The gut vagal sensory pathway drives postprandial sleep via activation of PVH-projecting GABAergic neurons in the NTS

Domain: neuroscience
Published: 2025-12-17
Editorial outcome: major_revision

Abstract
Feeding status bidirectionally modulates sleep; however, the neural circuitry that integrates the sensing of gastrointestinal (GI) state and sleep remains poorly understood. The afferent fibers of the vagus nerve extensively innervate the GI tract, transmitting postprandial satiety signals to the brain. This study investigates the key role of the upper gut-innervating vagal sensory neurons in modulating sleep-wake states and promoting postprandial sleep, uncovering the underlying circuit mechanisms. Both feeding and activation of stomach/duodenum-innervating vagal sensory neurons reduce wakefulness and increase NREM sleep in male mice. Conversely, chemogenetic inhibition abolished the sleep-promoting effects of feeding. Using anterograde transsynaptic tracing, single-nucleus RNA sequencing combined with optogenetic manipulation, we identified a vagal ascending pathway connecting the upper gut to the paraventricular nucleus of the hypothalamus (PVH) via GABAergic neurons in the nucleus of solitary tract (NTS). Stomach/duodenum-innervating vagal sensory neurons project directly to and functionally activate NTS GABAergic neurons. Activation of these neurons and their projections to the PVH suppressed wakefulness and prolonged NREM sleep. Overall, our study reveals a vagal sensory pathway that integrates satiety signals to modulate sleep. It reveals the direct neural circuitry mechanisms driving postprandial sleep and offers distinctive perspectives into the development of innovative interventions for sleep disorders.

Introduction
Sleep and food consumption are two essential biological processes that interactively maintain the homeostasis of the human body. Food ingestion triggers an integrated system of hormonal, nutritional, and neural signals to inform the brain about energy levels, leading to the cessation of food intake and a frequently occurring phenomenon known as postprandial somnolence1. Theories exploring the mechanistic links between food consumption and sleep include the redistribution of blood flow, hormonal responses, and a rise in plasma glucose; however, the neural mechanisms, particularly at the neural circuit level, remain poorly explored.

The afferent fibers of the vagus nerve extensively innervate the gastrointestinal tract, serving as a direct gut-brain communication pathway and conveying postprandial satiety signals to the brain2,3. During feeding, physical cues from the stomach wall, such as tension and tactile stimuli, stimulate the gastric vagal afferents via mechanosensors, particularly subtypes of IGLEs within the myenteric plexus that express GLP1R or OXTR4. In parallel, chemical cues, such as nutrients entering the intestine including glucose, fatty acids, and amino acids activate these neurons either through direct sensing at vagal terminals or via enteroendocrine cell-mediated pathways. In addition, the presence of food in the gut induces the release of a series of hormones, such as CCK, PYY, and GLP1, which act on their respective receptors expressed by vagal terminals in the submucosal layer of the intestinal wall to further stimulate the gut vagal afferents3,5.

Numerous brain regions respond to stimulation of vagal nerve as revealed by large-scale neuronal mapping5,6, expanding the possibility that the gut vagal pathway may be involved in the regulation of multiple brain functions beyond satiety feedback. Indeed, recent studies have progressively revealed the role of the gut vagal pathway in modulating higher brain functions, including nutrient preference7–9, reward10, learning and memory11,12, and mood11,13,14.

The gut vagal afferents predominantly terminate in the nucleus of the solitary tract (NTS) within the brainstem, where satiety signals are further conveyed to higher brain regions. As a critical integration center for viscerosensory information, the NTS projects to several brain areas involved in energy homeostasis, thus positioning the gut vagal pathway as a key regulator of feeding and feeding-related behaviors15–19. Interestingly, several projection areas of the NTS, including the locus coeruleus, paraventricular nucleus of the hypothalamus (PVH), ventral tegmental area (VTA), and central and extended amygdala, have also been implicated in the sleep-wake modulation20–24. Earlier studies have shown that both electrical and chemical stimulation of the NTS elicited electroencephalographic (EEG) patterns similar to those observed during slow-wave sleep25,26, as well as synchronized EEG activity characterized by an increase in theta wave power (4–6 Hz) in anesthetized animals27. These observations position the gut vagal pathway as a potential regulator of both feeding and sleep-wake states.

