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

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 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.

Keywords: NREM sleep; REM sleep; hypothalamus; narcolepsy; optogenetic; wakefulness.

Figure 1.

Archaerhodopsin (Arch) expression in Hcrt/orexin neurons in the orexin/Arch transgenic mouse brain. A, Schematic of transgene used to express Arch downstream of the prepro-orexin promoter. B, Top, Immunostaining of Hcrt/orexin-IR neurons (Alexa-594, red) located in the LHA. Middle, GFP-IR neurons (Alexa-488, green), reporting Arch expression, are also observed in the LHA. Bottom, Merged picture showing expression of Arch within Hcrt/orexin neurons in the LHA of the orexin/Arch transgenic mice. Scale bars: left column, 300 μm; right column, 50 μm. Arch expression was not observed in nonorexin neurons in any brain region. C, Arch expression was observed in the dendrites as well as somata of identified orexin-A-IR (red channel) and orexin-B-IR neurons (blue channel). Scale bar, 50 μm. Approximately 80% of Hcrt/orexin neurons colocalized with Arch. D, Immunohistochemistry demonstrates Arch expression in orexin-innervated regions, such as the tuberomammillary nucleus (TMN), locus coeruelus (LC), and dorsal raphe nucleus (DRN). E, In vitro functionality and sensitivity of the Arch proton pump expressed in Hcrt/orexin cells of orexin/Arch mice. Representative examples demonstrating that 575 ± 25 nm illumination of Hcrt/orexin neurons from orexin/Arch mice results in a reduction in firing rate in both (Ei) cell-attached and (Eii) current-clamp configurations. F, Yellow light stimulation (575 ± 25 nm, orange bar) significantly reduced the firing activity of orexin/Arch neurons in cell-attached configuration. G, Photoactivation of Arch similarly reduced electrical firing with concomitant hyperpolarization of the membrane potential in current-clamp recordings of Hcrt/orexin neurons. *p < 0.05; ***p < 0.001. Values are mean ± SEM

Figure 2.

Bilateral in vivo green light stimulation activates Arch and silences Hcrt/orexin neurons. Ai, Acute 1 min photoillumination applied at random intervals during wake bouts between ZT8 and ZT11 decreased the probability of wake (Aii) and increased NREM sleep (Aiii) but not REM sleep (Aiv). Bi, Longer 1 h photoillumination between ZT8 and ZT9 did not change the total time spent in sleep versus wake (Bii) compared with baseline conditions. Ci, In contrast, when 1 h green light was applied between ZT16 and ZT17, a significant increase in NREM sleep and concomitant reductions in both wake and LMA (Cii) occurred during photoillumination. The EEG spectra during photoillumination at any time of day were indistinguishable from sleep–wake of WT mice (Fig. 2-1). Di, One hour photoillumination beginning at ZT12 resulted in a decrease of c-FOS in orexin-IR neurons. Top, Black triangles represent double-labeled c-FOS⁺ Hcrt/orexin neurons. Bottom, Open triangles represent c-FOS⁻ Hcrt/orexin neurons. Open triangles represent orexin⁻ neurons. Dii, Bar graph represents the percentage of Fos⁺ orexin neurons (n = 11 for control, n = 12 for light illumination). Values are mean ± SEM. *p < 0.05 versus prelight application.

Figure 3.

Differential Arch expression levels in orexin/Arch mice cause aberrant electrophysiological properties of Hcrt/orexin neurons and result in a gradient of photoinhibition. A, qPCR results distinguished two subgroups of orexin/Arch mice, low- (aLE) or high- (aHE) Arch expressers, based on gene copy number. B, Cell-attached recordings in response to 5 min photoillumination (orange line, 575 ± 25 nm) grouped by age (juvenile: P14–P27; adult: >8 weeks) and Arch expression. In contrast to juvenile aLE mice, adult orexin/Arch mice exhibited photoinhibition throughout the illumination period. Upon cessation of photoillumination, there was a significant rebound in spiking activity, before the firing rate returned toward prestimulation levels. C, Summary data indicate that photoillumination silenced Hcrt/orexin neuronal activity. However, Hcrt/orexin neurons in aHE mice of both ages (purple) had hyperpolarized RMPs (V_m) and (D) a lower basal firing rate. E, Photoillumination evoked a significant outward current (I_o) with different light durations (Ei). Photoillumination also decreased sEPSCs onto Hcrt/orexin neurons in aLE mice (Eii). Further characterization of basal sEPSC activity (Eiii) indicated no difference in sEPSC amplitude between Hcrt/orexin neurons from orexin/Arch and orexin/eGFP mice, although there was a significant reduction in the frequency of glutamatergic events that was more pronounced in aHE mice. F, When photoillumination was applied in conditions to measure sIPSCs, yellow light (575 ± 25 nm) evoked an outward current in all groups (Fi), but sIPSC activity did not change (Fii). Nevertheless, basal GABAergic tone was altered in orexin/Arch mice relative to orexin/eGFP mice, with a larger amplitude of basal events and greater sIPSC frequency (Fiii). G, Bath application of oxytocin (300 nm), a known activator of Hcrt/orexin neurons, had differential effects on the excitability of Hcrt/orexin neurons in (Gi) aLE and (Gii) aHE orexin/Arch mice: oxytocin significantly depolarized V_m of Hcrt/orexin neurons from aLE mice but not aHE mice (Giii). Values are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; n.s., non-significant as in legends to Figures 4 and 5.

