Disrupted temperature-sleep coupling mechanism in a Dravet syndrome mouse model

Domain: chemistry
Published: 2026-02-26
Editorial outcome: major_revision

Abstract
Dravet syndrome (DS) is associated with epilepsy, developmental delays, thermal dysregulation, and sleep disturbances. While seizures have been linked to hippocampal dysfunction, what drives sleep disturbances and thermal dysregulation is poorly understood. Using DS mice (Scn1aA1783V), we identified a link between sleep and thermoregulation. We found that DS mice exhibited lower core body temperature. Next, using electrocorticography, local field potential recordings, and core temperature monitoring, we showed that DS mice exhibited a lack of core temperature change during the transition from waking to non-rapid eye movement sleep. This is in contrast to wild-type (WT) mice, in which sleep onset coincided with a temperature drop. Additionally, warmth promoted sleep in WT, but not in DS mice. Vector-mediated expression of SCN1A or chemogenetic stimulation of the anterior hypothalamus restored the warmth-induced somnogenesis in DS mice. These findings highlight a connection between sleep and thermal dysregulation in DS, implicating altered neuronal activity of the hypothalamus.

Introduction
Dravet syndrome (DS) is a developmental and epileptic encephalopathy caused by loss-of-function mutations in an SCN1A allele, encoding the α1 subunit of the voltage-gated sodium channel, NaV1.1 1. In addition to severe epilepsy, developmental delay and cognitive impairment, autonomic dysfunction and sleep problems are also common in patients with DS 2,3. Autonomic dysfunctions include temperature dysregulation, diaphoresis, and intolerance to cold 4,5. Sleep problems manifest as nocturnal seizures, trouble falling and staying asleep, and drowsiness 6–9. Mouse models of DS (DS mice) recapitulate many of the multifaceted human phenotypes 10–12, including impaired autonomic dysfunction, deficits in respiration, thermoregulation 13–15, circadian dysfunction, and abnormal sleep architecture 16,17. Among the many existing models, DS mice harboring the missense A1783V Scn1a mutation exhibit severe epilepsy and various DS-associated behavioral phenotypes, including spatial memory and motor deficits 12,18–21. Sleep properties or thermoregulation have not been investigated in this DS model.

Despite their distinct phenotypic manifestations, sleep and thermoregulation are closely associated. In rodents and humans, increased ambient temperature promotes sleep initiation, the onset of non-rapid eye movement (NREM) sleep coincides with a reduction in core temperature, abnormally elevated body temperatures are linked to abnormal sleep homeostasis, and sleep deprivation leads to elevated Tbody and brain temperatures 22–25. The close ties between these two homeostatic physiological responses require a well-coordinated and regulated brain function, especially during the transition states. Indeed, complex neural networks centered in the hypothalamus govern the transitions from wakefulness to sleep and the temperature shifts during these transitions 23,25,26. However, less is known about the features and mechanisms underlying sleep and temperature transitions in the context of DS. Identifying the neuronal basis of these deficits, as well as developing therapeutic strategies, could improve sleep quality and temperature homeostasis in numerous pathologies.

Here, we demonstrate that sleep and thermoregulation are dysregulated in DS mice (Scn1aA1783V). We show deficits in thermal adaptation, a lack of temperature reduction that is normally seen in the transition from wake to NREM sleep, and decreased sleepiness in response to warmth. Moreover, we find that canine adenovirus (CAV2)-mediated SCN1A delivery to, or chemogenetic activation of, anterior hypothalamic neurons rectifies sleepiness induced by a warm temperature. Together, these findings underscore a critical effect of the dysregulated activity of the anterior hypothalamus in the disturbed sleep and thermoregulation phenotype in DS.

Results
Dysregulation of body temperature of DS mice in changing ambient temperatures
The ability to stabilize core temperature was tested in DS mice at postnatal day (P) 21-25. At this age, all the mice experience spontaneous seizures 12,18,27. Interestingly, we noted a lower baseline temperature in DS mice compared to that of wild-type (WT) littermates (Fig. 1a, b). To examine the response to a changing ambient temperature, mice were transferred to a beaker on a heated pad set to 30°C for 30 min (yielding a moderate warm ambient temperature of 26°C) and back to RT for another 15 min. Despite starting from a lower temperature, at the end of the 30 min period, the core temperature of WT and DS mice was similar. Yet, upon return to RT, DS mice returned to a lower temperature than that of WT mice (Fig. 1c - e, Supplementary Fig. 1). Of note, while both WT and DS mice reached a temperature that was lower compared to their initial core temperature, the overall temperature of DS mice 15 min after recovery at RT was lower again lower than that of WT animals (Fig. 1e).

Febrile seizures at relatively low temperatures are a hallmark of Scn1aA1783V mice during their 4th week of life 10,27,28, which guided our decision to use a moderate heat stimulus. Despite that, ~25% of the tested DS mice experienced seizures, which led to the termination of the test and exclusion from further analyses (Supplementary Fig. 1).

Thermoregulation is an important step in maintaining homeostasis, especially in preparation for sleep, while nesting contributes to preserving body temperature during sleep 23,29. When placed overnight with nesting material, juvenile WT mice built complex nests. By contrast, 75% of DS mice did not create nests, and those that did, built less complex nests (Fig. 1f-h), demonstrating a reduced ability to perform this innate behavior, which serves as a primary step in preparation for sleep. Nevertheless, nesting is a multifactorial behavior that can also be influenced by DS-associated comorbidities, including motor or social deficits, which may contribute to this phenotype.

Together, these data show that DS mice have a lower baseline temperature and display a reduced ability, or possibly a reduced drive, to build a functional nest, which may disturb their preparation for sleep.

Temperature dysregulation in DS mice during wake to NREM sleep transitions
While sleep deficits occur in DS mice 16,17, an association with temperature dysregulation has not been reported. To explore the link between these physiological processes, we focused on the transition from wake to NREM sleep. This transition is normally associated with reduced core temperature, and the drop in temperature at NREM onset is important for effective sleep 23,25,29. To study this transition here, we performed 2-4 hours of video electrocorticography/electromyography (ECoG/EMG) recordings in conjunction with continuous measurements of core temperature in WT and DS mice during their 4th week of life (Fig. 2a, b). To identify the transitions to NREM, two investigators analyzed the vigilance states independently, basing their classification on the ECoG and EMG signals, as well as video analysis. Only epochs that were mutually agreed upon were considered for further analysis. Quantification of the average duration of wakefulness and NREM within these short-term recordings revealed a similar duration of wakefulness and NREM sleep in WT and DS, with at least one transition from wake to NREM (Supplementary Fig. 2). Next, we examined the power spectral densities (PSD) of the ECoG during the transition from wakefulness and NREM. The PSD of wakefulness was calculated two minutes prior to the transition to NREM, and the PSD of NREM was measured on the first minute into NREM. In WT mice, a comparison of the PSDs of these states showed that the transition to NREM was accompanied by a clear increase in ECoG delta power. Conversely, while in WT mice this translated in the contribution of delta power to the overall ECoG being larger during NREM sleep than waking, in the DS mice, the delta ratio was similar during wake and NREM (Fig. 2c-d). Examination of the core temperature during these transitions to NREM revealed that in WT mice, transitions to NREM were typically (65%) associated with a reduction in body temperature. In contrast, in DS mice, the core temperature remained, on average, stable during transitions, and fewer (21%) transitions were accompanied by a reduction in temperature (Fig. 2e-f).

The anterior hypothalamus is involved in temperature control and the regulation of sleep-wake cycles, also through its influence on the delta frequency band 25,30,31.
To examine features of hypothalamic delta activity during the transition to NREM, we implanted depth electrodes in the anterior hypothalamus and recorded the LFP signal 32 (Supplementary Fig. 3), along with the EMG and continuous core temperature measurement. While the ECoG and LFP signals were overall similar, the contribution of the delta frequency band, which is strongly associated with NREM sleep, tended to be more prominent in the LFP recording (Supplementary Fig. 3d). Of note, as these depth surgeries are more invasive, we used 5-week-old mice. Similar to the ECoG recordings, the hypothalamic depth electrode LFP data showed that in WT mice, transitions from wake to NREM were associated with an increased contribution of the delta frequency band and a reduction in their core temperature. By contrast, as observed with the ECoG recordings in DS mice, the contribution of delta to the overall LFP was similar during wake and NREM, and their temperature was globally stable through these transitions (Fig. 2g-k).

Together, ECoG, LFP, EMG, and core temperature recordings demonstrate that the transition to NREM sleep in DS mice is associated with a diminished increase in delta power at NREM onset and unaltered core temperature.

Hypothalamic SCN1A expression restores warm-temperature induced sleep promotion in DS mice
A warm ambient temperature (32-36C) promotes sleep through a mechanism that involves the activation of heat-sensing neurons in the hypothalamus 29,33. Given the deficits we observed in DS mice in temperature regulation upon the rise to a moderate ambient temperature of 30°C, and the sleep transition-related changes in delta power in DS mice, we set out to investigate whether warmth-induced somnogenesis is impaired in DS mice. Depth LFP and EMG activity were recorded in 5-6-week-old WT and DS mice. Recordings were first obtained for one hour at room temperature, followed by an additional hour at elevated ambient temperature achieved by placing the cage on a heating pad set to 36°C, resulting in a temperature of 29°C at the bottom of the recording beaker (Fig. 3a-c). Because DS mice at this age show reduced susceptibility to febrile seizures 10,27, we reasoned that a somewhat stronger warming stimulus could be applied compared to the 30°C condition that was used for the experiments in younger animals (Fig. 1). However, to minimize seizure risk, we avoided the higher temperatures (32-36°C air temperature) used in previous studies 29,33 making 29°C a deliberate compromise between physiological relevance and safety in this model.

Vigilance state analyses of the whole recording indicated that the warm temperature increased the time spent in NREM sleep in WT mice (Fig. 3d). Furthermore, during transitions from wakefulness (2 min prior to NREM onset) to NREM (1 min into NREM), WT mice showed a comparable increase in the relative contribution of delta-band activity to the overall LFP power at both RT and elevated temperature (Fig. 3e). By contrast, warm temperature did not promote NREM sleep in DS mice (Fig. 3d), and the delta ratio during NREM sleep in DS mice was similar at RT and 36°C (Fig. 3f).

Of note, none of the tested DS mice had a thermally-induced seizure during these recordings. Moreover, the number of interictal spikes in DS mice did not increase for the majority of mice when placed in warm ambient temperature (Supplementary Fig. 5), indicating that increased epileptic activity does not account for the lack of a sleep-promoting effect of a warm environment.

Next, we asked if this is directly related to the DS-associated Scn1a loss-of-function mutation, such that increasing NaV1.1 levels in the hypothalamus could ameliorate this phenotype. To address this possibility, we injected a canine adenovirus type 2 (CAV2) vector harboring an SCN1A expression cassette (CAV2-SCN1A) 28 bilaterally into the anterior hypothalamus of WT and DS mice. Hypothalamic LFP and EMG electrodes were implanted 1 - 2 weeks later. After 3 - 5 days of recovery, we tested the effect of increased ambient temperature (from RT to 36˚C) on somnogenesis. In WT mice injected with CAV2-SCN1A, similar to untreated WT mice, the change to 36˚C increased the duration of NREM sleep (Fig. 3j, k). However, a significant increase in the delta contribution at NREM was only observed at RT, likely due to the relatively low number of examined mice in this control cohort (Fig. 3k).

In DS mice, CAV2-SCAN1A injection into the hypothalamus had a limited impact on the frequency of interictal spikes (Supplementary Fig. 5). This is in contrast to the strong ameliorating effect on epileptiform activity following CAV2-SCN1A injection into the hippocampus or the thalamus 28. Nonetheless, CAV2-SCN1A injection in the hypothalamus of DS mice improved temperature-related sleep promotion as measured by enhanced percentage of NREM, and enhanced delta ratio during NREM at RT or warm temperatures (Fig. 3j, l). These data demonstrate that the impaired somnogenesis effect of ambient warmth in DS mice can be corrected by CAV2-mediated expression of NaV1.1 in the anterior hypothalamus.

To directly control for the effects of the hypothalamic CAV2 injections, a separate cohort of WT and DS mice was injected with a control vector containing a reporter gene CAV2-GFP, or with CAV2-SCN1A. The biodistribution was examined in mice following CAV2-GFP injection (WT and DS, n = 3 for each genotype), which were also used for behavioral and electrophysiological examinations, as well as in a separate group of three WT mice injected with the control vector CAV2-mCitrine, which were only used for immunohistochemistry. The biodistribution analysis demonstrated expression in excitatory and inhibitory neurons in the hypothalamus and, due to CAV2’s retrograde transport properties, also in interconnected brain regions (Fig. 4a, b, Supplementary Figs. 8 and 9).

In the cohort of CAV2-GFP or CAV2-SCN1A injected mice, we also evaluated nest-building behavior. Seven to ten days after vector injection, CAV2-SCN1A did not rescue the impaired nest-building abilities of DS mice (Supplementary Fig. 10). Next, ECoG electrodes were implanted, and the mice were allowed to recover for 2-3 days before we proceeded with ~4 h ECoG and temperature recordings at RT. Also in this cohort of mice, CAV2-SCN1A expression in the hypothalamus restored the increase in delta ratio during the transition to NREM sleep, which was absent in DS mice injected with the control vector (CAV2-GFP, Fig. 4a-e). Of note, these analyses were conducted using cortical ECoG rather than the hypothalamic LFP recordings used for the CAV2-SCN1A experiments shown in Fig. 3, confirming that the correction of sleep-related oscillatory activity by CAV2-SCN1A is also reflected at the cortical level. Core temperature measurements were included in this new cohort of mice to test whether hypothalamic SCN1A expression may also correct the drop in body temperature at NREM onset. However, in contrast to the data from naïve untreated mice shown in Fig. 2, no significant temperature decrease was detected after NREM onset in either WT or DS mice injected hypothalamically with either CAV2-GFP or CAV2-SCN1A. The discrepancy between these results and those presented in Fig. 2 may be attributable to the different experimental procedures, as the mice depicted in Fig. 2 underwent only one surgical process, for electrode implantation (ECoG at the age of 4 weeks, or LFP at the age of 5 weeks), while the mice shown in Fig. 4 underwent two surgeries, one for vector injection at 4 weeks and a second one for ECoG implantation at 5 weeks.

The warmth-induced somnogenesis experiment was also performed in this cohort of mice, 1-4 days after the ECoG recordings at RT, and confirmed that NREM sleep was promoted in DS mice injected with CAV2-SCN1A, whereas in DS mice injected with the control CAV2-GFP vector, the switch to warm temperature did not induce NREM sleep (Fig. 4f-j).

Together, the data shown in Figs 3 and 4 show that CAV2-mediated SCN1A expression in the hypothalamus increases the delta contribution to NREM sleep and corrects somogenesis in a warm environment.

Chemogenetic activation of hypothalamic neurons restores warm-temperature-induced sleep promotion in DS mice
The prevailing view is that DS-associated Scn1a mutations lead to reduced neuronal excitability due to impaired NaV1.1 channel function 34,35. The hypothalamus comprises a heterogeneous population of excitatory and inhibitory neurons, both expressing comparable levels of Scn1a mRNA (Supplementary Fig. 8) 36. If the primary effect of CAV2-SCN1A is to enhance the activity of transduced neurons, we wondered whether artificially increasing neuronal excitability, through chemogenetic activation, could mimic the effect of exogenous NaV1.1 expression. Specifically, we tested whether chemogenetic activation of hypothalamic neurons could restore temperature-dependent sleep induction, similar to the correction observed following CAV2-SCN1A injections.

