Role of pontine sub‐laterodorsal tegmental nucleus (SLD) in rapid eye movement (REM) sleep, cataplexy, and emotion

Abstract Pontine sub‐laterodorsal tegmental nucleus (SLD) is crucial for REM sleep. However, the necessary role of SLD for REM sleep, cataplexy that resembles REM sleep, and emotion memory by REM sleep has remained unclear. To address these questions, we focally ablated SLD neurons using adenoviral diphtheria‐toxin (DTA) approach and found that SLD lesions completely eliminated REM sleep accompanied by wake increase, significantly reduced baseline cataplexy amounts by 40% and reward (sucrose) induced cataplexy amounts by 70% and altered cataplexy EEG Fast Fourier Transform (FFT) from REM sleep‐like to wake‐like in orexin null (OXKO) mice. We then used OXKO animals with absence of REM sleep and OXKO controls and examined elimination of REM sleep in anxiety and fear extinction. Our resulted showed that REM sleep elimination significantly increased anxiety‐like behaviors in open field test (OFT), elevated plus maze test (EPM) and defensive aggression and impaired fear extinction. The data indicate that in OXKO mice the SLD is the sole generator for REM sleep; (2) the SLD selectively mediates REM sleep cataplexy (R‐cataplexy) that merges with wake cataplexy (W‐cataplexy); (3) REM sleep enhances positive emotion (sucrose induced cataplexy) response, reduces negative emotion state (anxiety), and promotes fear extinction.

Human cataplexy is mostly characterized by a sudden loss of muscle tone with intact consciousness often triggered by positive emotions such as laughter. Loss of orexin (or hypocretin) containing neurons in the lateral hypothalamus (LH) is a direct cause of human narcolepsy with cataplexy (NC). 5 Similarly, loss of orexin neurons, peptide, or receptors caused NC symptoms in mice. 6,7 Because REM sleep-like EEG and muscle atonia during cataplexy strikingly resemble REM sleep in mice, 8 it is widely believed that these two states may be controlled by a common neural circuit. Consistent with this idea, activation of glutamatergic neurons in the SLD in wildtype mice induced "cataplexy"-like state whereas their activation in 20-25 g male orexin-null mice (OXKO mice; mouse models of NC) increased the amounts of cataplexy. In contrast, inhibition of neither the SLD nor specific glutamatergic neurons prevented the baseline cataplexy in these mice, suggesting that cataplexy may occur in absence of the SLD. 9 Moreover, cataplexy and REM sleep behavior disorder (RBD) may occur in the same human subjects. 10

THE S LD IS NECE SSARY FOR REM S LEEP
To investigate whether the SLD is necessary for of REM sleep and cataplexy, we made bilateral lesions in the SLD using a diphtheria toxin-based lesion approach and examined the changes in sleep and cataplexy in OXKO mice (male, 20-25 g, Jackson Laboratory). For this, we injected a mixture of two AAVs-AAV containing the gene for Cre recombinase and (AAV-Cre) and a Cre-dependent AAV containing the gene for diphtheria toxin A (Flex-DTA)-into the SLD of OXKO mice (N = 10) and implanted them with EEG/EMG electrodes.
Another group of OX-KO (N = 10) received sham surgery and served as the control.
Three weeks after the surgeries, we performed EEG/EMG with concurrent video recordings for 48 h. The lesion areas around the SLD were mapped and confirmed by NeuN staining in Figure 1A.
Compared with SLD intact, the SLD lesions (SLDx) completely eliminated REM sleep and significantly increased wake amounts but did not alter NREM sleep significantly ( Figure 1D). REM sleep behavior (RBD)-like movements were not present in SLDx or control mice.

REM S LEEP ELIMINATION REDUCE S BA SAL AND SUCROS E (RE WARD) INDUCED C ATAPLE X Y WITH ALTERED FF T
EEG spectra of the baseline or sucrose induced cataplexy in OXKO controls were almost identical to REM sleep in terms of extreme high theta/delta ratio and atonia with only exception of theta peak reduced by about 1.0 Hz (REM sleep 7-8 Hz vs. cataplexy 6-7 Hz) ( Figure 1B,C). After SLDx, cataplexy EEG spectra displayed wake-like EEG with higher wide peak around 4.0 Hz than wake FFT but the overall delta power was only about a half of NREM sleep delta power ( Figure 1B,C). Despite of FFT alteration after SLDx, cataplexy episodes could be clearly identified by a combination of EMG, arrest behavior (video recording) and unique FFT from active motor behaviors and high and uneven EMG in wake. SLDx OXKO mice exhibited both baseline and sucrose induced cataplexy, but their levels were significantly reduced, compared with OXKO controls ( Figure 1E). SLDx reduced the baseline cataplexy amounts by about 40% time mostly from reduction in cataplexy numbers. Sucrose produced only ~10% increase in cataplexy time in SLDx OXKO mice, compared to ~100% increase in the OXKO controls ( Figure 1E). Sucrose induced cataplexy that was mostly due to an increase in average cataplexy duration and less due to cataplexy number in OXKO mice was blunted by SLD lesions ( Figure 1E).

