behaviors in the Fmr1 KO, we observe greater effects upon mGluR5 reduction.

Genetic reduction of group 1 metabotropic glutamate receptors alters select behaviors in a mouse model for fragile X syndrome.

INTRODUCTION: Genetic heterogeneity likely contributes to variability in the symptoms among individuals with fragile X syndrome (FXS). Studies in the Fmr1 knockout (KO) mouse model for FXS suggest that excessive signaling through group I metabotropic glutamate receptors (Gp1 mGluRs), comprised of subtypes mGluR1 and mGluR5, may play a role. Hence, Gp1 mGluRs may act as modifiers of FXS. Currently no studies have addressed whether manipulation of mGluR1 activity may alter Fmr1 KO behavioral responses, and only a few have reported the effects of mGluR5 manipulation. Therefore, the goals for this study were to extend our understanding of the effects of modulating Gp1 mGluR activity on Fmr1 KO behavioral responses. METHODS: The present study determined if genetically reducing mGluR1 or mGluR5 by 50% affects an extensive array of behaviors in the Fmr1 KO. RESULTS: Reduction of mGluR1 moderately decreased Fmr1 KO activity. Reduction of mGluR5 caused an analgesic response in the Fmr1 KO and decreased active social behavior. Modulation of either mGluR1 or mGluR5 did not significantly alter audiogenic seizures, anxiety- and perseverative-related responses, sensorimotor gating, memory, or motor responses. CONCLUSIONS: Genetic reduction of mGluR1 or mGluR5 modified a few select Fmr1 KO behaviors, although these modifications appeared to be subtle in nature and/or limited to select behaviors. This may indicate that 50% reduction of either mGluR1 or mGluR5 is insufficient to produce behavioral changes, and therefore, these receptors may not be dominant modifiers of a number of Fmr1 KO behavioral phenotypes.

Modifying behavioral phenotypes in Fmr1KO mice: genetic background differences reveal autistic-like responses.

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability in humans. In addition to cognitive impairment, patients may exhibit hyperactivity, attention deficits, social difficulties and anxiety, and autistic-like behaviors. The degree to which patients display these behaviors varies considerably and is influenced by family history, suggesting that genetic modifiers play a role in the expression of behaviors in FXS. Several studies have examined behavior in a mouse model of FXS in which the Fmr1 gene has been ablated. Most of those studies were done in Fmr1 knockout mice on a pure C57BL/6 or FVB strain background. To gain a better understanding of the effects of genetic background on behaviors resulting from the loss of Fmr1 gene expression, we generated F1 hybrid lines from female Fmr1 heterozygous mice on a pure C57BL/6J background bred with male Fmr1 wild-type (WT) mice of various background strains (A/J, DBA/2J, FVB/NJ, 129S1/SvImJ and CD-1). Male Fmr1 knockout and WT littermates from each line were examined in an extensive behavioral test battery. Results clearly indicate that multiple behavioral responses are dependent on genetic background, including autistic-like traits that are present on limited genetic backgrounds. This approach has allowed us to identify improved models for different behavioral symptoms present in FXS including autistic-like traits.