CAV2-hM3D-IRES-mCitrine was injected into the anterior hypothalamus of 4-week-old WT or DS mice. CAV2-hM3D-IRES-mCitrine encodes hM3D, a CNO (clozapine N-oxide)-activatable DREADD and an mCitrine expression cassette. 1-2 weeks post-injection, LFP and EMG electrodes were implanted, and the mice were examined 3-5 days later for the sleep-promoting effect of increased ambient temperature (~6 weeks old during the recording). As a control for the CNO injection, mice were first tested following the injection of saline just before the cage was placed on a 36 C heating pad. A few days later, the experiment was repeated using CNO injection (10 mg/kg) (Fig. 5a, b). This dosage of CNO did not alter locomotor activity, as assessed in an open field test in WT mice without DREADD expression (Supplementary Fig. 12), indicating the absence of a nonspecific sedation effect at this dose 37.

In WT mice, the warm temperature promoted NREM sleep after saline or CNO injection (Fig. 5c-f), similarly to untreated WT mice (Fig. 3d, e). The higher delta ratio during NREM sleep compared to waking was observed in some of the conditions, but not all (Fig. 5f). By contrast, in saline-injected DS mice, similar to untreated DS mice (Fig. 3d, f), or CAV2-GFP injected DS mice (Fig. 4f), the warm temperature did not promote NREM sleep (Fig. 5g, i). Conversely, in the same DS mice, following CNO administration, the warm temperature promoted NREM sleep, as well as an increase in the contribution of delta power at NREM, but only in warm temperature after CNO injection (Fig. 5i-j). Thus, acute chemogenetic activation of hypothalamic neurons was sufficient to restore warm temperature-induced sleep promotion in DS mice.

Methods
Study approval
All animal experiments were approved by the Institutional Care and Use Committee of Tel Aviv University.

Animals
WT and DS mice harboring the global Scn1aA1783V/WT mutation were generated by crossing conditional floxed Scn1aA1783V/WT males (The Jackson Laboratory; stock #026133, C57BL/6J) with CMV-Cre females (The Jackson Laboratory; stock #006054, C57BL/6J), as described before 12,27,28. The mice were kept on the pure C57BL/6J genetic background.Mice were housed in a standard animal facility at a constant temperature of 22°C, on a 12-hour light/dark cycle, with ad libitium access to food and water. Both male and female WT and DS mice were used in all of the experiments, and the data were combined.

Temperature regulation in a changing ambient temperature
WT and DS mice at the age of P21-P25 were used (4th week). The mice were handled for 5 minutes prior to the test. To measure the core temperature of the mice, we used a rectal thermal probe connected to a digital temperature controller (RET-4 probe, connected to TCAT-2DF, Physitemp Instruments, Clifton, NJ USA). The baseline temperature was measured 30-60 sec after the probe was inserted and continuously for the duration of the experiment using a video recording of the digital temperature controller. The mice were given an additional 5-10 minutes to habituate to the rectal probe at room temperature (RT), and the experiment started as the mouse was moved to the test beaker, which was placed on a heated pad set to 30°C (controlled by a second digital temperature controller, TCAT-2DF, Physitemp Instruments, Clifton, NJ USA). Under these conditions, the temperature at the bottom of the beaker was 26°C. We used the moderate heat of 30°C in DS mice at their fourth week of life due to their high sensitivity to thermally induced seizures 10,27. Next, the mouse was moved to a different beaker at RT, and the body temperature was measured for another 15 minutes. This was the protocol for ten WT mice and six DS mice (Fig. 1). Three more WT mice and six more DS mice were only followed when placed over the heated pad, and not during the cooling stage (Supplementary Fig. 1). The core temperature was extracted from the video in one-minute intervals. The test was terminated immediately if DS mice showed any signs of seizures (Supplementary Fig. 1).

Nest building assay
Two hours before the dark period, mice aged P21-22 (Fig. 1) or P31-33 (Supplementary Fig. 10) were placed in individual cages with wet food and water. The bedding materials were weighed and placed in the middle of the cage. The next morning, the unused nesting materials were weighed. In addition, the nests were given a score on a scale from 1-5, with a score of 1 in case the bedding materials were untouched and a score of 5 if most (>95%) of the materials were used 75.

ECoG surgery
Electrode implantation was performed in mice as described before 12,28,76. Briefly, mice were injected with ketamine/xylazine (191/4.25 mg/kg) for anesthesia. Carprofen (5 mg/kg) was injected for analgesia prior to the procedure. A midline incision was made above the skull, and two electrodes of fine (130 m bare diameter; 180 m coated diameter, cat # 785500 A-M systems, Carlsborg WA, USA) silver wires were placed under the dura at visually identifiable locations over the somatosensory cortex. A reference electrode was placed over the cerebellum. Two electrodes with ball tips were used for EMG, which was placed above the neck musculature on one side, and a ground electrode, which was placed under the skin over the shoulder on the other side. The millmax connector was secured with dental cement, and the skin was closed with sutures. For the ECoG recordings in Fig. 2 we used mice at their 4th week of life. For the ECoG recordings in Fig. 4, the mice were 5-6 weeks old. The mice were given 2-3 days for recovery after surgery before recording.

LFP depth electrode surgery
Five to six week old mice were anesthetized as described for the ECoG surgery and were mounted on a stereotaxic frame (Ultra Precise Stereotaxic Instruments, Stoelting, Wood Dale, IL, USA). Fine silver wire electrodes (same type as used for ECoG) were inserted into the lateral hypothalamus (AP:+1 mm, ML:±1 mm, DV:-5.2 mm). A reference electrode was placed over the cerebellum. An EMG electrode was placed above the neck musculature on one side and a ground electrode was placed under the skin over the shoulder on the other side. In a subset of animals, the electrodes were coated with Dil (1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate, 10% in ethanol) to verify their location. In others, after the recordings, the brains were extracted to 4% PFA and sectioned to verify the electrode location. The mice were given 3-5 days for recovery after surgery before recording.

Viral injection into the hypothalamus
WT and DS mice, at the age of P22-26, were injected with CAV2-NSE-SCN1A, CAV2-NSE-GFP, CAV2-NSE-mCitrine (CAV2-SCN1A, CAV2-GFP or CAV2-mCitrine 28), or CAV2-hM3D-IRES-mCitrine (Plateforme de Vectorologie de Montpellier, IGMM Montpellier, France). The mice were anesthetized using ketamine/xylazine (191/4.25 mg/kg), and carprofen (5 mg/kg) was used for analgesia. The mice were placed in a stereotaxic device (Ultra Precise Stereotaxic Instruments, Stoelting, Wood Dale, IL, USA). A midline incision was made above the skull, and holes were made using a 25G needle at the site of injection. We used the following coordinates for: AP +0.9 mm; ML ± 0.9 mm; DV -5.2 mm. The injections were done at a rate of 100 nl/min. One microliter containing 1 × 109 physical particles was injected (0.5 μl on each side) using a 1 μl beveled needle Hamilton syringe (7000 series, Hamilton, Reno, NV, USA). The needle was kept in place for 10 minutes following injection on each side. Then, the needle was retracted slowly to prevent leakage of the vector. The mice were allowed to recover from the surgery and were kept in isolation for 1-week following the injection. 7-14 days later, the same mice underwent surgery to implant the LFP depth electrode (Figs. 3, 5), or ECoG (Fig. 4) as described above.
For the histological analyses, the mice were perfused and sliced as described before 28.

Video-ECoG /LFP- and core temperature recordings
Video-ECoG/LFP recordings were performed in a separate room during the light period (8 am-6 pm) for 2-5 h per mouse in a padded Plexiglas box (Fig. 2 and Fig. 4d) or a 2-liter beaker (Fig. 3, Fig. 4g-j, Fig. 5) with wet food provided. The mice were connected to a T8 Headstage (gain X100 Triangle BioSystems, Durham, NC, USA), using PowerLab 8/35 acquisition hardware and LabChart 8 software for analyses (ADInstruments, Colorado Springs, CO, USA). The sampling rate was 1 kHz with a notch filter at 50 Hz, and a band-pass filter of 0.5-100 Hz. For the recordings depicted in Fig. 2 and Fig. 4d, the core temperature was continuously measured using a rectal probe connected to a digital temperature controller (RET-4 probe, connected to TCAT-2DF, Physitemp Instruments, Clifton, NJ USA), of which the digital display was monitored continuously by video. The recordings were long enough, and the conditions were suitable for the vast majority of the mice (except for one DS mouse, which was excluded from the analysis) to reach NREM sleep, but not REM sleep.

Depth electrode LFP or ECoG recordings in a changing ambient temperature
LFP recordings were performed as indicated above, with the exception of a subset of data that are presented in Fig. 5, which were performed using Neuralynx Digital Lynx data acquisition system (Neuralynx, Inc., Bozeman, MT, USA) using the same configurations as those recorded using PowerLab 8/35. To test the effect of temperature on sleep promotion, the mice were recorded in two blocks: the first one at room temperature for one hour, after which the recording chamber was moved and placed over a heated pad set to 36°C (TCAT-2DF, Physitemp Instruments, Clifton, NJ USA), and a second block was recorded for another hour. Under these conditions, the air at the bottom of the beaker was at ~29°C. Note that for these experiments, since the DS mice were older than five weeks, a higher ambient temperature of 36°C was used (and not the 30°C used for the younger DS mice in Fig. 1), given their reduced sensitivity to thermally induced seizures at this age. For the experiments described in Fig. 5, immediately prior to the placement of the heated pad, the mice were given an IP saline injection, and when the experiment was repeated 3-4 days later, an IP injection with 10 mg/kg clozapine-N-oxide (CNO, Angene, London, URK). We analyzed the entire recording after saline injection, but excluded the first 15 minutes following CNO injection to allow for CNO absorption 77,78. Locomotor activity of WT mice, without DREADD expression, was not affected by 10 mg/kg CNO. Specifically, 15 min after CNO injection, locomotor activity of the mice was monitored for 10 minutes in an open field of 50 by 50 cm (Supplementary Fig. 12).

Vigilance state analysis
ECoG or depth electrode LFP signals were analyzed in Spike2 software (Cambridge Electronic Design Limited, Cambridge, UK) or AccuSleep MATLAB-based interface 79, by segmenting the data into 5-second epochs, followed by visual inspection of the ECoG/LFP and EMG signals. Wakefulness was identified by desynchronized ECoG/LFP patterns and relatively high levels of EMG activity. Non-rapid-eye-movement (NREM) sleep was marked by high-amplitude, slow wave patterns accompanied by low EMG activity. Virtual channels derived from the ECoG/LFP signal, particularly delta power (0.5-4 Hz), were used to support NREM stage identification. However, in DS mice, as the increased delta activity during NREM is diminished, the EMG signal contributed to the classification. To distinguish rapid-eye-movement (REM) sleep from wakefulness, a theta (4-8 Hz) to delta power ratio of >2.5, along with low EMG activity relative to NREM, was applied in the classification. Periods where wakefulness and NREM sleep alternated within 20 seconds were classified as transition states. Example recordings with hypnograms including all stages are shown Supplementary Fig. 3, whereas the hypnograms in Figs. 3-5 are shown without indication of these transitions for improved clarity. Transition periods, which did not allow for classification, were excluded from analysis. Two investigators analyzed the vigilance states independently and only sections that were mutually agreed upon were considered for further analysis. For the data shown in Fig. 2, in WT mice, over 90% of the transitions from wake to NREM were agreed upon by the two investigators (n = 34 transitions from recordings of 6 WT mice). In DS mice, the two investigators agreed on ~70% of the transitions (n = 37 transitions from recordings of 8 DS mice). Artifacts, sleep arousals (defined as increased short gamma activity accompanied by active EMG within sleep epochs), and epileptiform activities were carefully excluded to ensure accurate vigilance state scoring. Video recordings were consulted to verify sleep-wake classifications in cases of uncertainty.

ECoG/LFP analyses of power spectral density
Power spectral density (PSD) was calculated using a fast Fourier transform with Hann (cosine-bell) data window of 50% overlap (LabChart 8 software, AD Instruments, Colorado Springs, CO, USA). For each mouse, we calculated the PSD of the ECoG, or depth electrode LFP, during wakefulness and NREM, using the average value of the bilateral electrodes. The PSD of wakefulness was calculated from 30 seconds of recording, starting two minutes prior to the transition to NREM (in some cases, slight modifications of the timing were used to avoid movement artifact). The PSD of NREM sleep epochs was calculated from 30 seconds of recordings starting from one minute into NREM. Data from all transitions for each mouse were averaged prior to averaging across different mice, such that each mouse contributed one PSD value to the analysis. The ratio of delta was calculated as the sum of the power between 0.9 and 3.9 Hz, divided by the sum of the total power between 0.9 and 99 Hz.

Immunohistochemistry, immunofluorescence and in situ hybridization
Immunohistochemistry, immunofluorescence, and in situ hybridization using RNAscope technology were performed as described previously 28. Primary and secondary antibodies included chicken anti-GFP (1:1000, Abcam, ab13970, RRID:AB_300798), and Alexa Fluor® 488 donkey anti-chicken IgG (1:200, Jackson ImmunoResearch, Cat#703-545-155, RRID:AB_2340375). For colorimetric immunohistochemistry, sections were incubated with an avidin–biotin complex (Vector Laboratories, PK-6100, RRID:AB_2336819) and peroxidase activity was visualized using 0.05% 3,3′-diaminobenzidine (Sigma, D5637), and visualized using and visualized using Nanozoomer 2 Hamamatsu ,Nikon Eclipse NI-E with a DS-Ri2 camera (4908 × 3264 px, 7.3 μm), and Leica Thunder microscope with a Leica K3C USB3 color camera (3072 × 2048, 2.4 μm pixels). Immunofluorescence images were acquired with a Zeiss LSM980 Airyscan microscope. RNAscope™ Multiplex Fluorescent V2 Assay (Advanced Cell Diagnostics, Cat#323100) was used to detect Gad1 (Cat#4009 51), and Scn1a (Cat#434181).

