Activity of putative orexin neurons during cataplexy

Ethics approval

All experiments were conducted at Kagoshima University according to the guiding principles for the care and use of animals in the field of physiological sciences, published by the Physiological Society of Japan (2015), and were approved by the Experimental Animal Research Committee of Kagoshima University (MD17105 and MD20004).

Animals

We used two genetically engineered animal models of narcolepsy, which express the calcium indicator GCaMP6 in their putative orexin neurons. First, prepro-orexin-knockout mice [2] (ORX−/−) were crossed with transgenic mice carrying a tetracycline-controlled transactivator transgene (tTA) under the control of the orexin promoter [11] (ORX-tTA). The resulting ORX−/−; ORX-tTA mice were expected to express tTA, but not orexin, in their “orexin” neurons (Fig. 1A1). By injecting AAV-GCaMP6 (stereotaxic injection of AAV section) into the hypothalamus of these mice, we obtained ORX−/−; ORX-GCaMP6 (model 1) mice. The second model was constructed by crossing ORX−/−; ORX-tTA mice with TetO-GCaMP6 mice (Fig. 1A2). In TetO-GCaMP6 knock-in mice, the beta-actin gene was modified to convey a gene encoding tetracycline operator (TetO)-GCaMP6 [33] (B6; 129-Actb < tm3.1(tetO-GCaMP6)Kftnk > , obtained from RIKEN Bio Resource Research Center, RBRC09552). We used these two models to examine whether these two models, which are conceptually the same but technically different, would yield similar results.

All mice were housed in a room maintained at 22–24 °C with lights on at 19:00 and off at 7:00 for at least 2 weeks before experimentation began. We selected a reversed light/dark cycle so that the experimenters could observe mice in their active nocturnal phase of behavior during the daytime. We used male mice to avoid possible effects from an estrous cycle, as female hormonal cycles, at least in humans, may have some effect on sleep-related disorders, including narcolepsy with cataplexy [34]. We housed mice individually from the start of the reversed light/dark cycle to avoid any possible social rank effect on male behavior [35].

Stereotaxic injection of AAV

Under isoflurane anesthesia (2%, inhalation) using a stereotaxic instrument (ST-7, Narishige, Tokyo, Japan), a viral mixture consisting of recombinant AAV-tetO(3G)-G-CaMP6 (serotype: DJ; 600 nl/injection, 3 × 1012 copies/ml) and AAV-tetO(3G)-mCherry (serotype: DJ; 600 nl/injection, 6 × 1012 copies/ml) was stereotaxically injected into the left side of the hypothalamic perifornical area in ORX−/−;ORX-tTA mice (Fig. 1A1). All AAVs used in this study were produced by Yamanaka’s Laboratory at Nagoya University, Japan [11]. The injection site was as follows: 1.5-mm posterior from the bregma, 0.8-mm lateral, and 5.0-mm ventral from the dura.

In vivo recordings of neuronal activity using a fiber photometry system and cardiovascular parameters using a radio-telemetry system

We used our previously reported method [11]. In brief, 2–3 weeks after viral injection (model 1) or 1 week before the measurement (model 2), mice were anesthetized with 2% isoflurane and were surgically implanted with a guide cannula (diameter: 600 µm, length of guide: 8 mm, made by LUCIR, Tsukuba, Japan) to place the optical fiber immediately above the hypothalamus (1.5 mm posterior to the bregma, 0.8 mm lateral, 4.0 mm ventral) to record orexin neuronal activity. Immediately after performing the guide cannula implantation, we performed additional surgery to implant a radio-telemetry transducer (TA11PA-C20, Data Sciences International, St. Paul, MN, USA) into the abdominal cavity of the mice to record electrocardiograms (lead II). During all surgeries, care was taken to maintain body temperature. After surgery, the mice were treated with penicillin, an analgesic, and buprenorphine. For recovery, mice were individually housed and monitored and had access to food and water ad libitum for at least 1 week.

A fiber photometry system (COME2-FTR/OPT, LUCIR, Tsukuba, Japan) was used to record the activity of orexin neurons in freely moving mice, as previously described [11]. During the measurement, a dummy fiber was removed and a 400-μm silica fiber (LUCIR) was inserted through the guide cannula into the brain. G-CaMP6 fluorescence and mCherry fluorescence were collected using the same silica fiber. The respective G-CaMP6 or mCherry fluorescence was guided to individual photomultipliers. The signal was digitized at 100 Hz using a data acquisition system (PowerLab16/35, ADInstruments, New South Wales, Australia) and was recorded using LabChart software version 8 (ADInstruments Inc., Bella Vista, NSW, Australia).