Figure 4.

Aberrant circadian and metabolic features of orexin/Arch mice compared with orexin/Ataxin-3 (ATAX) and WT mice. A, Hourly metabolic rate over a 48 h period (Ai) and summed for the light, dark, and 24 h periods (Aii) revealed reduced FI, z activity, and RER during the dark phase in ATAX mice and reduced FI and RER in aHE mice during the light phase relative to other strains. Both aLE and aHE mice had greater x activity and RER during the dark phase relative to other strains. B, By 12 weeks of age, the body weight of orexin/Arch aHE mice exceeded that of both WT and ATAX mice. Inset, Body weight trajectory with age across the different strains using a line of best fit for each genotype (0.83 > R² > 0.90). C, Age-matched mice were used to assess postmortem body composition; aHE orexin/Arch mice had significantly elevated adiposity, particularly increased epididymal and subcutaneous fat mass. D, Male mice (9 weeks) were fasted overnight for a glucose tolerance test; no significant differences were observed across genotypes. E, Representative wheel-running actograms represent comparable locomotor rhythms between WT and aLE orexin/Arch mice under both LD (yellow bar: gray bar) and DD (gray shaded area) conditions. In contrast, aHE orexin/Arch mice have greatly reduced running wheel activity. Two-way ANOVA with Bonferroni post hoc test: *p < 0.05; **p < 0.01; ***p < 0.001. Abd, Abdominal fat; EpD, epididymal fat pad; SubQ, subcutaneous fat pad.

Figure 5.

EEG phenotyping of male orexin/Ataxin-3 (ATAX) and orexin/Arch mice reveals distinct differences in sleep–wake architecture and EEG spectral composition. A, Diurnal distribution of wakefulness (Ai) and NREM sleep (Aii) in both aLE and aHE orexin/Arch mice is comparable with age-matched male WT mice but distinct from that of ATAX mice. However, the number of REM bouts in aHE orexin/Arch mice was significantly increased over the 24 h period relative to the other strains (Aiii). Spontaneous cataplexy was only observed in ATAX mice (Aiv). Repeated-measures ANOVA revealed strain differences in hourly LMA (Fig. 5-1Ai) without a significant effect on T_b (Fig. 5-1Aii). B, The chocolate-cataplexy assay was used to determine whether cataplexy could be elicited in orexin/Arch mice under rewarding conditions. Presentation of chocolate at ZT12 significantly increased time spent awake (Bi) and reduced NREM sleep (Bii) during the dark phase for all strains. The percent time in REM sleep also decreased, except in ATAX mice (Biii). Chocolate also robustly increased cataplexy in ATAX mice but did not elicit cataplexy in orexin/Arch mice (Biv). Neither LMA (p = 0.12; Fig. 5-1Bi) nor T_b (p = 0.21; Fig. 5-1Bii) was significantly different among genotypes in the CCA. C, Spectral analysis of normalized EEG power across the active phase (ZT12-ZT23) and inactive phase (ZT24-ZT11) indicated significant changes in different bands across sleep–wake states in orexin/Arch mice during baseline recordings. During the 4 h SD, neither LMA (p = 0.87; Fig. 5-1Ci) nor T_b (p = 0.089; Fig. 5-1Cii) differed across strains. Bout D., Bout duration. Two-way ANOVA with Bonferroni post hoc test: *p < 0.05; **p < 0.01; ***p < 0.001 relative to WT.

Figure 6.

Sleep homeostasis parameters are normal in orexin/Arch mice. A, Percentage of wake, NREM, and REM sleep during a 4 h SD period initiated at ZT0 (blue shading) and the subsequent recovery period from ZT4 to ZT12. B, The latencies to NREM and REM sleep were significantly shorter in orexin/Arch mice under baseline conditions, but all genotypes had similar latencies after 4 h SD. C, After 4 h SD, all mice increased EEG delta power during NREM sleep, indicating comparable sleep homeostasis across genotypes. D, An mMSLT, comprised of five 20 min SD sessions interspersed with five 20 min “nap” opportunities, was initiated at ZT3 to assess sleepiness of each genotype. E, Latencies to NREM and REM sleep for all 4 genotypes during each “nap” opportunity of the mMSLT indicated that ATAX mice had significantly shorter REM latencies during the third and fourth nap opportunities. F, After initiation of access to chocolate at ZT12, total FI (i.e., consumption of chocolate and chow) was measured over the subsequent 4 h. Orexin/Arch mice had food preference and consumption levels comparable with WT and ATAX mice. Mice were then killed at ZT16, and assessment of c-FOS expression in Hcrt/orexin neurons indicated that orexin/Arch mice had levels of cFOS in Hcrt/orexin neurons comparable with WT mice, and that cFOS expression was not specific to Hcrt/orexin neurons within the LHA. Values are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 as in legends to Figures 4 and 5.