Statistical analyses
Data are reported as mean ± standard error (SE). All statistical tests were carried out via GraphPad Prism. The details of the specific test and the p-values are listed in the Figure legend, with the full description listed in Supplementary Data 1. Differences were considered significant at p < 0.05. The illustrations in Fig. 1a; Fig. 3c,i; Fig. 4a,e,f; Fig. 5a; Supplementary Fig. 6a and Supplementary Fig. 11a were created in BioRender. Rubinstein, M. (2026) https://BioRender.com/ol3g4vn

Data availability
Source data are provided with this paper

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "I liked this manuscript, and I think the results are interesting and give important insights into aspects of the pathology of\nDravet syndrome. The basic idea that the authors discover is that in mice that have only one functioning SCN1A allele (a\nDravet model) the phenomenon of nest building, warmth-induced NREM sleep and the concurrent hypothermia as the mice\nenter NREM is missing; this sleep-temperature phenotype can be rescued by expressing the SCN1A channel back into the\npreoptic hypothalamus. In general, the manuscript is well written, and the experimental logic and description are easy to\nfollow, and the Intro and Discussion give balanced viewpoints. Nevertheless, I do have some specific\nsuggestions/comments:\n1. line 89, line 105. Heated pad set to thirty degrees. Does this actually make the air temperature in the beaker thirty\ndegrees, or is the air just warmer but not at 30 degrees C? I guess the key thing is that the skin of the mice is being warmed\nrather than that the air temperature being at 30 degrees? And later experiments, line 358, are done at 36 degrees on the\nwarming pad, considerably hotter. Why are the different temperatures used? Please clarify.\n2. Does SCN1A re-expression in the hypothalamus of SCN1A mice rescue the nesting phenotype? Do the mice now build\nnests? Maybe not, but it would be interesting to know.\n3. Does SCN1A re-expression in the hypothalamus of SCN1A mice rescue the “temperature decrease with sleep”\nphenotype that is shown in Fig. 2F and L? Again, if not, fine, but it would be good to know.\n4. I found the “sleepiness index” quite confusing. A number of parameters are condensed into a “sleepiness scale”, but I was\nnot convinced how useful this was. It was unclear how all the factors blend in, and what gets under or overestimated. Maybe\na rethink is needed here, or simply take that part out.\nI don’t really understand the graphs in Fig. 3F, and Fig. 4M. They left me confused. In Fig. 3F what is the x axis? It is not\nlabelled. The simple graphs and parameters in e.g. Fig. C and J and Fig. 4J are straightforward in contrast.\n5. In Fig. 4, I think expressing SCN1 A in wild-type hypothalamus and comparing it with SCN1A re-expression in Dravet\nmice is the wrong comparison. Overexpressing a sodium channel might give a phenotype in itself. I would suggest, instead,\nthat Fig. 4A to G (the WT results) could be a Supp. Figure. On the other hand, parts H through to N of Fig 4 (expression of\nSCN1A in Dravet hypothalamus) should be moved into Fig 3, so that we have wild-type (top part), Dravet phenotype middle\npart, and rescue of phenotype with SCN1A in hypothalamus in bottom panel. This seems to be the correct comparison. So, I\nsuggest redesigning Fig. 3 and getting rid of the current Fig. 4\n6. CNO expts.\na. 10 mg/kg CNO is at the top-end and I would think too high to be sure the effect is not due to conversion to clozapine,\nwhich induces sedation. What does 10 mg/kg CNO do to a control mouse? Important to check it does not induce, to some\nextent, cause sleepiness.\nb. Fig. 5B. More detail is needed for the anatomical expression of the virus. It seems surprising that expression is just in a\nmicropatch of LPO? I would think that might be possible in a rat, but not a mouse hypothalamus. The photomicrograph just\nlooks odd to me. More extensive anatomical characterization should be given of viral expression. In the schematic in part A,\nplease indicate the timing of when CNO or saline is given prior to the mice being placed in a warm environment.\nc. Given that the hM3Dq receptor is expressed pan-neuronally in the preoptic hypothalamus, it seems surprising that the\nsame result is always produced. I would think that sometimes there would be more expression in wake-promoting preoptic\nneurons, depending on each animal where the virus is transduced exactly, and wakefulness is produced with CNO. There\nmust be quite a lot of variability here? (glutamate neurons in the preoptic can produce wake when excited).\nSigned: William Wisden", "reviewer_2": "In this novel study, Dr. Rubinstein and colleagues shed light on the understudied yet crucial problems of thermal\ndysregulation and sleep disturbances in Dravet syndrome. Notably, they establish a connection between these two deficits\nin a mouse model of the disorder. Their findings reveal that Dravet mice have a lower baseline body temperature.\nFurthermore, through integrated EcoG, LFP, and temperature monitoring, they discovered that, unlike wild-type mice, which\nexperience a temperature drop transitioning from wakefulness to NREM sleep, Dravet mice do not. Intriguingly, increasing\nbody temperature induced sleep in wild-type mice but failed to do so in Dravet mice. Interestingly, when they restored\nNav1.1 expression using a CAV2-Nav1.1 vector that they previously developed or induced neuronal activation using\nchemo-genetics in the anterior hypothalamus, warmth-induced sleep was rescued in the Dravet mice.\nThis work is novel, well-designed, and well-written and contributes to the field by highlighting\nthe contribution of previously unexplored brain areas to the multifaceted phenotypes of Dravet\nsyndrome. Publication of this important paper will be very valuable to the field. That said, there are some issues that need to be integrated or better characterized to maximize the\ninformative power and the impact of the work.\n1. The authors focused on the transition from wakefulness to NREM sleep to investigate the link between temperature\ndysregulation and sleep disturbances. However, to have a complete picture, it would be worth to study additionally the link\nbetween core body temperature alterations and the transition from NREM to REM phase. This is likely something not so\ndifficult to do as they have already performed the recordings.\n2. The results of the two rescue experiments with injection of CAVs expressing either Nav1.1 or hM3D are impressive.\nHowever, they could be more informative if the authors could better characterize the percentage and extent of neuronal\ntransduction in the hypothalamus and interconnected brain areas, considering the retrograde properties of CAVs.\n3. In which neuronal cell types is Nav1.1 expressed in the hypothalamus? RNAscope or analysis of available single cell\ndata set of adult mouse brain could help.", "reviewer_3": "In this report, Fadila et al., seek to establish a connection between sleep homeostasis and thermoregulation in an SCN1A\nhaplo-insufficient mouse, carrying a Dravet Syndrome-associated mutation. Mouse models of DS recapitulate several of\nfeatures of disease including seizures, impaired sleep and poor thermoregulation. Changes in core body temperature\ncoincide with sleep/wake state transitions and increasing ambient temperature can promote NREM sleep. The authors\nhypothesize that sleep and temperature homeostasis disturbances are linked in DS mice and seek to understand the\nmechanisms by which they are coordinated. There is potentially dual value in this approach as it stands to be informative\nregarding the relationship between sleep and thermoregulation in the disease but also could reveal principles\nmechanistically linking SCN1A function to sleep and temperature homeostasis. While this is an interesting manuscript with\nsome very nice preliminary findings, there are many methodological concerns (detailed below) including a lack of\nappropriate controls, inappropriate statistical analyses, and a lack of experimental detail as well as very limited\nmechanistic insight rendering the manuscript of limited impact in its current form.\nOur specific concerns and suggestions are laid out below more or less organized by Figures.\nFigure 1\n// The authors claim that “DS mice increased their core more rapidly than WT mice” (line 90), however no quantification of\nthe rate of change in core body temperature is provided. When mice are returned to RT to recover, the genotypes have\ndifferent core body temperatures. However, it is curious that the authors do not highlight that both genotypes ‘overshoot’ their\ntemperature recovery, ending up at a temperature below the baseline core body temperature shown in 1B and 1C. Do both\nWT and DS mice have impaired recovery in this model? .\nThe authors next evaluate nesting behavior in WT vs DS mice because nests can stabilize core body temperature during\nsleep. DS mice may not build nests as complex as their WT littermates however, the significance of these findings at his\njuncture (or anywhere for that matter in the manuscript) is unclear. While pre-sleep behaviors in mice are now well\nrecognized. In the context of the current manuscript, a defect in these behaviors raises the possibility of important confounds:\nfor example, perhaps DS mice defect in nesting contributes to a lack of movement that would raise body temperature prior to\nsleep. This possibility is not addressed.\n//Moreover, the localization of these pre-sleep behaviors is still not well circumscribed in the brain.\n// It is unclear how these findings fit with previous and subsequent data in the manuscript. How does nesting behavior\nrelated to core body temperature in DS mice, sleep and hypothalamic activity? If nesting behavior is abnormal in DS mice,\ncould this argue that SCN1A mutations disrupt circuits involved in behaviors which regulate temperature? This line of\nquestioning is distinct from hypothesis that SCN1A acts in key brain regions directly involved in regulating core body\ntemperature (i.e. hypothalamus).\nFigure 2\nThe authors next evaluate sleep and the change of core body temperature during wake -> NREM transitions in WT and DS\nmice. They find that while WT mice have a higher delta power in NREM compared to wake, DS mice do not exhibit any\nchange in delta power. In addition, while WT mice experience a decrease in core body temperature at wake -> NREM\ntransitions, there is no change in DS mouse core body temperature at transition states.\n//There are major technical concerns with the sleep recordings. It is unclear from the methods section at what time of day the\nrecordings were done and how the mice were housed during the recording session.\n// Were animals protected from environmental disturbances? Were they acclimated to the recording chambers or temperature\nprobes before recording sleep?\n// Additionally, the recovery period post-surgery/implantation seems relatively short (2-3 days).\n// The statistical analysis of the PSD is not appropriate. It is advised to conduct a 2-way ANOVA, comparing WT-WAKE, WT-\nNREM, DS-WAKE and DS-NREM. From Figure S1, it appears that the authors instead conducted t-tests to compare within\ngenotypes only. The authors state “ In DS mice, the increase in delta power was smaller than in WT animals” (line 160). With\nthe current statistical analysis, they cannot make such a claim (unless we missed this somewhere).\n// As a general comment for all sleep recordings done in the report, there are often poor representative ECoG & EMG\nrecording traces. Figure 2A, for example, displays ECoG and EMG traces in ‘wake’ and ‘NREM’ which are entirely\nindistinguishable from each other.\nFigure 3\n// the authors argue that WT mice exhibit an increase in delta power after heating, but it should be addressed whether this\neffect is driven by one mouse which seems to have a significant change (3D).\n// The authors also employ a ‘sleepiness scale’ to evaluate the relative sleepiness of WT and DS mice after heating.\nHowever, it is unclear as to why the development or use of this ‘sleepiness scale’ is required. We questions whether the use\nof EMG signal as a marker for sleepiness, in itself, is arbitrary and inappropriate. For example, a mouse with a severed\nspinal cord or with absence of spinal motor neurons may have different/absent EMG signals but no change in “sleepiness”\nbut this scale would render a “sleepier” mouse simply as a function of immobility. Without validation or generalizability of\nsuch a scale, the data from it is hard to understand. Furthermore, this scale not used in Figure 2 recordings -- why not? For\nthis and subsequent figures (3, 4 and 5) the authors also no longer separate a PSD analysis from wake vs NREM and it is\nunclear why (in line 227). It seems that the main effect of PSD delta power was in NREM stages (figure 2) and they may be\nmissing an opportunity to have stronger effects.\nFigure 4\nThe authors test whether expression of SCN1A in the anterior hypothalamus can restore warmth-mediated sleep in DS\nmice. They argue that CAV-mediated SCN1A expression in DS mice restores the increased NREM time and delta power\ninduced by heating.\n// However, this experiment is lacking appropriate control (empty virus not “untreated”) and statistical analyses. While it\nappears the authors had an ‘untreated’ group (showed in supplements) they do not include these groups in their statistical\nanalyses.\n// And again, the authors fail to statistically compare WT and DS mice against each other. They could do this by statistically\ncomparing the change in delta ratio from RT -> 36°C in each injection x genotype group.\nFigure 5\nThe authors test whether chemogenetic activation of the anterior hypothalamus using activating DREADDS can rescue\nwarmth-mediated sleep in DS mice.\n//The rationale for the experiment is only partially clear. What is known about the activity of the neurons in anterior\nhypothalamus in DS mice? Is the activity in the haplo-insufficient model lower or higher than WT mice? In which cells?\nTesting this would support the DREADD experiments.\n// There is again a lack of proper control (empty vector) and appropriate approach to statistical analysis. Including a CNO\nonly control would be ideal as the effects observed could be CNO or hypothalamus-mediated.\n// there is no confirmation that CNO administration actually had an effect on anterior hypothalamus activity.\nOther major concerns\n- Lack of description about the haplo-insufficient DS mouse model\n- Varied ages for the mouse studies\n- baseline temperature difference is gone in older animals\n- many seizures in the younger animals, do any of them die?\n- Lack of investigation and discussion about potential metabolic contributions to differences in basal and sleep-state\ntransition associated thermoregulation\n- No investigation into the activity of the hypothalamus in DS mice, this could link hypothalamic involvement to the\ntemperature-sleep transitions\n- There is also occasional inconsistency in the PSD findings – sometimes the main significant effect of the experimental\nvariable is seen in delta power and sometimes it is in delta ratio, but there is a lack of discussion about this.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to Reviewers’ comments on Fadila et al. NCOMMS-25-18755.\nWe thank the Reviewers for their insightful and helpful comments and provide point-by-point\nresponses below in blue font, with references to changes in the main text indicated in grey\nfont. In the revised manuscript, textual changes based on the comments are indicated with\ntracked changes. The reference to line numbering below refers to the revised manuscript\nwithout tracked changes active.\nReviewer #1 (Remarks to the Author):\nI liked this manuscript, and I think the results are interesting and give important insights into\naspects of the pathology of Dravet syndrome. The basic idea that the authors discover is that\nin mice that have only one functioning SCN1A allele (a Dravet model) the phenomenon of nest\nbuilding, warmth-induced NREM sleep and the concurrent hypothermia as the mice enter\nNREM is missing; this sleep-temperature phenotype can be rescued by expressing the\nSCN1A channel back into the preoptic hypothalamus. In general, the manuscript is well\nwritten, and the experimental logic and description are easy to follow, and the Intro and\nDiscussion give balanced viewpoints. Nevertheless, I do have some specific\nsuggestions/comments:\nAnswer: Thank you for your helpful comments and suggestions, which we carefully addressed\nin the revised manuscript.\n1. line 89, line 105. Heated pad set to thirty degrees. Does this actually make the air\ntemperature in the beaker thirty degrees, or is the air just warmer but not at 30 degrees C? I\nguess the key thing is that the skin of the mice is being warmed rather than that the air is just\nwarmer but not at 30 degrees C? And later experiments, line 358, are done at 36 degrees on\nthe warming pad, considerably hotter.