On the experimental day, mice were individually placed into a recording chamber in a soundproof box with a 12-h:12-h reversed light/dark cycle for 10 h, from 8:00 (lights off at 7:00) to 18:00. The chamber was illuminated with a far infrared lamp (940 nm, SA2-IR, World Musen, Hong Kong), and a piece of chocolate was placed in the chamber at the start of observation to increase the episodes of cataplexy [21]. Mouse behavior was continuously recorded with a video camera (CBK21AF04, The Imaging Source Asia, Taipei, Taiwan) and was monitored on a personal computer located outside the soundproof box using the video capture function in LabChart. Electrocardiograms of mice were sampled through a radio-frequency receiver (RLA1020, Data Sciences International), digitally converted at 1000 Hz, and transferred to a computer with PowerLab (ADInstruments). The heart rate was calculated using the cyclic variable function in LabChart. The video movie, G-CaMP6 and mCherry fluorescence signals, electrocardiogram, and calculated heart rate were stored on a personal computer. The fluorescence signal intensity at its nadir during the 10-h observation period was defined as 0%. At this moment, the mouse seemed to be in a sleeping/rest state, as judged through video recordings. To exclude individual differences in fluorescence signal intensity, the average value from − 60 to − 30 s from the onset of cataplexy was defined as 100%.

Stress stimulation

To test whether the putative orexin neuron in this study responded to an aversive stimulus, as did the orexin-producing neurons, we used an intruder stress test, as in our previous study [11]. This stressor was applied by placing an age-matched wild-type mouse (intruder mouse) in a small polypropylene cage into the experimental cage for 2 min. The polypropylene cage was constructed such that the intruder and resident (experimental) mice were unable to contact each other physically, but visual, auditory, and olfactory communications were possible.

Behavioral observation of cataplexy

Cataplexy was determined according to the established criteria for mice [20], which are defined by several observable features. The first feature is an abrupt episode of nuchal atonia lasting at least 10 s. Atonia was determined to occur when mice were in a prone position with their head and belly down in the bedding with their limbs and tail typically situated straight out from the trunk. This posture shows a clear contrast to a normal sleeping position in which mice are curled up and fold their limbs and tail underneath their trunk. Furthermore, in atonia, the mouse was immobile besides the movements associated with breathing during an episode. Finally, there had to be at least 40 s of active wakefulness (moving) preceding the atonia episode. The original criteria recommended recordings of EEG, but we did not adopt EEG to avoid heavy attachments to the head with the photometry fiber and EEG cables. Therefore, we refer to “cataplexy-like behavior” instead of “cataplexy” in this manuscript.

Immunohistochemistry

Mice were deeply anesthetized with urethane (2.0 g/kg, i.p.) and were transcardially perfused with Ringer’s solution (containing CaCl2), followed by 4% paraformaldehyde in 0.1 M Tris (pH 7.4) + 3 mM CaCl2. We added calcium to the ordinal washing and fixative solutions because in our preliminary experiment, we found that the fluorescence of GCaMP6 was better preserved with calcium supplementation. The brain was removed, post-fixed in 4% paraformaldehyde + Ca solution at 4 °C overnight, and was subsequently immersed in phosphate buffered saline (PBS) at 4 °C for at least 2 days. A series of 40-μm sections were obtained using a vibratome (SuperMicroSlicer Zero1; DOSAKA EM, Kyoto, Japan). For staining, coronal brain sections were immersed in blocking buffer (1% normal horse serum and 0.3% Triton-X in PBS) and were then incubated with an anti-orexin A rabbit antibody (1:1000, 14346v, Peptide Institute, Osaka, Japan) at room temperature (about 20 °C) for 1 h. The sections were washed with PBS and were incubated in a CF568-conjugated anti-rabbit IgG antibody (1:500, catalogue #20098, Biotium, Heyward, CA, USA) for 2 h at room temperature. These brain sections were mounted on slides and were imaged using a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan). For counting, we used one slice in which the optic fiber tract was most evident in the animal (Fig. 1C).

Statistical analysis

Statistical analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA). To compare the two groups of data, we used the Student’s t-test. To compare three or more groups of means, we used one-way or two-way analysis of variance followed by Sidak’s multiple comparison tests depending on the data structure. Data were reported as means and standard error of the mean. Statistical significance was set at P < 0.05.

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