All experiments were conducted at Kagoshima University following the guiding principles for the care and use of animals in physiological sciences published by the Physiological Society of Japan (2015) and were approved by the Experimental Animal Research Committee of Kagoshima University (MD17105).
For the animal model of narcolepsy, 17 male and 17 female orexin neuron-ablated (ORX-AB) mice [7, 9, 10] were used in a balanced number of sexes for each experiment. They were 8–16 weeks old at the start of the experiment and weighed 22–32 g. The original mating pairs were a generous gift from Prof. Yamanaka at Nagoya University and bred in Kagoshima University’s animal facility. A method for selective ablation of orexin neurons has been previously reported . Essentially, orexin-tTA mice that express tetracycline transactivator (tTA) exclusively in orexin neurons under the control of the human prepro-orexin promoter  were bred with tetO diphtheria toxin A fragment (DTA) mice (B6.Cg-Tg (tetO DTA) 1Gfi/J, The Jackson Laboratory) to generate orexin-tTA; tetO DTA mice. In these double-transgenic mice (called ORX-AB in this paper), doxycycline is removed from their chow starting from birth so that by four weeks of age, almost all (> 97%) of orexin neurons are ablated . Our previous studies confirmed the ablation of orexin neurons [7, 9, 22]. All mice were housed in a room maintained at 22–24 °C with lights on at 19:00 and off at 7:00 at least two weeks before experimentation started. We selected a reversed light/dark cycle so experimenters could observe mice in their active nocturnal phase of behavior during the daytime. Mice had food and water available ad libitum. The mice were grown in a group before the experiment. During the experimental period, they were singly housed to avoid any possible social rank effect on behavior .
Behavioral observation of cataplexy
A far-infrared lamp illuminated the experimental chamber in a light-controlled soundproof box (940 nm, SA2-IR, World Musen, Hong Kong). Mouse behavior was continuously recorded with a video camera (CBK21AF04, Imaging Source Asia, Taipei, Taiwan) and monitored on a computer outside the soundproof box. Cataplexy was determined according to the established criteria for mice , 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 prone with their head and belly down in the bedding with their limbs and tail typically situated straight out from the trunk. This posture contrasts with a normal sleeping position in which mice are curled up and fold their limbs and tail underneath their trunk. Second, the mouse is immobile aside from the movements associated with breathing during an episode. Finally, at least 40 s of active wakefulness (moving) preceding the atonia episode. The original criteria recommend recordings of EEG, but we did not adopt EEG to avoid possible obstruction of mice movement by the recording cable. Therefore, we use “cataplexy-like behavior” instead of “cataplexy” when describing current results in this manuscript.
Conditioned place preference test
We used a homemade place preference apparatus to examine the possible effect of memory on the number of cataplexies (Fig. 1). Two plastic chambers (14 × 20 × 15 cm) were connected via a tube that allowed the animal to move back and forth freely. The floor of one chamber was filled with wood chip bedding and the other with a plastic board, so there was a clear difference between the two. Since our test was performed during the dark period, visual cues by wall color would not be as effective as a popular conditioned place preference test that uses both visual and tactile cues by floor texture for memory recall. The test chambers were thoroughly cleaned after each test to avoid any odorous cues.
The mice were first allowed to explore the apparatus for acclimatization for 60 min freely. The next day, we tested whether the mice preferred a specific side chamber or a specific floor material while both chambers were empty, except for floor material, for one hour. The mice's position was judged by the tip of the nose and assigned to one chamber, even if the part of the body was in the connecting tube. The time spent in each chamber was recorded to assess the basal preference (pre-value). The preference score was calculated by dividing the time spent in the treatment-paired chamber by the total time spent in both chambers. Conditioning sessions were conducted for two consecutive days. In the first conditioning session at ZT14-17 for mice, mice were confined to one chamber for 60 min with a piece of milk chocolate (Hershey’s Kiss; Hershey). At ZT19-22 for mice, we confined the mice to the other chamber with a regular chow. During the two conditioning sessions, mice were returned to their home cage in the dark room. On the second day, the first session was a regular chow treatment, and the second was a chocolate treatment. A combination of floor texture and treatment was randomly assigned to the mice. On the day after the last conditioning session, the preference test was conducted in the same manner as the basal preference assessment at ZT19-22. Throughout all sessions, cataplexy behavior was blindly detected from video recordings to the treatment.