\nWhy are the different temperatures used? Please clarify.\nAnswer: When the heated pad was set to 30˚C, the temperature at the bottom of the beaker\nwhere the mouse was placed reached approximately 26˚C. When the pad was set to 36˚C,\nthe temperature at the bottom of the beaker reached ~29˚C.\nThe rationale for using two different temperature settings relates to the age-dependent\nsensitivity of DS mice to heat-induced (‘febrile’) seizures, which varies between the third and\nfifth weeks of life. In the experiment shown in Fig. 1, we examined DS mice at their 4th week\nof life (P21-P25), when their susceptibility to febrile seizures is highest. For this reason, a\nmodest increase in temperature (i.e., with the heating pad set at 30˚C, yielding an ambient\ntemperature of 26˚C at the bottom of the beaker) was chosen. Even under these moderate\nwarming conditions, approximately 25% of the mice experienced heat-induced seizures. By\ncontrast, for the experiments shown in Figs. 3-5, the mice were older, five to six weeks old, at\nwhich point their sensitivity to febrile seizures is greatly reduced. Therefore, we chose to use\na higher heating pad temperature (i.e., 36˚C, yielding ~29˚C at the bottom of the beaker) to\nprovide a more robust warmth stimulus that more closely mimics conditions used in prior\nstudies investigating the sleep-promoting effects of elevated ambient temperature.\nThis information has now been included in the respective Methods section (page 18, lines 5-\n12)\nthe first one at room temperature for one hour, after which the recording chamber was moved\nand placed over a heated pad set to 36°C (TCAT-2DF, Physitemp Instruments, Clifton, NJ\nUSA), and a second block was recorded for another hour. Under these conditions, the air at\nthe bottom of the beaker was at 29°C. Note that for these experiments, since the DS mice\nwere older than five weeks, a higher ambient temperature of 36°C was used (and not the 30°C\nused for the younger DS mice in Fig. 1), given their reduced sensitivity to thermally induced\nseizures at this age.\nand Results sections (page 7, lines 16-32).\nA warm ambient temperature (32-36°C) promotes sleep through a mechanism that involves\nthe activation of heat-sensing neurons in the hypothalamus (Harding et al., 2019; Kroeger et\nal., 2018). Given the deficits in temperature regulation upon the rise to a moderate ambient\ntemperature of 30°C, and the sleep transition-related changes in delta power in DS mice, we\nset out to investigate whether warmth-induced somnogenesis is impaired in DS mice. Depth\nLFP and EMG activity were recorded in 5-6-week-old WT and DS mice. Recordings were first\nobtained for one hour at room temperature, followed by an additional hour under elevated\nambient temperature achieved by placing the cage on a heating pad set to 36°C, resulting in\na temperature of 29°C at the bottom of the recording beaker (Fig. 3A-C). Because DS mice at\nthis age show reduced susceptibility to febrile seizures (Almog et al., 2021; Mavashov et al.,\n2023), we reasoned that a somewhat stronger warming stimulus could be applied compared\nto the 30°C condition used in younger animals (Fig. 1). However, to minimize seizure risk, we\navoided the higher temperatures (32-36°C) used in previous studies (Harding et al., 2019;\nKroeger et al., 2018) making 29°C a deliberate compromise between physiological relevance\nand safety in this model.\n2. Does SCN1A re-expression in the hypothalamus of SCN1A mice rescue the nesting\nphenotype? Do the mice now build nests? Maybe not, but it would be interesting to know.\nAnswer: We did not assess nesting behavior in the original dataset of hypothalamic CAV2-\nSCN1A-injected mice. To address this question, we performed a new set of experiments, in\nwhich the effects of hypothalamic injection of CAV2-SCN1A were compared with those of the\ncontrol vector CAV2-GFP in both WT and DS mice. Seven to ten days after vector injection,\nwe tested the nest-building abilities of the mice. As shown in the newly added Supplementary\nFig. 10, and mentioned in the Results section on page 14, lines 1-12, CAV2-SCN1A injection\ninto the hypothalamus failed to correct this DS-associated phenotype. (pages 8-9, lines 28-7)\nTo directly control for the effects of the hypothalamic CAV2 injections, a separate cohort of\nWT and DS mice was injected with a control vector containing a reporter gene CAV2-GFP, or\nwith CAV2-SCN1A.\n…\nIn the cohort of CAV2-GFP or CAV2-SCN1A injected mice, we also evaluated nest-building\nbehavior. Seven to ten days after vector injection, CAV2-SCN1A did not rescue the impaired\nnest-building abilities of DS mice (Supplementary Fig. 10).\nCAV2-SCN1A injection into the hypothalamus does not correct nest-building abilities in DS mice.\nExamples of the nesting material after a night in the home cage (left) and nest-building scores (right) showing\nreduced nest-building behavior in DS mice injected with CAV2-GFP or CAV2-SCN1A (WT: CAV2-GFP, n = 7. WT:\nCAV2-SCN1A, n = 8. DS: CAV2-GFP, n = 6. DS: CAV2-SCN1A, n = 6. p < 0.0001, 2-way ANOVA. The results of\nHolm-Sidak post hoc analyses are depicted on the graph).\n3. Does SCN1A re-expression in the hypothalamus of SCN1A mice rescue the “temperature\ndecrease with sleep” phenotype that is shown in Fig. 2F and L? Again, if not, fine, but it would\nbe good to know.\nAnswer: Unfortunately, in the original experiments of hypothalamic CAV2-SCN1A-injected\nmice, shown in Fig. 3, we did not include body temperature monitoring. We now included\ntemperature recordings in the additional experiments, referred to also in our answer to the\nprevious question, that were performed in mice injected in the hypothalamus with CAV2-\nSCN1A or CAV2-GFP.\nFor these experiments, WT and DS mice were injected in the hypothalamus with the control\nvector, CAV2-GFP, at the age of P22-P26, similar to the experiments shown in the original\nFig. 3. After assessing their nest-building abilities (as described above), the mice underwent\nanother surgery to implant the ECoG electrodes. To address whether the “temperature\ndecrease with sleep” is restored by hypothalamic SCN1A expression, we assessed the effect\nof hypothalamic CAV2-SCN1A on sleep properties and temperature at RT using 4-h\nrecordings at RT in 5-6 week-old mice. These recordings, similar to the original experiments\nin naïve mice shown in Fig. 2, were performed 2-3 days after ECoG electrode implantation.\nFirst, we assessed the contribution of delta frequencies to the overall ECoG spectral power\nduring wakefulness (2 minutes prior to the transition to sleep) and during the first minute of\nNREM sleep, and examined changes in core body temperature across these transitions. In\nthis set of experiments, however, the characteristic temperature decrease at sleep onset was\nnot evident in any of the experimental groups, including WT mice injected with either CAV2-\nGFP or CAV2-SCN1A. Since similar effects were observed across all experimental groups,\nregardless of genotype or vector type (CAV2-GFP or CAV2-SCN1A), the discrepancy\nbetween these findings and those of the untreated WT and DS mice presented in Fig. 2\nappears attributable to differences in experimental procedures. The mice in Fig. 2 underwent\na single surgical intervention (either ECoG implantation at 4 weeks of age, or LFP implantation\nat 5 weeks), whereas the mice of the additional set of experiments injected with CAV2-GFP\nor CAV2-SCN1A, underwent two surgeries, i.e., one for vector injection at 4 weeks of age and\n3\na second one for ECoG implantation at 5 weeks of age. Furthermore, in the experiments\nshown in Fig. 2, invasive LFP electrode implantation was performed in 5-week-old mice,\nwhereas viral injections targeting the same region were carried out in younger mice (4 weeks\nold). Thus, although both procedures involved mechanical disturbance of the hypothalamus,\nit is possible that younger mice are more vulnerable to such perturbations, leading to\ndisruptions in temperature drop at NREM onset, independent of SCN1A expression.\nA description of these findings has been added to the Results section (pages 8-9 , lines 28-\n24), and is shown in new figures Fig. 4a-e, Supplementary Fig. 11.\nTo directly control for the effects of the hypothalamic CAV2 injections, a separate cohort of\nWT and DS mice was injected with a control vector containing a reporter gene CAV2-GFP, or\nwith CAV2-SCN1A\n.…\nIn the cohort of CAV2-GFP or CAV2-SCN1A injected mice, we also evaluated nest-building\nbehavior. Seven to ten days after vector injection, CAV2-SCN1A did not rescue the impaired\nnest-building abilities of DS mice (Supplementary Fig. 10). Next, ECoG electrodes were\nimplanted, and the mice were allowed to recover for 2-3 days before we proceeded with ~4 h\nECoG and temperature recordings at RT. Also in this cohort of mice, CAV2-SCN1A\nexpression in the hypothalamus restored the increase in delta ratio during the transition to\nNREM sleep, which was absent in DS mice injected with the control vector (CAV2-GFP, Fig.\n4a-e). Of note, these analyses were conducted using cortical ECoG rather than the\nhypothalamic LFP recordings used for the CAV2-SCN1A experiments shown in Fig. 3,\nconfirming that the correction of sleep-related oscillatory activity by CAV2-SCN1A is also\nreflected at the cortical level. Core temperature measurements were included in this new\ncohort of mice to test whether hypothalamic SCN1A expression may also correct the drop in\nbody temperature at NREM onset. However, in contrast to the data from naïve untreated mice\nshown in Fig. 2, no significant temperature decrease was detected after NREM onset in either\nWT or DS mice injected hypothalamically with either CAV2-GFP or CAV2-SCN1A. The\ndiscrepancy between these results and those presented in Fig. 2 may be attributable to the\ndifferent experimental procedures, as the mice depicted in Fig. 2 underwent only one surgical\nprocess, for electrode implantation (ECoG at the age of 4 weeks, or LFP at the age of 5\nweeks), while the mice shown in Fig. 4 underwent two surgeries, one for vector injection at 4\nweeks and a second one for ECoG implantation at 5 weeks.\n4. I found the “sleepiness index” quite confusing. A number of parameters are condensed into\na “sleepiness scale”, but I was not convinced how useful this was. It was unclear how all the\nfactors blend in, and what gets under- or overestimated. Maybe a rethink is needed here, or\nsimply take that part out.\nAnswer: Thank you for this constructive feedback. Following your advice, we removed this\nindex and instead quantified the percentage of NREM sleep at RT and a warm environment\n(i.e., when the mice are placed on a heating pad set to 36°C). These data are now shown in\nFigs. 3-5. In addition, following the advice of Reviewer #3 (and to align with our measurements\nshown in Fig. 2), we have added in Figs. 3-5: a quantification of the delta ratio during\nwakefulness and NREM. To align with the results of Fig. 2, the analyses of wakefulness were\nconducted for the period 2 minutes prior to the transition to NREM, and the NREM analyses\nused the 1-minute period into NREM.\nwhat is the x axis? It is not labelled. The simple graphs and parameters in e.g. Fig. C and J\nand Fig. 4J are straightforward in contrast.\nAnswer: These graphs depicted the sleepiness index in individual mice (so the x-axis\nrepresented different mice). As noted above, these ‘sleepiness index’ measurements have\nnow been replaced with measurements of the percentage of NREM and the delta ratio at wake\nand NREM.\n5. In Fig. 4, I think expressing SCN1A in wild-type hypothalamus and comparing it with SCN1A\nre-expression in Dravet mice is the wrong comparison. Overexpressing a sodium channel\nmight give a phenotype in itself. I would suggest, instead, that Fig. 4A to G (the WT results)\ncould be a Supp. Figure. On the other hand, parts H through to N of Fig 4 (expression of\nSCN1A in Dravet hypothalamus) should be moved into Fig 3, so that we have wild-type (top\npart), Dravet phenotype middle part, and rescue of phenotype with SCN1A in hypothalamus\nin bottom panel. This seems to be the correct comparison. So, I suggest redesigning Fig. 3\nand getting rid of the current Fig. 4\nAnswer: Thank you for this suggestion. As advised, we are now showing untreated and CAV2-\nSCN1A-injected DS mice in the same Figure (i.e., Fig. 3). Moreover, we added a direct\nstatistical comparison between untreated WT, untreated DS, and CAV2-SCN1A-injected DS\nmice, which is shown in Supplementary Fig. 6b. As shown in this figure, there was no statistical\ndifference between naïve and CAV2-SCN1A-injected WT mice.\nSupplementary Fig. 6\nA full statistical comparison between different experimental groups shown in Figs. 3, 4.\n(a). The experimental paradigm, used for examining the effect of warmth on sleep promotion.\n(b-c). The same data shown in Figs. 3d and 3j are replotted here to highlight genotype differences. (2-way ANOVA.\nThe results of Holm-Sidak post hoc analyses are depicted on the graph).\n5\n(d). The data shown in Figs. 4f are shown are depicted again here with the inclusion of the full statistical analysis\nto highlight also genotype differences. (2-way ANOVA. The results of Holm-Sidak post hoc analyses are depicted\non the graph).\n* p < 0.05; ** p < 0.01; *** p < 0.001\n6. CNO expts.\na. 10 mg/kg CNO is at the top-end and I would think too high to be sure the effect is not due\nto conversion to clozapine, which induces sedation. What does 10 mg/kg CNO do to a control\nmouse? Important to check it does not induce, to some extent, cause sleepiness.\nAnswer: We selected this dosage based on a previous paper that tested the effect of\nchemogenetic on seizure threshold (Mattis et al., 2022; PMID: 35212623). To directly test the\npossibility that this dosage may cause sleepiness, we examined the effect of 10 mg/kg CNO\non locomotor activity in four untreated awake WT mice assessed for a period of 10 minutes,\nstarting 15 minutes after CNO injection. This time frame aligns with the exclusion from analysis\nobtained from the first 15 minutes after CNO injection (Jendryka et al., 2019, PMID:30872749;\nVardy et al., 2015,PMID:25937170) that we adhered to for the warmth-induced somnogenesis\nexperiments, information which we have now included in the Methods section (page 18, lines\n17-20).\nLocomotor activity of WT mice, without DREADD expression, was not affected by 10 mg/kg\nCNO. Specifically, 15 min after CNO injection, locomotor activity of the mice was monitored\nfor 10 minutes in an open field of 50 by 50 cm (Supplementary Fig. 12).\nAnd the Results section (page 18, lines 1-3):\nThis dosage of CNO did not alter locomotor activity, as assessed in an open field test in WT\nmice without DREADD expression (Supplementary Fig. 12), indicating the absence of a\nnonspecific sedation effect at this dose 37.\n10000\n8000\n6000\n4000\n2000\n0\ng/ k\ng)\nali n\ne\nm S\n0\n(\nN\nC\nSupplementary Fig. 12\nCNO administered at 10 mg/kg had no effect on the locomotor activity of WT mice. WT mice were placed in a 50\nx 50 cm open field arena 15 min after CNO injection, and their locomotor activity within the arena was recorded for\n10 min. Four days later, the same mice were injected with saline, and their locomotor activity was recorded again\n15 min after saline injection over 10 min. The similar distance covered under both conditions indicates that CNO\nat this dose does not have a non-specific sedative effect.\n6\n)mc(\nderevoc\necnatsiD\nAnd the Discussion section (page 14, lines 21-25) :\nA limitation of the present study is that we did not directly assess how the effects of the DS\nScn1a mutation and hypothalamic CAV2-mediated SCN1A expression, or alternatively\nchemogenetic stimulation, influence neuronal firing and excitability in the anterior\nhypothalamic region. Future studies addressing these questions could provide deeper\nmechanistic insight into the neuronal basis of sleep and thermoregulation deficits in DS.\nMoreover, while DS mice generally showed an enhanced delta contribution in NREM after\nCNO administration, we detected variability in the magnitude and the direction of the response\nto CNO, with one mouse displaying a reduction in NREM delta power at 36°C (Fig. 5J green),\nsuggesting that the effect differ, as proposed by the Reviewer (see below) based on the\nspecific neuronal population which was transduced, rather than a non specific effect.\nb. Fig. 5B. More detail is needed for the anatomical expression of the virus. It seems surprising\nthat expression is just in a micropatch of LPO? I would think that might be possible in a rat,\nbut not a mouse hypothalamus. The photomicrograph just looks odd to me. More extensive\nanatomical characterization should be given of°expression.