REM S LEEP LOSS IN CRE A S E S NEG ATIVE EMOTION
Next, to investigate whether REM sleep elimination produced anxiety-like behavior, we conducted open field test (OFT) and elevated plus maze (EPM) test in SLDx-OXKO mice and OXKO mice three weeks after EEG/EMG recording. Age and sex-matched wildtype mice (N = 10) were used as an additional control. In OFT, two control groups did not differ in the total travel distance or in time spent in the central area. In contrast, the SLDx-OXKO mice traveled significantly longer total distance (restless behavior) but spent less time in the central area than both sets of controls ( Figure 1F). Similarly, in the EPM SLDx OXKO mice spent significantly more time in closed arms and less time in open arms than two control groups ( Figure 1G).
We then assessed the aggressive behavior in SLDx-OXKO mice, as defensive aggression is often seen in anxious animals. Consistent with the increased anxiety, SLDx mice were more aggressive than two controls. The number of attacks and the total time engaged in aggressive behavior were significantly higher in SLDx-OXKO mice than in two controls ( Figure 1H). These data indicate that REM sleep loss increases anxiety-like behaviors.
Finally, we examined fear extinction memory in these SLDx

DISCUSS ION
Our key findings are that SLD lesions eliminate REM sleep, reduce basal and reward induced cataplexy with EEG transformation from REM sleep-like to wake-like; and REM sleep elimination increases anxiety-like behaviors and impairs fear extinction and memory. and sucrose induced cataplexy (by 75%) and sucrose induced cataplexy from 100% increase over the baseline in control to only 10% increase in SLDx. WT, OXKO and SLDx OXKO mice in OFT (E), EPM (F) and aggression test (G) show that REM sleep elimination increases anxiety-like behaviors. (I) Fear extinction test in OXKO and SLDx OXKO mice demonstrate that REM sleep elimination significantly impairs fear extinction (D2) and extinction fear memory consolidation (D3). *p < 0.05. ** p < 0.01, *** p < 0.001. 4 Hz, two state merging may produce 6-7 Hz theta EEG. If cataplexy is indeed a mixed state, one may predict that direct single cell recording in the hippocampus in OXKO mice would show identical firing patterns in REM sleep and cataplexy. W-cataplexy overall delta power was higher than wake delta power but only about 50% delta power of NREM sleep, thus it is a distinct atonia state. Similar EEG pattern with high 2-4 Hz originated from the frontal cortex has been reported in freezing behavior in mice and cataplexy-like behavior by the mesencephalic locomotor region (MLR) lesions in rats. [21][22][23] Rats of orexin-ataxin that produces incomplete loss of orexin neurons exhibit wake-like FFT cataplexy. 24 W-cataplexy as a conscious state may well be due to incomplete loss of orexin neurons. Small amounts of orexin may be sufficient to prevent R-cataplexy. We predict that human with the extreme low-level orexin displays R-cataplexy intrusions with rapid eye movements and dream-like hallucination into W-cataplexy. Indeed, there is a case report that REM-sleep like eye movements and dreamlike hallucination intrusion into cataplexy. 25 Regarding the neural circuit of W-cataplexy, our previous studies suggest that the MLR reticulospinal neurons that contain orexin 2 receptors may mediate W-cataplexy as MLR lesions produce cataplexy-like behavior with a high delta wake EEG in rats. 21,26 It is not clear how two distinct states, R-cataplexy and W-cataplexy, mix up in cortical activity.
One possibility is that they merge at a small time scale, so that EEG FFT appears to be evenly and continuous theta dominant, producing hallucination (REM sleep) and awareness (wake) simultaneously. We hypothesize that orexin system through the SLD and MLR simultaneously prevents R-cataplexy and W-cataplexy. Meta-analyses on associations between REM sleep and anxiety using sleep deprivation in a large number of animal findings are inconsistent. 15,16,28 We speculate that inconsistent and various level stress by sleep deprivation methodology produces different outcomes on anxiety and fear emotion, which may cause the inconsistent outcomes. It appears that human sleep deprivation and insomnia consistently display high anxiety, 16 while sleep deprivations have inconsistent effects on in fear memories in both animals and human. 13 Although our animal experiment could not resolve whether REM sleep regulates conscious vs subconscious emotion and memory, a recent study of human sleep apnea occurred during REM sleep or during NREM sleep suggests that REM sleep may particularly regulate subconscious negative emotion and memory. 29 Our results suggest that REM sleep regulates cataplexy in two aspects: being a part of cataplexy and emotion perception. REM sleep loss would remove R-cataplexy and blunt emotion.
In conclusions: the SLD is the sole generator for REM sleep and responsible for R-cataplexy; REM sleep increases positive emotion and reduces negative emotion and memory.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.