To examine the possible effect of aversive memory on cataplexy-like behavior, we performed a similar conditioned place preference (avoidance) test using an instinctively aversive odorant, 2-methyl-2-thiazoline (2MT; 2346-00-1, Henan Alfa Chemical, Qinyang City, China) , in place of chocolate described above. In this experiment, 0.5 ml of 2MT in a glass bottle (50 ml) that has a metal lid with small holes was placed in the chamber. The control was distilled water. To avoid a possible carry-over effect, we used a different set of naïve animals for this experiment.
After each observation, place preference apparatus cages were thoroughly washed with cleaner, sterilized with hypochlorous acid, and air dried. Observation using male and female mice was performed on different days to avoid possible cross-over effects from smell and sound.
To examine whether spontaneous cataplexy activates the NAc as chocolate-induced cataplexy did , we measured a cellular activation marker, pERK. We selected to measure pERK but not c-Fos since pERK is a cellular activation marker with a more rapid and narrow time window than other activation markers . Our previous study examined brain regions activated at the initiation of chocolate-induced cataplexy. We found that the rostral part of the NAc was the only region out of 33 brain regions examined when pERK was used as an activation marker . Therefore, we focused on NAc in this study. We examined five groups of mice brains that were sampled at ZT14-17. (1) Chocolate-induced cataplexy-like behavior group: When the mice showed cataplexy-like behavior within one min after the chocolate bite, they were immediately euthanized with a lethal dose of urethane (1.8 g/kg, i.p.) and bleeding, transcardially perfused with phosphate-buffered saline (PBS, 0.01 M, pH 7.4), followed by 4% paraformaldehyde (PFA) in PBS and the brain was removed. (2) Spontaneous cataplexy group: When the mice showed cataplexy-like behavior without chocolate in his/her home cage, their brain was sampled as described above. (3) The control group: The mice were euthanized after confirmation of no cataplexy for 10 min. (4) 2MT-related cataplexy-like behavior group: We did not define 2MT- “induced” cataplexy because mice did not show any cataplexy-like behavior within one minute after smelling the 2MT bottle, while they had when smelling and biting chocolate. Nevertheless, mice sometimes showed cataplexy-like behavior in the cage where the bottle of 2MT was settled. Therefore, we call such cataplexy-like behavior 2MT- “related.” When the mice showed cataplexy-like behavior, their brain was sampled as described above. (5) No cataplexy in the 2MT group: To examine the possible effect of 2MT alone on NAc activity, the mice were euthanized after no cataplexy occurred for 10 min in the cage with 2MT. In all five cases, the mice's behavior in the home cage was recorded by a video camera in a soundproof box and monitored on a computer screen outside the soundproof box . The experimenter manually judged cataplexy-like behavior, and the brain was sampled within approximately five min after injection of the anesthetic. The brains were post-fixed in 4% PFA solution at 4 °C overnight and immersed in 30% sucrose in PBS at 4 °C for two days. Coronal sections, including the NAc, were cut at 40 µm thickness using a vibratome, and every fourth Section (10 slices from each specimen) was used for immunostaining. The brain sections were immersed in a blocking solution (1% normal horse serum and 0.3% Triton-X in 0.01 M PBS) for 1 h at room temperature. The sections were then incubated with anti-pERK rabbit antibody (4370, Cell Signaling Technology, RRID: AB_2315112) at 1/400 diluted in blocking solution overnight. The sections were washed with PBS and then incubated with CF488-labelled donkey anti-rabbit IgG (1:500, 20015, Biotium, Heyward, CA, USA, RRID: AB_10559669) for 90 min. The sections were then mounted on a glass slide and examined using a fluorescence microscope (BZ-X700, Keyence Corp., Osaka, Japan). Among the 10 slices from each specimen, the most representative slice near the rostral NAc (1.2 mm anterior to bregma) (Fig. 4A) was selected and photographed using the same exposure time for all the slices from 5 experimental groups. The number of pERK-positive cells in the rectangle (400 × 1000 µm) in the NAc was bilaterally counted (Fig. 4B) with the assistance of the counting function in Photoshop software in a blinded manner to the treatment.
The number of cataplexy-like behaviors during pre- and post-conditioning periods was normalized by the stay time in the chamber. There was no need for such data normalization during conditioning-1 and conditioning-2 because the mice were confined to one chamber during the conditioning period. Statistical analyses were performed using Prism software v.9 (GraphPad). Two-way (test day x gender) ANOVA was used to compare the preference score between pre- and post-values in the conditioning test. Repeated measure design one-way ANOVA with the Geisser-Greenhouse correction, followed by Sidak’s multiple comparison test, was used to compare cataplexy-related scores. One-way ANOVA followed by Sidak’s multiple comparison test was used to examine the number of pERK-positive cells. P < 0.05 was considered statistically significant. Data are presented as mean ± SEM.