\nAnswer: Unfortunately, we did not obtain better expression images with CAV2-hM3D-IRES\nmiCitrine in the brain. The amount of mCitrine, which is the ORF after the IRES, is very low\n(Mizuguchi et al. 2000; PMID:10933956). And, like numerous labs, we also attempted to use\ncommercial antibodies against hM3D, but in our hands, the signal was weak and not specific.\nWe are also unaware of any study showing convincing hM3D immunoreactivity in vivo.\nUsing CAV2-hM3D-IRES- mCitrine in E1-transcomplementing cells (DK-E1), we observe\nmCitrine expression (added as Supplementary Fig. 9i-n). Detection of mCitrine in DK-E1 cells\nseeded as a monolayer, with transduction of the majority of cells on the coverslip, is easier\nthan detecting transduced neurons in a brain following in vivo expression. These data indicate\nthat the vector quality (physical to infectious particle ratio) is excellent and that transgenes\nfrom CAV2-hM3D-IRES-mCitrine are expressed. Briefly, DK-E1 cells were incubated with 50\nparticles of CAV2-hM3D IRES-miCitrine/cell. 24 h post-incubation, mCitrine and hM3D\nimmunofluorescence were examined.\nSupplementary Fig. 9\n(i-n). CAV2-hM3D IRES miCitrine expression in the DK-E1 cells. DK-E1 cells were seeded as a monolayer and\nincubated with CAV2-hM3D-IRES-mCitrine (50 particles/cell) for 24 h, followed by immunofluorescence. mCitrine\nimmunofluorescence (green), and hM3D (red).\n7\n(i) .mCitrine; (j) hM3D; (k) mCitrine and hM4D; (l) DAPI and mCitrine; (m) DAPI and hM3D, (n) DAPI, mCitrine and\nhM3D). Anti-GFP (1:1000, Abcam, ab13970, RRID:AB_300798) was used to detect mCitrine. HPA024106 (1:100,\nSigma) was used to detect hM3D.\nScale bar: 10 µM\nIn addition, we injected three WT mice, in the\nhypothalamus, with a reporter vector, CAV2-\nCMV-mCitrine, which harbors the CMV\npromoter driving mCitrine. CAV2-CMV-\nmCitrine injections led to robust mCitrine\nimmunoreactivity in the hypothalamus and\nconnecting regions.\nThese data were not added to the manuscript\nto avoid confusion, as the expression levels\nof mCitrine using CAV2-CMV-mCitrine\nmCitrine expression in the hypothalamus of a WT\nreporter vector are much higher than those of\nmouse following injection of CAV2-CMV-mCitrine\nCAV2-hM3D-IRES miCitrine (which enabled the\nto the anterior hypothalamus\neasy detection of the vector in the brain). Yet it\nindicates the expected biodistribution. See the full distribution of the biodistribution for this\nvector in our response to Reviewer #2, point 2; the panel depicted here corresponds to D).\nAdditionally, in response to the other Reviewer’s comments, we have added a more detailed\nhistological analysis using CAV2-NSE-mCitrine (mCitrine is driven by the NSE promoter,\nwhich also drives the expression of SCN1A in CAV2-SCN1A). Both promoters drive transgene\nexpression in excitatory and inhibitory neurons. These data using CAV2-NSE-mCitrine are\npresented in Fig. 4B,C, Fig. S8, and Fig. S9, and corresponding statements have been added\nto the Results (pages 8-9, lines 30-3)\nThe biodistribution was examined in mice following CAV2-GFP injection (WT and DS, n = 3\nfor each genotype), which were also used for behavioral and electrophysiological\nexaminations, as well as in a separate group of three WT mice injected with the control vector\nCAV2-mCitrine, which were only used for immunohistochemistry. The biodistribution analysis\ndemonstrated expression in excitatory and inhibitory neurons in the hypothalamus and, due\nto CAV2’s retrograde transport properties, also in interconnected brain regions (Fig. 4a, b,\nSupplementary Figs. 8 and 9).\nWith the technical difficulties to demonstrate CAV2-hM3D-IRES miCitrine, we would like to\nshare additional evidence to support the functional expression of this vector in the brain\nThis evidence comes from a separate project in which the same DREADD vector, CAV2-\nhM3D-IRES-mCitrine, was injected into the thalamus and hippocampus of DS mice. The goal\nin that project is to compare the effects of acute DREADD activation to those of CAV2-SCN1A\nin these regions. CAV2-SCN1A injections into the thalamus and hippocampus were previously\nshown to correct both epileptic and cognitive deficits in DS mice (Fadila et al., 2023; PMID:\n37192002). The experimental procedures in this other project differ with respect to the\ntargeted brain regions thalamus & hippocampus, vs. hypothalamus, recording configuration\n(i.e., ECoG in this other project vs. hypothalamic LFP for the DREADD experiments in the\n8\ncurrent manuscript) and the DREADD agonist that was used (DCZ in this other project vs.\nCNO in the current manuscript).\nIn addition, the overall outcome in this other project concerned an aggravation of DS\nphenotypes following DREADD activation, instead of an improvement as observed for the\ncorrection of warmth-induced somnogenesis in the present manuscript. Despite the\ndifferences, however, these ongoing studies demonstrate that the same DREADD vector can\nelicit DS-specific effects based on behavioral and ECoG spectral power features, supporting\nthe functional expression of this vector in the brain, as shown in the Figure below. Specifically,\nin DS mice, chemogenic stimulation resulted in increased anxiety, shown as reduced time at\nthe center of the open field arena, as well as a reduction in the contribution of delta frequencies\nto the overall ECoG spectrum, along with an increase in the contribution of beta frequencies.\nThe observation that no change in open-field activity or ECoG activity was observed in WT\nmice following chemogenetic stimulation, while these changes were observed in DS mice,\nindicates a specific effect of DREADD activation in hippocampal and thalamic regions,\nsupporting the functional effect of CAV2-hM3D-IRES-mCitrine in vivo.\nData from a parallel project involving DREADD injection into the thalamus and hippocampus. A, B)\nFollowing saline or DCZ (0.1 mg/kg) , the locomotor activity (A, WT: n = 9; DS: n = 7 p = 0.4, 2-way ANOVA), and\nthe location of the mice within the center of the arena (B) were examined (p = 0.002, 2-way ANOVA. The results\nof Holm-Sidak post hoc analyses are depicted on the graph)\nC,D) The power of ECoG in WT (C, WT: n = 5; p = 0.7, 2-way ANOVA) and DS mice (D, DS: n = 3, p = 0.03, 2-\nway ANOVA The results of Holm-Sidak post hoc analyses are depicted on the graph), at the indicated frequency\nbands, following DREADD activation.\nIn the schematic in part A, please indicate the timing of when CNO or saline is given prior to\nthe mice being placed in a warm environment.\nAnswer: We have added to the legend of Fig. 5 that CNO or saline was administered\nimmediately (t=0 min) prior to placing the mouse in the warm environment, as was stated\nalready in the Methods section, and we added this information also to the legend of Fig.5.\n(a) A schematic representation of the experimental design. Saline injection was given right before the mouse was\nplaced on the heated pad. Two to three days later, the experiment was repeated, and CNO injection was\nadministered just before the mouse was placed on the heated pad. The first 15 minutes following CNO injection\nwere excluded from the analyses.\nMethods section (page 18, lines 12-17)\nFor the experiments described in Fig. 5, immediately prior to the placement of the heated pad,\nthe mice were given an IP saline injection, and when the experiment was repeated 3-4 days\nlater, an IP injection with 10 mg/kg clozapine-N-oxide (CNO, Angene, London, UK). We\nanalyzed the entire recording after saline injection, but excluded the first 15 minutes following\nCNO injection to allow for CNO absorption 77,78.\nc. Given that the hM3Dq receptor is expressed pan-neuronally in the preoptic hypothalamus,\nit seems surprising that the same result is always produced. I would think that sometimes\nthere would be more expression in wake-promoting preoptic neurons, depending on each\nanimal where the virus is transduced exactly, and wakefulness is produced with CNO. There\nmust be quite a lot of variability here? (glutamate neurons in the preoptic can produce wake\nwhen excited).\nAnswer: Indeed, the overall effect we observed with CNO was sleep-promoting across all\ntested mice (WT and DS). However, as also indicated in our answer to question 6a of this\nReviewer, we did detect variability in the magnitude and the direction of the response to CNO\nwith respect to the delta power contribution to wake and NREM at 36°C. While DS mice\ngenerally showed an enhanced delta contribution, this effect was not consistently seen in all\nof the animals, with one mouse even displaying a reduction in NREM delta power at 36°C\n(Fig. 5J green). We agree that such variability likely reflects differences in the specific cell\npopulations of transduced in each mouse, and we have now included a statement discussing\nthis potential source of variability in the revised Discussion (pages 12-13, lines 23-11).\nIn light of the intricate neuronal population of the hypothalamus, which includes both sleep-\nand wake-promoting neurons 57, pinpointing the cellular basis of these DS-associated sleep\nand thermoregulatory deficits remains challenging. This complexity is further illustrated by the\npresence of hypothalamic neuron types with unique properties, such as wake-promoting\n10\nglutamatergic parvalbumin-positive neurons 60. Moreover, since Scn1a is expressed in both\nexcitatory and inhibitory hypothalamic neurons 36, identifying the precise cellular mechanisms\ninvolved becomes even more difficult. However, the observation that hypothalamic neuronal\nCAV2-mediated SCN1A expression or chemogenetic stimulation restored the warmth-induced\nsleep-promoting response suggests that reduced neuronal excitability, resulting from Scn1a\nloss-of-function mutation, is a plausible mechanism for the observed DS-related deficits.\nNevertheless, CAV2-mediated expression of SCN1A or hM3D is not restricted to a specific\nclass of neurons. CAV2-SCN1A contains the neuronal-specific enolase (NSE) promoter, and\nCAV2-hM3D-IRES-mCitrine harbors the CMV (cytomegalovirus early enhancer) promoter.\nBoth promoters lead to transgene expression in excitatory as well as inhibitory neurons 28.\nMoreover, with the effective retrograde transport properties characteristic of CAV2 61,62,\ninjection of the CAV2-based vectors to the anterior hypothalamus also transduced neurons in\nregions that project to the hypothalamus. Thus, it is possible that the anterior hypothalamic\ninjection of CAV2-SCN1A leads to a correction in the activity of multiple types of neurons in\nthis region, as well as additional connecting networks that hub on the anterior hypothalamus,\n11\nthat together contribute to the observed improvement in sleep and thermoregulation in DS\nmice.\nAnd pages 14, lines 21-25\nA limitation of the present study is that we did not directly assess how the effects of the DS\nScn1a mutation and hypothalamic CAV2-mediated SCN1A expression, or alternatively\nchemogenetic stimulation, influence neuronal firing and excitability in the anterior\nhypothalamic region. Future studies addressing these questions could provide deeper\nmechanistic insight into the neuronal basis of sleep and thermoregulation deficits in DS.\nSigned: William Wisden\nIn this novel study, Dr. Rubinstein and colleagues shed light on the understudied yet crucial\nproblems of thermal dysregulation and sleep disturbances in Dravet syndrome. Notably, they\nestablish a connection between these two deficits in a mouse model of the disorder. Their\nfindings reveal that Dravet mice have a lower baseline body temperature. Furthermore,\nthrough integrated EcoG, LFP, and temperature monitoring, they discovered that, unlike wild-\ntype mice, which experience a temperature drop transitioning from wakefulness to NREM\nsleep, Dravet mice do not. Intriguingly, increasing body temperature induced sleep in wild-\ntype mice but failed to do so in Dravet mice. Interestingly, when they restored Nav1.1\nexpression using a CAV2-Nav1.1 vector that they previously developed or induced neuronal\nactivation using chemo-genetics in the anterior hypothalamus, warmth-induced sleep was\nrescued in the Dravet\nmice.\nThis work is novel, well-designed, and well-written and contributes to the field by highlighting\nthe contribution of previously unexplored brain areas to the multifaceted phenotypes of Dravet\nsyndrome. Publication of this important paper will be very valuable to the field.\nAnswer: Thank you for your helpful comments and suggestions, which we have addressed in\nthe revised manuscript.\nThat said, there are some issues that need to be integrated or better characterized to\nmaximize the informative power and the impact of the work.\n1. The authors focused on the transition from wakefulness to NREM sleep to investigate the\nlink between temperature dysregulation and sleep disturbances. However, to have a complete\npicture, it would be worth to study additionally the link between core body temperature\nalterations and the transition from NREM to REM phase. This is likely something not so difficult\nto do as they have already performed the recordings.\nAnswer: Unfortunately, in the relatively short recordings of one hour that we performed for\nthese experiments, only very few epochs consistent with REM sleep were observed, and these\nwere not consistently identified by both independent scorers. As unequivocal REM periods\ncould therefore hardly be confirmed, this sleep stage was not suitable for reliable analysis. We\nare planning a follow-up study with extended 24 - 48-hour recordings, which will allow robust\ndetection of REM sleep and enable us to address this important aspect in detail.\nA statement on the lack of REM sleep data being available for analysis has now been included\nin the Methods section (page 17, lines 28-30).\nThe recordings were long enough, and the conditions were suitable for the vast majority of the\nmice (except for one DS mouse, which was excluded from the analysis) to reach NREM sleep,\nbut not REM sleep.\n2. The results of the two rescue experiments with injection of CAV2s expressing either Nav1.1\nor hM3D are impressive. However, they could be more informative if the authors could better\ncharacterize the percentage and extent of neuronal transduction in the hypothalamus and\ninterconnected brain areas, considering the retrograde properties of CAV2s.\nAnswer: We appreciate the suggestion and underscore the value of providing more\ninformation on the extent of neuronal transduction in the hypothalamus and interconnected\nbrain areas. With endogenous Nav1.1 expression in WT and DS mice, the biodistribution could\nonly be examined using a reporter protein.\nmCitrine, where the transgene is driven by the NSE promoter (Fadila et al. 2023: PMID\n37192002). As shown in the newly added Supplementary Fig. 9a-g, mCitrine immunoreactivity\nwas observed across multiple brain regions, consistent with the retrograde transport\nproperties of CAV2 vectors. At the injection site (a), mCitrine-positive cells and fibers were\nobserved in the preoptic region of the hypothalamus, as well as the anterior and lateral\nhypothalamus. Thalamic regions also contained numerous labeled cells. Some cortical\nlabeling was observed in the agranular and piriform cortices, although the number of positive\ncells was relatively low. The amygdala displayed mCitrine-positive cells and fibers, mainly\nwithin the medial and basolateral nuclei.\nSupplementary Fig. 9\n(a-g). Biodistribution of CAV2-mCitrine (in which the mCitrine reporter is driven by the. NSE promoter) following\nstereotaxic injection into the hypothalamic preoptic region. Due to CAV2’s retrograde transport properties, injection\ninto the hypothalamus resulted in expression in multiple regions of the hypothalamus, thalamus, amygdala, and\nsome cortical regions. (a-g) mCitrine immunoreactivity was observed at the injection site (a), and mCitrine-positive\ncells and fibers were observed across the preoptic region, including the lateral and medial preoptic areas, as well\nas the anterior and lateral hypothalamus (a-h). Thalamic regions also contained numerous labeled cells (c-g).\nSome cortical labeling was observed in the agranular and piriform cortices (a-g), although the number of positive\ncells in those regions was relatively low. The amygdala displayed mCitrine-positive cells and fibers, mainly within\nthe medial and basolateral nuclei.Scale bars: 1 mm.\nThese data are referred to in the Results section (pages 8-9, lines 30-4):\nThe biodistribution was examined in mice following CAV2-GFP injection (WT and DS, n = 3\nfor each genotype), which were also used for behavioral and electrophysiological\nexaminations, as well as in a separate group of three WT mice injected with the control vector\nCAV2-mCitrine, which were only used for immunohistochemistry. The biodistribution analysis\ndemonstrated expression in excitatory and inhibitory neurons in the hypothalamus and, due\nto CAV2’s retrograde transport properties, also in interconnected brain regions (Fig. 4a, b,\nSupplementary Figs. 8 and 9).