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Transgenic Archaerhodopsin-3 Expression in Hypocretin/Orexin Neurons Engenders Cellular Dysfunction and Features of Type 2 Narcolepsy.
J Neurosci 2019JN

Abstract

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 STATEMENTOptogenetics 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 (NT2), a sleep disorder for which no good animal model currently exists.

Authors+Show Affiliations

Center for Neuroscience, Biosciences Division, SRI International, Menlo Park, CA 94025, USA. Institute for Neurogenomics, Helmholtz Zentrum München, German Research Centre for Environmental Health, Neuherberg 85764, Germany.Research Institute of Environmental Medicine, Nagoya University, Nagoya, 464-8601, Japan.Center for Neuroscience, Biosciences Division, SRI International, Menlo Park, CA 94025, USA.Center for Neuroscience, Biosciences Division, SRI International, Menlo Park, CA 94025, USA.Research Institute of Environmental Medicine, Nagoya University, Nagoya, 464-8601, Japan thomas.kilduff@sri.com yamank@riem.nagoya-u.ac.jp.Center for Neuroscience, Biosciences Division, SRI International, Menlo Park, CA 94025, USA thomas.kilduff@sri.com yamank@riem.nagoya-u.ac.jp.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31628177

Citation

Williams, Rhîannan H., et al. "Transgenic Archaerhodopsin-3 Expression in Hypocretin/Orexin Neurons Engenders Cellular Dysfunction and Features of Type 2 Narcolepsy." The Journal of Neuroscience : the Official Journal of the Society for Neuroscience, 2019.
Williams RH, Tsunematsu T, Thomas AM, et al. Transgenic Archaerhodopsin-3 Expression in Hypocretin/Orexin Neurons Engenders Cellular Dysfunction and Features of Type 2 Narcolepsy. J Neurosci. 2019.
Williams, R. H., Tsunematsu, T., Thomas, A. M., Bogyo, K., Yamanaka, A., & Kilduff, T. S. (2019). Transgenic Archaerhodopsin-3 Expression in Hypocretin/Orexin Neurons Engenders Cellular Dysfunction and Features of Type 2 Narcolepsy. The Journal of Neuroscience : the Official Journal of the Society for Neuroscience, doi:10.1523/JNEUROSCI.0311-19.2019.
Williams RH, et al. Transgenic Archaerhodopsin-3 Expression in Hypocretin/Orexin Neurons Engenders Cellular Dysfunction and Features of Type 2 Narcolepsy. J Neurosci. 2019 Oct 18; PubMed PMID: 31628177.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Transgenic Archaerhodopsin-3 Expression in Hypocretin/Orexin Neurons Engenders Cellular Dysfunction and Features of Type 2 Narcolepsy. AU - Williams,Rhîannan H, AU - Tsunematsu,Tomomi, AU - Thomas,Alexia M, AU - Bogyo,Kelsie, AU - Yamanaka,Akihiro, AU - Kilduff,Thomas S, Y1 - 2019/10/18/ PY - 2019/02/07/received PY - 2019/10/08/revised PY - 2019/10/09/accepted PY - 2019/10/20/entrez JF - The Journal of neuroscience : the official journal of the Society for Neuroscience JO - J. Neurosci. N2 - 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 STATEMENTOptogenetics 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 (NT2), a sleep disorder for which no good animal model currently exists. SN - 1529-2401 UR - https://www.unboundmedicine.com/medline/citation/31628177/Transgenic_Archaerhodopsin-3_Expression_in_Hypocretin/Orexin_Neurons_Engenders_Cellular_Dysfunction_and_Features_of_Type_2_Narcolepsy L2 - http://www.jneurosci.org/cgi/pmidlookup?view=long&pmid=31628177 DB - PRIME DP - Unbound Medicine ER -