\nUnfortunately, we did not obtain better expression images with CAV2-hM3D-IRES miCitrine\nin the brain. The amount of mCitrine, which is the ORF after the IRES, is very low (Mizuguchi\net al. 2000; PMID:10933956). And, like numerous labs, we also attempted to use commercial\nantibodies against hM3D, but in our hands, the signal was weak and not specific. We are also\n13\nunaware of any study showing convincing hM3D immunoreactivity in vivo. Despite its\nlimitations, the current pictures in Supplementary Fig. 9h and Fig. 5b show the injection site.\nAs shown in the new Supplementary Fig. 9i-n, using CAV2-hM3D-IRES-mCitrine in E1-\ntranscomplementing cells (DK-E1), we observe mCitrine expression. Detection of mCitrine in\nDK-E1 cells seeded as a monolayer, with transduction of the majority of cells on the coverslip,\nis easier than detecting transduced neurons in a brain following in vivo expression. These data\nindicate that the vector quality (physical to infectious particle ratio) is excellent and that\ntransgenes from CAV2-hM3D-IRES-mCitrine are expressed. Briefly, DK-E1 cells were\nincubated with 50 particles of CAV2-hM3D IRES-miCitrine/cell. 24 h post-incubation, mCitrine\nand hM3D immunofluorescence were examined.\nIn addition, we injected three WT mice, in the hypothalamus, with a reporter vector, CAV2-\nCMV-mCitrine, which harbors the CMV promoter driving mCitrine. CAV2-CMV-mCitrine\ninjections led to robust mCitrine immunoreactivity in the hypothalamus and connecting\nregions.\nThese data were not added to the manuscript to avoid confusion, as the expression levels of\nmCitrine using CAV2-CMV-mCitrine reporter vector are much higher than those of CAV2-\nhM3D-IRES miCitrine (which enabled the easy detection of the vector in the brain). Yet it\nindicates the expected biodistribution in the hypothalamus and connecting regions, including\nthalamic and cortical areas.\n(A-M) CAV2-CMV-mCitrine biodistribution in the brain. CAV2-CMV-mCitrine was injected into the hypothalamus\n(D was the site of injection), leading to expression in multiple connecting brain regions, including the hypothalamus\n(C, D: MnPO: median preoptic,vLPO: ventrolateral preoptic, MPO: medial preoptic); thalamus (J,K: DLG: dorsal\nlateral geniculate nucleus, VLG: ventral lateral geniculate nucleus; IGL: intergeniculate leaflet, Rt: Reticular nucleus\nof the thalamus, Hb: Habenula), some neurons in cortical regions (A-K: Cortical Regions: Pir: piriform cortex, Icj:\nIslands of Calleja, EnDo: Endopiriform nucleus, Ac: agranular cortex), and the amygdala (E-F). scale, 1mm.\n3. In which neuronal cell types is Nav1.1 expressed in the hypothalamus? RNAscope or\nanalysis of available single cell data set of adult mouse brain could help.\nAnswer: We added a combined RNAscope and immunofluorescence analysis in WT mice\ninjected with CAV2-mCitrine. These data demonstrate the expression of mCitrine in both\n14\nexcitatory and inhibitory neurons in the hypothalamus, as well as Scn1a mRNA expression in\nthese two neuronal populations.\nMoreover, we extracted the Scn1a mRNA expression from hypothalamic single-cell RNA-seq\ndatasets (Steuernagel et al., 2022: PMID:36266547). These data are shown in Supplementary\nFig. 8.\nSupplementary Fig. 8\nScn1a expression in excitatory and inhibitory neurons in the hypothalamus\na-g. A Representative section of the hypothalamus of a WT mouse injected with CAV2-mCitrine. mCitrine\nimmunofluorescence (green), and RNAScope (Gad in magenta, Scn1a in red) showing the expression of Scn1a\nmRNA in both excitatory and inhibitory neurons.\na.mCitrine and DAPI. b. Gad and DAPI, c. Scn1a and DAPI. d. mCitrine and Gad. e. Scn1a and mCitrine. f. Scn1a.\ng. mCitrine, Gad and Scn1a. Scale bar: 1 µM\nh. Single-cell RNA-sequencing data restructured based on the open-access hypothalamic single-cell RNA-seq data\nfrom Steuernagel et al. (Steuernagel et al., 2022), showing Scn1a transcript levels (nCount RNA) in multiple\nclusters of GABAergic (blue) and glutamatergic (green) neurons. The dashed lines represent the average\nexpression level across all types of GABAergic neurons or all types of glutamatergic neurons.\nReviewer #2 (Remarks on code availability):\nI don't have the experience to test the code.\n---------------------------------------------------------------------------------------------------------------------------\nIn this report, Fadila et al., seek to establish a connection between sleep homeostasis and\nthermoregulation in an SCN1A haplo-insufficient mouse, carrying a Dravet Syndrome-\nassociated mutation. Mouse models of DS recapitulate several of features of disease including\nseizures, impaired sleep and poor thermoregulation. Changes in core body temperature\ncoincide with sleep/wake state transitions and increasing ambient temperature can promote\nNREM sleep. The authors hypothesize that sleep and temperature homeostasis disturbances\nare linked in DS mice and seek to understand the mechanisms by which they are coordinated.\nThere is potentially dual value in this approach as it stands to be informative regarding the\nrelationship between sleep and thermoregulation in the disease but also could reveal\nprinciples mechanistically linking SCN1A function to sleep and temperature homeostasis.\nWhile this is an interesting manuscript with some very nice preliminary findings, there are\nmany methodological concerns (detailed below) including a lack of appropriate controls,\ninappropriate statistical analyses, and a lack of experimental detail as well as very limited\nmechanistic insight rendering the manuscript of limited impact in its current form.\nThank you for your valuable comments and suggestions, which we have addressed in the\nrevised manuscript.\nOur specific concerns and suggestions are laid out below more or less organized by Figures.\nFigure 1\n// The authors claim that “DS mice increased their core more rapidly than WT mice” (line 90),\nhowever no quantification of the rate of change in core body temperature is provided.\nAnswer: We have removed references to the rate of core temperature increase, as this\nparameter was indeed not formally quantified.\nWhen mice are returned to RT to recover, the genotypes have different core body\ntemperatures. However, it is curious that the authors do not highlight that both genotypes\n‘overshoot’ their temperature recovery, ending up at a temperature below the baseline core\nbody temperature shown in 1B and 1C. Do both WT and DS mice have impaired recovery in\nthis model? .\nAnswer: Thank you for this relevant observation and concern. It is correct that for both WT\nand DS mice, upon the return to RT, show a reduction in core body temperature that is lower\nthan the baseline measurement at RT. This overshot has been described before and is a part\nof an adaptive homeostatic response (Romanovsky, 2018, PMID: 30454596).\nWe added a clearer depiction of these results in Fig. 1e, showing that both WT and DS mice\nreached a lower body temperature, compared to baseline temperature. Yet, the absolute\ntemperature in DS mice reached a lower body temperature than WT animals (p = 0.0009,\nTwo-Way ANOVA followed by Holm-Sidak post-hoc analysis). We have adapted the\ndescription of these findings in the Results (page 5, lines 11-15) accordingly to address this\naspect.\nYet, upon return to RT, DS mice returned to a lower temperature than that of WT mice (Fig.\n1c - e, Supplementary Fig. 1). Of note, while both WT and DS mice reached a temperature\nthat was lower compared to their initial core temperature, the overall temperature of DS mice\n15 min after recovery at RT was lower again lower than that of WT animals (Fig. 1e).\nof 10 WT and 6 DS mice was monitored for body temperature during both the heating and\ncooling phases, while the other cohort of 3 WT and 6 DS mice (all together 13 WT mice and\nsubsequent cooling phase after removal from the pad. These data were shown in the original\nFig. We realized that combining data from the two different cohorts of mice in a single\nvisualization (despite stating the number of mice in each panel in the legend) could be\nconfusing, especially when addressing the overshoot aspect discussed above. To improve\nclarity, the datasets are now shown separately: the revised Fig. 1c-e that was explained above,\nthus only presents the mice that were followed across both the heating and cooling phases.\nNew Supplementary Fig. 1b-c now depicts the graph that was shown in the original version of\nthe manuscript, showing both cohorts, mice that were monitored only during the heating\nphase, as well as the mice shown in Fig. 1C-E that were monitored during both heating and\ncooling. The legends of both figures have been adapted accordingly.\nThe authors next evaluate nesting behavior in WT vs DS mice because nests can stabilize\ncore body temperature during sleep. DS mice may not build nests as complex as their WT\nlittermates however, the significance of these findings at his juncture (or anywhere for that\nmatter in the manuscript) is unclear. While pre-sleep behaviors in mice are now well\nrecognized. In the context of the current manuscript, a defect in these behaviors raises the\npossibility of important confounds: for example, perhaps DS mice defect in nesting contributes\nto a lack of movement that would raise body temperature prior to sleep. This possibility is not\naddressed.\n//Moreover, the localization of these pre-sleep behaviors is still not well circumscribed in the\nbrain.\nAnswer: We acknowledge that nest building is an innate but multifactorial behavior that can\nbe affected by other DS-associated comorbidities, such as motor or social deficits. We also\nagree with the Reviewer that reduced pre-sleep nesting could, in principle, influence\nthermoregulation, as diminished movement or insulation may attenuate the typical pre-sleep\nincrease in body temperature. This possibility provides an additional behavioral context in\nwhich the observed alterations in sleep-related temperature dysregulation in DS mice may\narise.\nAdditionally, we concur that the neural substrates controlling pre-sleep behaviors like nesting\nremain poorly delineated, which limits definitive attribution of the observed phenotype to\nspecific upstream mechanisms. Following an additional set of experiments conducted in\nresponse to question 2 from Reviewer #1, we determined that vector-mediated expression of\nSCN1A in the hypothalamus did not normalize nesting behavior. The findings are consistent\nwith the DS-related Scn1a mutation affecting broader neural networks or additional\ncomorbidities that contribute to pre-sleep behavior, while not excluding a contribution from\nhypothalamic dysfunction.\nThe following text has been added to the Results section (page 5, lines 25-27)\nNevertheless, nesting is a multifactorial behavior that can also be influenced by DS-associated\ncomorbidities, including motor or social deficits, which may contribute to this phenotype.\nAnd the Discussion (page 13, lines 17-24)\nNesting, which is linked to preoptic hypothalamic activity 63–66, also remained abnormal in DS\nmice despite CAV2-SCN1A expression in the hypothalamus, indicating that, in the context of\nnesting may further contribute to sleep temperature dysregulation. Conversely, as CAV2-\nSCN1A injection into the hippocampus or thalamus corrected the epileptic and cognitive\nphenotypes of DS 28, it remains to be determined whether CAV2-SCN1A injection to these\nsites could also correct the sleep and thermoregulation deficits described here.\n// It is unclear how these findings fit with previous and subsequent data in the manuscript. How\ndoes nesting behavior related to core body temperature in DS mice, sleep and hypothalamic\nactivity? If nesting behavior is abnormal in DS mice, could this argue that SCN1A mutations\ndisrupt circuits involved in behaviors which regulate temperature? This line of questioning is\ndistinct from hypothesis that SCN1A acts in key brain regions directly involved in regulating\ncore body temperature (i.e. hypothalamus).that SCN1A acts in key brain regions directly\ninvolved in regulating core body temperature (i.e. hypothalamus).\nAnswer: The evaluation of nesting behavior was included as it relates to sleep preparation,\nthermoregulation, and neuronal activity in hypothalamic circuits known to be involved in the\nregulation of these processes. Prior studies have shown that nesting activates preoptic\nGABAergic and glutamatergic neurons (Tagawa et al., 2024, PMID:38594484), lesions to the\npreoptic area reduce nesting (Lee et al., 1999, PMID:10212050; Olazábal et al., 2002,\nPMID:11855898), and optogenetic activation of Vgat+ neurons in the preoptic area promotes\nnesting (Li et al., 2019, PMID:30459220). These observations, in our view, collectively support\na link between nesting and hypothalamic activity relevant to thermoregulation and sleep onset.\nIn our study, DS mice displayed a less complex nesting behavior compared to WT, together\nwith altered sleep-wake parameters and temperature regulation. This convergence suggests\npotential interaction between these processes; however, we agree that the directionality and\ncausality remain uncertain. A defect in nesting could, as indicated in the previous answer and\nin the adapted Discussion text, in principle, alter sleep thermoregulation, but this remains\nspeculative. With respect to the role of hypothalamic SCN1A in regulating nesting behavior,\nas noted in the previous comment, vector-mediated expression of SCN1A in the hypothalamus\ndid not normalize nesting behavior. While these findings do not exclude a contribution of\nhypothalamic dysfunction to impaired nesting behavior in DS, the observations are supportive\nof other neural networks or comorbidities contributing to nesting behavior pre-sleep. As\nindicated in the previous answer, statements concerning these aspects have been included in\nthe Results and Discussion sections, as described above.\nFigure 2\nThe authors next evaluate sleep and the change of core body temperature during wake ->\nNREM transitions in WT and DS mice. They find that while WT mice have a higher delta power\nin NREM compared to wake, DS mice do not exhibit any change in delta power. In addition,\nwhile WT mice experience a decrease in core body temperature at wake -> NREM transitions,\nthere is no change in DS mouse core body temperature at transition states.\n//There are major technical concerns with the sleep recordings. It is unclear from the methods\nsection at what time of day the recordings were done and how the mice were housed during\nthe recording session.\n// Were animals protected from environmental disturbances? Were they acclimated to the\nrecording chambers or temperature probes before recording sleep?\nsufficiently detailed, which has caused uncertainty about the procedures. All recordings were\nperformed during the light phase between 8am-6pm, with similar time-of-day variation across\ngenotypes and experimental groups. No additional habituation steps were included before\nrecording. All ECoG/LFP sleep recordings were performed in a separate room, in a padded\nPlexiglas box (for the sleep related temperature recordings) or 2-liter glass beaker (for the\nwarmth-induced experiments) with wet food provided. All of the mice (except for one DS\nmouse, which was not included in the analyses as we focused on the transitions from wake to\nNREM) were observed to fall asleep under these conditions, indicating that the environment\nwas adequately calm and suitable for natural sleep onset.\nWe have now clarified in the Methods section that (pages 17, lines 19-30):\nVideo-ECoG/LFP recordings were performed in a separate room during the light period (8 am-\n6 pm) for 2-5 h per mouse in a padded Plexiglas box (Fig. 2 and Fig. 4d) or a 2-liter beaker\n(Fig. 3, Fig. 4g-j, Fig. 5) with wet food provided.\n…\nThe recordings were long enough, and the conditions were suitable for the vast majority of the\nmice (except for one DS mouse, which was excluded from the analysis) to reach NREM sleep,\nbut not REM sleep.\n// Additionally, the recovery period post-surgery/implantation seems relatively short (2-3 days).\nAnswer: We regret that the post-surgical recovery period is judged as too brief. Based on the\nmouse’s behavioral and neurophysiological features in the recording setup, including wake\nand NREM cycles and related characteristic power properties of the ECoG/LFP signals in the\nWT animals, we consider a 2-3 day interval after ECoG surgery sufficient for full recovery\nbefore initiating recordings. Note that for recovery after LFP surgery, a 3-5 days recovery\nperiod was used, which we now added to the Method section (page 16, lines 18,20).\nFor the ECoG recordings in Fig. 2 we used mice at their 4th week of life. For the ECoG\nrecordings in Fig. 4, the mice were 5-6 weeks old. The mice were given 2-3 days for recovery\nafter surgery before recording.\n…(page 16 line 31)\n(for the LFP) The mice were given 3-5 days for recovery after surgery before recording.\nIn our experience, animals reliably regained normal activity, grooming, and weight within this\nperiod, and (as indicated above) show good LFP/ECoG quality, including sleep onset.\n// The statistical analysis of the PSD is not appropriate. It is advised to conduct a 2-way\nANOVA, comparing WT-WAKE, WT-NREM, DS-WAKE and DS-NREM. From Figure S1, it\nappears that the authors instead conducted t-tests to compare within genotypes only.\nThe authors state “ In DS mice, the increase in delta power was smaller than in WT animals”\n(line 160). With the current statistical analysis, they cannot make such a claim (unless we\nmissed this somewhere).\nAnswer: We regret that our initial description of the statistical analysis may have been unclear.\nIn fact, all the PSD data were originally analyzed using a Two-Way repeated-measures\nANOVA to compare wake vs NREM within each genotype (WT and DS, depicted in Fig. 2).\n4, which includes a comparison between genotypes.\nMoreover, as advised, Two-Way ANOVA (considering that the delta power was measured in\nthe same mouse during the wake and NREN stages), with Holm Sidak post-hoc analysis, is\nused throughout the Figures. This analysis demonstrates that the difference in % delta did not\nreach statistical significance when comparing WT vs DS. We have included these details in\nthe revised Methods (page 20, lines 7-10)\nData are reported as mean ± standard error (SE). All statistical tests were carried out via\nGraphPad Prism. The details of the specific test and the p-values are listed in the Figure\nlegend, with the full description listed in Supplementary Table 1. Differences were considered\nsignificant at p < 0.05.\nResults sections (page 6, lines 17-22)\nThe PSD of wakefulness was calculated two minutes prior to the transition to NREM, and the\nPSD of NREM was measured on the first minute into NREM. In WT mice, a comparison of the\nPSDs of these states showed that the transition to NREM was accompanied by a clear\nincrease in ECoG delta power. Conversely, while in WT mice this translated in the contribution\nof delta power to the overall ECoG being larger during NREM sleep than waking, in the DS\nmice, the delta ratio was similar during wake and NREM (Fig. 2c-d).\nIn addition, we updated the corresponding figure legends to explicitly state which test was\nused.\n// As a general comment for all sleep recordings done in the report, there are often poor\nrepresentative ECoG & EMG recording traces. Figure 2A, for example, displays ECoG and\nEMG traces in ‘wake’ and ‘NREM’ which are entirely indistinguishable from each other.\nAnswer: We understand this Reviewer’s observation and regret that the presentation of the\nLFP/ECoG and EMG traces did not provide a convincing impression of the wake-NREM\ndifferentiation. This limitation is especially apparent in the short-duration examples shown in\nFig. 2 and Fig. 4, which display only a few minutes of recording, as we are focusing on the\ntransition from wakefulness to NREM sleep.\nTo address this, we changed the example in Fig. 2a, selecting an example that better\nexemplifies the transition, which also shows changes in the EMG activity (that were not too\nprominent in the previous example, in which the detection of the wake-NREM transition relied\nmostly on delta power and ECoG). As described in the Methods, a virtual channel derived\nfrom the ECoG/LFP signal displaying delta power was used to support the identification of\nNREM epochs, as well as the EMG channel. An example of this channel was added to Figs.\n2 and 4. We would further like to note that the EMG channel was used to provide additional\nsupport for classifying NREM sleep, particularly in DS mice, which exhibit lower delta activity\nincreases during this stage, while accounting for residual muscle activity that can persist\nduring NREM sleep. The poor quality of NREM sleep in DS mice, reflected by the less\npronounced increase in delta power compared to wakefulness, poses an inherent challenge\nfor accurate sleep classification in this model. To address this, two independent investigators\nmanually identified sleep transitions, and only transitions on which both agreed were included\nin the analysis.\nFigs. 3, 4, and 5, we included hypnogram depictions of the wake and NREM epochs, leaving\nout the transition periods for clarity. Supplementary Fig. 3 contains a detailed depiction of all\nidentified stages, including the transitions.\nWe have clarified our vigilance state analysis approach in the revised Methods section (pages\n18-19, lines 25-4):\nWakefulness was identified by desynchronized ECoG/LFP patterns and relatively high levels\nof EMG activity. Non-rapid-eye-movement (NREM) sleep was marked by high-amplitude, slow\nwave patterns accompanied by low EMG activity. Virtual channels derived from the ECoG/LFP\nsignal, particularly delta power (0.5-4 Hz), were used to support NREM stage identification.\nHowever, in DS mice, as the increased delta activity during NREM is diminished, the EMG\nsignal contributed to the classification. To distinguish rapid-eye-movement (REM) sleep from\nwakefulness, a theta (4-8 Hz) to delta power ratio of >2.5, along with low EMG activity relative\nto NREM, was applied in the classification. Periods where wakefulness and NREM sleep\nalternated within 20 seconds were classified as transition states. Example recordings with\nhypnograms including all stages are shown Supplementary Fig. 3, whereas the hypnograms\nin Figs. 3-5 are shown without indication of these transitions for improved clarity. Transition\nperiods, which did not allow for classification, were excluded from analysis.\nFigure 3\n// the authors argue that WT mice exhibit an increase in delta power after heating, but it should\nbe addressed whether this effect is driven by one mouse, which seems to have a significant\nchange (3D).\n// The authors also employ a ‘sleepiness scale’ to evaluate the relative sleepiness of WT and\nDS mice after heating. However, it is unclear as to why the development or use of this\n‘sleepiness scale’ is required. We questions whether the use of EMG signal as a marker for\nsleepiness, in itself, is arbitrary and inappropriate. For example, a mouse with a severed\nspinal cord or with absence of spinal motor neurons may have different/absent EMG signals\nbut no change in “sleepiness” but this scale would render a “sleepier” mouse simply as a function of\nimmobility. Without validation or generalizability of such a scale, the data from it is hard to\nunderstand.\n//Furthermore, this scale not used in Figure 2 recordings -- why not? For this and subsequent\nfigures (3, 4 and 5) the authors also no longer separate a PSD analysis from wake vs NREM\nand it is unclear why (in line 227). It seems that the main effect of PSD delta power was in\nNREM stages (figure 2) and they may be missing an opportunity to have stronger effects.\nAnswer: We thank the reviewer for these thoughtful comments. We acknowledge that the\nconcept and presentation of the “sleepiness scale” raised confusion, as was also noted by\nReviewer #1. We fully agree that EMG activity alone does not uniquely define sleep or\nsleepiness, and that reduced muscle tone may occur in contexts other than sleep.\nIn light of this and the other Reviewer’s concerns, we have removed the “sleepiness scale”\nfrom the manuscript. Instead, as advised, we now rely on % NREM and ECoG/LFP delta\ncontribution during wake and NREM power to more objectively reflect sleep-wake dynamics.\nWe have added for all data-sets: (1) the percentage of time the mice spent in NREM sleep,\nand (2) the relative contribution of delta frequencies to the PSD during both wake (2 min before\nNREM onset) and NREM sections (1 min into NREM), as was shown in Fig. 2. The additional\nanalyses of relative delta power during the wake states and NREM are shown in Fig. 3e, 3f,\nFig. 3k, 3l, Fig. 4e, and Fig. 5f, 5j.\ninduced increase in % NREM, whereas in DS mice, a warm-induced increase in NREM is\nmissing (Figs. 3-5).\nMoreover, in WT mice, there is an increase in the contribution of delta power during as the\nmouse transitions to NREM sleep (Fig. 3e), whereas in DS, none of the examined cohorts\ndemonstrated this increase (untreated mice with sleep at RT, Fig. 2d, 2j, untreated mice at RT\nor 36 °C, Fig. 3f, or DS mice injected with CAV2-hM4D-mCitrine without CNO injection, Fig.\n5j blue).\nNevertheless, in WT mice in Fig. 3k at 36 °C and in Fig.5e at 36 °C or on the 2nd recording\nday at RT, the contribution of delta to NREM was not statistically different from that of wake.\nIn Fig. 3k, we suspect that this is related to the relatively small number of mice examined (4\nmice), with the possibility that the experimental procedure of viral injection or LFP surgery,\nin one of the mice, may have caused disruption in delta-driven brain regions in one of these mice\n(although also in this mouse the % NREM was increased by warmth.\nIn Fig 5f, 5 mice were tested, but since there are more conditions, reaching statistical\nsignificance is more difficult.\nThis was added to the Results section (page 8, lines 15-18)\nIn WT mice injected with CAV2-SCN1A, similar to untreated WT mice, the change to 36˚C\nincreased the duration of NREM sleep (Fig. 3j, k). However, a significant increase in the delta\ncontribution at NREM was only observed at RT, likely due to the relatively low number of\nexamined mice in this control cohort (Fig. 3k).\nWe hope that these updated analyses provide a clearer, validated assessment of sleep-wake\nbehavior without relying on the previously proposed scale.\nFigure 4\nThe authors test whether expression of SCN1A in the anterior hypothalamus can restore\nwarmth-mediated sleep in DS mice. They argue that CAV2-mediated SCN1A expression in\nDS mice restores the increased NREM time and delta power induced by heating.\n// However, this experiment is lacking appropriate control (empty virus not “untreated”) and\nstatistical analyses. While it appears the authors had an ‘untreated’ group (showed in\nsupplements) they do not include these groups in their statistical analyses.\nAnswer: Thanks. To address this concern, we performed an additional set of experiments in\nanother cohort of mice, in which we conducted ECoG recordings to compare warmth-induced\nsleep in WT and DS mice that were injected with either a control vector (CAV-GFP) or CAV-\nSCN1A.\nThe Methods section has been updated accordingly (page 17, lines 14-15):\nWT and DS mice, at the age of P22-26, were injected with CAV2-NSE-SCN1A, CAV2-NSE-\nGFP, CAV2-NSE-mCitrine (CAV2-SCN1A, CAV2-GFP or CAV2-mCitrine 28), or CAV2-hM3D-\nIRES-mCitrine (Plateforme de Vectorologie de Montpellier, IGMM Montpellier, France).\nIn the Results section (pages 8-9, lines 28-28), we added that:\nTo directly control for the effects of the hypothalamic CAV2 injections, a separate cohort of\nWT and DS mice was injected with a control vector containing a reporter gene CAV2-GFP, or\nwith CAV2-SCN1A. The biodistribution was examined in mice following CAV2-GFP injection\n(WT and DS, n = 3 for each genotype), which were also used for behavioral and\nelectrophysiological examinations, as well as in a separate group of three WT mice injected\n22\nwith the control vector CAV2-mCitrine, which were only used for immunohistochemistry. The\nbiodistribution analysis demonstrated expression in excitatory and inhibitory neurons in the\nhypothalamus and, due to CAV2’s retrograde transport properties, also in interconnected brain\nregions (Fig. 4a, b, Supplementary Figs. 8 and 9).\nIn the cohort of CAV2-GFP or CAV2-SCN1A injected mice, we also evaluated nest-building\nbehavior. Seven to ten days after vector injection, CAV2-SCN1A did not rescue the impaired\nnest-building abilities of DS mice (Supplementary Fig. 10). Next, ECoG electrodes were\nimplanted, and the mice were allowed to recover for 2-3 days before we proceeded with ~4 h\nECoG and temperature recordings at RT. Also in this cohort of mice, CAV2-SCN1A\nexpression in the hypothalamus restored the increase in delta ratio during the transition to\nNREM sleep, which was absent in DS mice injected with the control vector (CAV2-GFP, Fig.\n4a-e). Of note, these analyses were conducted using cortical ECoG rather than the\nhypothalamic LFP recordings used for the CAV2-SCN1A experiments shown in Fig. 3,\nconfirming that the correction of sleep-related oscillatory activity by CAV2-SCN1A is also\nreflected at the cortical level. Core temperature measurements were included in this new\ncohort of mice to test whether hypothalamic SCN1A expression may also correct the drop in\nbody temperature at NREM onset. However, in contrast to the data from naïve untreated mice\nshown in Fig. 2, no significant temperature decrease was detected after NREM onset in either\nWT or DS mice injected hypothalamically with either CAV2-GFP or CAV2-SCN1A. The\ndiscrepancy between these results and those presented in Fig. 2 may be attributable to the\ndifferent experimental procedures, as the mice depicted in Fig. 2 underwent only one surgical\nprocess, for electrode implantation (ECoG at the age of 4 weeks, or LFP at the age of 5\nweeks), while the mice shown in Fig. 4 underwent two surgeries, one for vector injection at 4\nweeks and a second one for ECoG implantation at 5 weeks.\nThe warmth-induced somnogenesis experiment was also performed in this cohort of mice, 1-\n4 days after the ECoG recordings at RT, and confirmed that NREM sleep was promoted in DS\nmice injected with CAV2-SCN1A, whereas in DS mice injected with the control CAV2-GFP\nvector, the switch to warm temperature did not induce NREM sleep (Fig. 4f-j).\n23\n(a-b) The control vector CAV2-mCitrine was injected into the hypothalamus. LPO: Lateral preoptic area, MPO: medial\npreoptic area, vLPO: ventrolateral Preoptic nucleus: anteroventral periventricular nucleus. Scale bar: 1 mm.\n(c) mCitrine expression in the vLPO. Scale bar 10 µM\n(d) Examples showing the ECoG signal, EMG, sonogram, and the virtual delta depicting delta (0.5-4 Hz) power in a WT: CAV2-\nGFP, WT: CAV2-SCN1A, DS: CAV2-GFP and DS: CAV2-SCN1A during a transition from wakefulness to NREM sleep.\n(e) About 2 weeks after viral injection, the mice were recorded for 4 h at RT. The relative contribution of delta power during\nthe analyzed wake and NREM periods (WT: CAV2-GFP, n = 7. WT: CAV2-SCN1A, n = 6. DS: CAV2-GFP, n = 6. DS: CAV2-\nSCN1A, n = 5. p = 0.023, Two-way ANOVA. The results of Holm-Sidak post hoc analyses are depicted on the graph).\n(f) The effect of warm temperature was tested in the same mice 4-6 days later, at this stage, the mice were recorded for 1h\nat RT, and then on a pad heated to 36°C. The percent of time the mice spent in NREM sleep at each temperature, is\ndepicted (WT: CAV2-GFP, n = 5. WT: CAV2-SCN1A, n = 6. DS: CAV2-GFP, n = 4. DS: CAV2-SCN1A, n = 5. p < 0.0001, two-\nway ANOVA. The results of Holm-Sidak post hoc analyses are depicted on the graph).\n(g-j) Examples of ECoG, EMG, sonogram and hypnogram of WT: CAV2-GFP (g), WT: CAV2-SCN1A (h), DS: CAV2-GFP (i),\nDS: CAV2-SCN1A (j).\n// And again, the authors fail to statistically compare WT and DS mice against each other.\nThey could do this by statistically comparing the change in delta ratio from RT -> 36°C in each\ninjection x genotype group.\nAnswer: We added the statistical comparison between genotypes for the %NREM related to\nthe data in Figs. 3 and 4 to the new Supplementary Fig. 6.\nSupplementary Fig. 6\nA full statistical comparison between different experimental groups shown in Figs. 3, 4.\n(a). The experimental paradigm, used for examining the effect of warmth on sleep promotion.\n(b-c). The same data shown in Figs. 3d and 3j are replotted here to highlight genotype differences. (2-way ANOVA.\nThe results of Holm-Sidak post hoc analyses are depicted on the graph).\n(d). The data shown in Figs. 4f are shown are depicted again here with the inclusion of the full statistical analysis\nto highlight also genotype differences. (2-way ANOVA. The results of Holm-Sidak post hoc analyses are depicted\non the graph).\n* p < 0.05; ** p < 0.01; *** p < 0.001\nThe authors test whether chemogenetic activation of the anterior hypothalamus using\nactivating DREADDS can rescue warmth-mediated sleep in DS mice.\n//The rationale for the experiment is only partially clear. What is known about the activity of\nthe neurons in anterior hypothalamus in DS mice? Is the activity in the haplo-insufficient model\nlower or higher than WT mice? In which cells? Testing this would support the DREADD\nexperiments.\nAnswer: Thank you for these comments. Little is known about the activity of the hypothalamus\nin DS, mainly because cellular studies are challenging in this region due to its diverse neuronal\npopulation. However, as indicated in our response to a related question from Reviewer #2\n(i.e., question 3, pages 14-15 of this rebuttal), RNAseq data, which are extracted from\nSteuernagel et al.(2022, PMID: 36266547) and replotted here, and our RNAscope analyses\nof three WT mice demonstrated comparable expression of Scn1a in both excitatory and\ninhibitory neurons. These data are presented in the new Supplementary Fig. 8 that we\nincluded on page 15 of this rebuttal.\nThe DS model used here harbors a missense Scn1a mutation; it is not a haplo-insufficient\nmodel . However, like other DS models, the Scn1a mutation causes a loss of function effect,\nand electrophysiological recordings in the hippocampus of these mice demonstrated reduced\nfiring on inhibitory neurons, reduced hippocampal inhibition, and altered firing and network\nconnections between excitatory neurons (Almog et al., 2022, PMID: 33271326, 2021, PMID:\n35370551; Fadila et al., 2023, PMID: 37192002; Layer et al., 2023, BioRxiv). Nevertheless,\nadditional studies are needed to directly examine the activity of specific classes of\nhypothalamic neurons in DS.\nThus, the rationale for testing chemogenetic activation of the hypothalamus is that DS-\nassociated Scn1a loss-of-function mutations are expected to reduce neuronal excitability. This\nis in line with the observation of reduced overall spectral power in DS, as seen in our\nhypothalamic LFP recordings (Supplementary Fig. 4). The findings presented in Fig. 5 indeed\nsupport the view that chemogenetic activation, similar to exogenous SCN1A expression,\nimproved warmth-induced NREM in DS mice.\nTo address that, we added to the Results section an expended explanation for our reasoning:\n(page 10, lines 4-12)\nThe prevailing view is that DS-associated Scn1a mutations lead to reduced neuronal\nexcitability due to impaired Na 1.1 channel function 34,35. The hypothalamus comprises a\nV\nheterogeneous population of excitatory and inhibitory neurons, both expressing comparable\nlevels of Scn1a mRNA (Supplementary Fig. 8) 36. If the primary effect of CAV2-SCN1A is to\nenhance the activity of transduced neurons, we wondered whether artificially increasing\nneuronal excitability, through chemogenetic activation, could mimic the effect of exogenous\nNa 1.1 expression. Specifically, we tested whether chemogenetic activation of hypothalamic\nV\nneurons could restore temperature-dependent sleep induction, similar to the correction\nobserved following CAV2-SCN1A injections.\nWe expanded the Discussion on these findings to underscore the complementary insight of\nthe CAV2-SCN1A vs DREADD experiments (pages 12-13, lines 23-11).\nIn light of the intricate neuronal population of the hypothalamus, which includes both sleep-\nand wake-promoting neurons 57, pinpointing the cellular basis of these DS-associated sleep\nand thermoregulatory deficits remains challenging. This complexity is further illustrated by the\npresence of hypothalamic neuron types with unique properties, such as wake-promoting\n26\nglutamatergic parvalbumin-positive neurons 60. Moreover, since Scn1a is expressed in both\nexcitatory and inhibitory hypothalamic neurons 36, identifying the precise cellular mechanisms\ninvolved becomes even more difficult. However, the observation that hypothalamic neuronal\nCAV2-mediated SCN1A expression or chemogenetic stimulation restored the warmth-induced\nsleep-promoting response suggests that reduced neuronal excitability, resulting from Scn1a\nloss-of-function mutation, is a plausible mechanism for the observed DS-related deficits.\nNevertheless, CAV2-mediated expression of SCN1A or hM3D is not restricted to a specific\nclass of neurons. CAV2-SCN1A contains the neuronal-specific enolase (NSE) promoter, and\nCAV2-hM3D-IRES-mCitrine harbors the CMV (cytomegalovirus early enhancer) promoter.\nBoth promoters lead to transgene expression in excitatory as well as inhibitory neurons 28.\nMoreover, with the effective retrograde transport properties characteristic of CAV2 61,62,\ninjection of the CAV2-based vectors to the anterior hypothalamus also transduced neurons in\nregions that project to the hypothalamus. Thus, it is possible that the anterior hypothalamic\ninjection of CAV2-SCN1A leads to a correction in the activity of multiple types of neurons in\nthis region, as well as additional connecting networks that hub on the anterior hypothalamus,\nthat together contribute to the observed improvement in sleep and thermoregulation in DS\nmice.\nWe also added to the Discussion that cellular electrophysiological studies within this region\nare challenging, and future studies are needed to address this important question (page 14,\nlines 21-25).\nA limitation of the present study is that we did not directly assess how the effects of the DS\nScn1a mutation and hypothalamic CAV2-mediated SCN1A expression, or alternatively\nchemogenetic stimulation, influence neuronal firing and excitability in the anterior\nhypothalamic region. Future studies addressing these questions could provide deeper\nmechanistic insight into the neuronal basis of sleep and thermoregulation deficits in DS.\n// There is again a lack of proper control (empty vector) and appropriate approach to statistical\nanalysis. Including a CNO only control would be ideal as the effects observed could be CNO\nor hypothalamus-mediated.\nAnswer: To directly test the possibility that CNO may cause sleepiness in mice without\nDREADD expression, we examined the effect of 10 mg/kg CNO on locomotor activity in four\nawake WT mice assessed for a period of 10 minutes in the open field starting 15 minutes after\nCNO injection. These data were added to the new Supplementary Fig. 10; the locomotor\nactivity was similar following CNO (10 mg/kg) and saline injections.\nWe added the following statements to:\nThe Methods section (page 18, lines 17-20):\nLocomotor activity of WT mice, without DREADD expression, was not affected by 10 mg/kg\nCNO. Specifically, 15 min after CNO injection, locomotor activity of the mice was monitored\nfor 10 minutes in an open field of 50 by 50 cm (Supplementary Fig. 12).\nAnd the Results section (page 10, lines 20-23):\nThis dosage of CNO did not alter locomotor activity, as assessed in an open field test in WT\nmice without DREADD expression (Supplementary Fig. 12), indicating the absence of a\nnonspecific sedation effect at this dose 37.\n27\n10000\n8000\n6000\n4000\n2000\n0\ng/ k\ng)\nali n\ne\nm S\n0\n(\nN\nC\nSupplementary Fig. 12\nCNO administered at 10 mg/kg had no effect on the locomotor activity of WT mice. WT mice were placed in a 50\nx 50 cm open field arena 15 min after CNO injection, and their locomotor activity within the arena was recorded for\n10 min. Four days later, the same mice were injected with saline, and their locomotor activity was recorded again\n15 min after saline injection over 10 min. The similar distance covered under both conditions indicates that CNO\nat this dose does not have a non-specific sedative effect.\nAnd the Discussion section (page 14, lines 21-25).\nA limitation of the present study is that we did not directly assess how the effects of the DS\nScn1a mutation and hypothalamic CAV2-mediated SCN1A expression, or alternatively\nchemogenetic stimulation, influence neuronal firing and excitability in the anterior\nhypothalamic region. Future studies addressing these questions could provide deeper\nmechanistic insight into the neuronal basis of sleep and thermoregulation deficits in DS.\n// there is no confirmation that CNO administration actually had an effect on anterior\nhypothalamus activity.\nAnswer: As noted also in our answer to a question from this Reviewer on Fig. 5 (as well as to\na related question from Reviewer #1), cellular electrophysiological studies of the\nhypothalamus are extremely challenging. Therefore, the effect of CNO was examined at the\nneuronal network and behavioral level only, focusing on the ability of CNO to promote NREM\nsleep in a warm environment in DS mice. As noted in our earlier answers, we addressed this\nlimitation in the Discussion.\nOther major concerns\n- Lack of description about the haplo-insufficient DS mouse model\nAnswer: We added to the Introduction previous studies of this animal model (page 3, lines 11-\n14).\nAmong the many existing models, DS mice harboring the missense A1783V Scn1a mutation\nexhibit severe epilepsy and various DS-associated behavioral phenotypes, including spatial\nmemory and motor deficits 12,18–21. Sleep properties or thermoregulation have not been\ninvestigated in this DS model.\n- Varied ages for the mouse studies\nAnswer: The use of two mouse age groups was dictated by technical considerations. DS mice\nat 4 weeks of age are at heightened risk for seizures and premature death. Therefore, for\n28\n)mc(\nderevoc\necnatsiD\nmore invasive procedures such as hypothalamic depth LFP electrode implantation, and for\nexperiments involving hypothalamic viral injections followed by depth LFP or ECoG surgery,\nwe performed the LFP and ECoG surgery in 5-week-old mice, which have a significantly lower\nrisk for surgical complications and SUDEP. Additionally, sufficient time for post-surgical\nrecovery and viral expression necessitated the use of an older cohort for the viral transfection\nstudies. Crucially, sleep and thermoregulation deficits were observed in both younger (4-\nweek-old) and older (5-week-old) DS mice, demonstrating that the phenomenon is not\nconfined to a single age group. This consistency across cohorts, in our view, supports the\nvalidity of our observations.\nWe included an adapted statement concerning the rationale for using 5-week-old mice for\nexperiments involving depth LFP recordings in the Results section (page 7, lines 2-3)\nOf note, as these depth surgeries are more invasive, we used 5-week-old mice.\nand included in the Discussion (page 14, lines 18-20)\nNonetheless, sleep and thermoregulation deficits were observed in both juvenile (4-week-old)\nand older (5-6-week-old) DS mice, demonstrating that the phenomenon is not confined to a\nsingle age group or disease stage.\n- baseline temperature difference is gone in older animals\nAnswer: The absence of a baseline temperature difference in older DS mice is not considered\na concern but rather reflects an age-related change in the disease phenotype. We hypothesize\nthat this age-related normalization of core body temperature reflects a reduction in seizure\nburden rather than an experimental confound.\nThis observation and a discussion on the interpretation has now been included into the\nDiscussion (page 14, lines 4-20):\nAnother observation we made was that juvenile DS mice, at the beginning of their fourth week\nof life, showed a lower baseline core temperature compared to WT mice. This difference could\nbe attributed to altered metabolic homeostasis described in DS 72, involving dysregulation of\nenergy metabolism and mitochondrial function, or to the severe epilepsy typical at this age.\nBoth factors may contribute to the reduced thermoregulatory stability observed across sleep-\nwake transitions. In the DS model used here, all the mice experience spontaneous seizures\nduring their fourth week of life 12,27. The lower baseline temperature may therefore be related\nto post-ictal hypothermia, which has been reported in both human epilepsy patients 73 and DS\nmice up to several hours after a seizure 74. In older DS mice at 5-6 weeks of age, when\nspontaneous seizures become less frequent 12,27, the baseline temperature difference\nbetween DS and WT mice was less pronounced, which is consistent with a reduced seizure\nburden, as well as a reduction in metabolic stress. Although we did not assess metabolic\nparameters in the present study, this will be an important direction for future investigations to\nbetter define the contribution of metabolic mechanisms to thermoregulatory regulation in DS.\n- many seizures in the younger animals, do any of them die?\nAnswer: We did not observe any deaths during our recording, but about 50% of our DS mice\nwere found dead in the cage following electrode implantation or viral injection. This is the rate\nwe see with untreated DS mice (Fadila et al, 2020, PMID:32865826, Fadila et al. 2023, PMID:\n37192002).\n- Lack of investigation and discussion about potential metabolic contributions to differences in\nbasal and sleep-state transition associated thermoregulation\ncontributions to the observed thermoregulatory differences. Although metabolic parameters\nwere not directly investigated in the present study, previous reports have documented altered\nmetabolic homeostasis in DS models, including changes in energy expenditure and\nmitochondrial function. Such alterations, together with the pronounced seizure burden in\njuvenile DS mice, may contribute to the reduced baseline core temperature and impaired\nthermoregulatory functions during sleep-wake transitions. This aspect has been incorporated\ninto the revised Discussion (page 14, lines 4-20):, where we also note that a direct assessment\nof metabolic involvement would be an important objective for future studies.\nAnother observation we made was that juvenile DS mice, at the beginning of their fourth week\nof life, showed a lower baseline core temperature compared to WT mice. This difference could\nbe attributed to altered metabolic homeostasis described in DS 72, involving dysregulation of\nenergy metabolism and mitochondrial function, or to the severe epilepsy typical at this age.\nBoth factors may contribute to the reduced thermoregulatory stability observed across sleep-\nwake transitions. In the DS model used here, all the mice experience spontaneous seizures\nduring their fourth week of life 12,27. The lower baseline temperature may therefore be related\nto post-ictal hypothermia, which has been reported in both human epilepsy patients 73 and DS\nmice up to several hours after a seizure 74. In older DS mice at 5-6 weeks of age, when\nspontaneous seizures become less frequent 12,27, the baseline temperature difference\nbetween DS and WT mice was less pronounced, which is consistent with a reduced seizure\nburden, as well as a reduction in metabolic stress. Although we did not assess metabolic\nparameters in the present study, this will be an important direction for future investigations to\nbetter define the contribution of metabolic mechanisms to thermoregulatory regulation in DS.\n- No investigation into the activity of the hypothalamus in DS mice, this could link hypothalamic\ninvolvement to the temperature-sleep transitions\nAnswer: We agree with the Reviewer that the lack of hypothalamic neuronal recordings is a\nlimitation of the current study and have addressed this in the revised Discussion (page 14,\nlines 21-25), as also noted above.\nA limitation of the present study is that we did not directly assess how the effects of the DS\nScn1a mutation and hypothalamic CAV2-mediated SCN1A expression, or alternatively\nchemogenetic stimulation, influence neuronal firing and excitability in the anterior\nhypothalamic region. Future studies addressing these questions could provide deeper\nmechanistic insight into the neuronal basis of sleep and thermoregulation deficits in DS.\n- There is also occasional inconsistency in the PSD findings – sometimes the main significant\neffect of the experimental variable is seen in delta power and sometimes it is in delta ratio, but\nthere is a lack of discussion about this.\nAnswer: Following your advice, we have now consistently examined the contribution of delta\nfrequencies to wake and NREM, as well as the percentage of NREM sleep, during RT and\nwarm environment. These analyses are now presented instead of the delta power, which was\npreviously shown in the text and was derived from sections that combined wake and NREM,\nresulting in less consistent results.\nIn the results shown in Figs. 2-5, we consistently see an increase in % NREM in warm\ntemperatures in WT mice and a lack of increase in % NREM sleep in DS mice.\n---------------------------------------------------------------------------------------------------------------------------\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\n31"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have made very good efforts to address all my points. I think the manuscript and presentation are improved and\nthe work is an interesting and important contribution.\nSigned: William Wisden", "reviewer_2": "The authors addressed all my concerns.", "reviewer_3": "The authors have made a series of commendable revisions that have satisfied my concerns with the initial manuscript.\nCongratulations."}, "author_response": ""}]

Decision letter