Within the NTS, glutamatergic, GABAergic, and catecholaminergic neurons are the predominant subtypes, working together to coordinate the homeostatic reflexes that regulate vital functions, including cardiovascular, respiratory, and gastrointestinal activities28. Interspersed throughout the NTS, GABAergic neurons form a predominant inhibitory network that maintain the balance of excitation and inhibition within the NTS and adjacent hindbrain regions29,30. Local infusion of GABA receptor agonists and antagonists has demonstrated the critical involvement of the NTS GABAergic network in regulating cardiorespiratory homeostasis and gastrointestinal motility31,32. Beyond their role in the local inhibitory network, a significant proportion of GABAergic neurons project to various subcortical and hypothalamic brain regions33. While the functions of NTS-local projecting GABAergic neurons are well-defined, the roles of long-range projecting GABAergic neurons remain largely unexplored. Studies have shown that GABAergic neurons are activated by feeding, and that selective activation of NTS GABAergic neurons suppresses food intake and body weight through projections to the arcuate nucleus of the hypothalamus, highlighting the important role of NTS long-range projecting GABAergic neurons in mediating postprandial behaviors34,35.

In the present study, we investigated the role of GI-vagal afferents in modulating sleep-wake states and explored the neural mechanisms underlying satiety-induced sleep. Using virus-assisted circuit tracing and chemogenetic/optogenetic manipulation, we further elucidate the neural circuitry involved. We showed that refeeding after overnight fasting induced an extension of NREM sleep with a complementary reduction in wakefulness. Chemogenetic activation of vagal afferent inputs innervating the stomach and duodenum, but not the small intestine, significantly promoted NREM sleep. Conversely, chemogenetic inhibition of these inputs ablated the satiety-induced extension of NREM sleep. Moreover, we found that vagal afferents innervating the stomach and duodenum could induce activation of GABAergic (NTSGABA) neuronal populations in the NTS. Chemogenetic or optogenetic activation of NTSGABA neurons, or their long-range projections to the PVH, led to a significant increase in NREM sleep. These results illustrated a distinct role for GI-vagal afferents in the modulation of sleep-wake states and revealed a vagal-dominant ascending pathway driving postprandial sleep.

Results
Acute feeding after overnight fasting induces sleep enhancement
To determine whether sleep-wake states are influenced by energy status, we recorded the electroencephalogram (EEG) and electromyogram (EMG) of mice that fasted for 16 h (Fasted) and of mice that were given unrestricted access to food pellets for 2 h after fasting (Refed) (Fig. 1A). The wakefulness, REM, and NREM states were classified based on EEG and EMG recordings (Fig. 1B). Refeeding resulted in a strong and significant increase in both the ratio (Fig. 1C, upper panel, *p<0.05, **p<0.01, two-way RMANOVA) and the duration (Fig. 1C, lower panel, **p<0.01, t-test) of NREM sleep and REM sleep, accompanied by a complementary decrease in wakefulness. The prolonged NREM duration was accompanied by a significant extension of the NREM bout duration (Fig. 1D, *p<0.05 t-test) with the number of bouts remaining unaltered (Fig. 1E). Correspondingly, the number of transitions between each pair of sleep states was not changed (Fig. 1F). These results suggest that refeeding immediately after fasting enhances sleep, resembling the state of postprandial sleep.

The gastrointestinal (GI) tract is densely innervated by the terminals of vagal sensory neurons, which respond to food-associated mechanical and chemical stimuli, transmitting satiety signals to the central nervous system via the NTS. We then evaluated the activation of NTS neurons upon refeeding using mice with either intact or ablated gastric vagal sensory neurons. The CCK-saporin method was employed to specifically ablate vagal afferent signaling in the GI tract36. Refeeding markedly increased the number of Fos-positive neurons in the NTS, which was dramatically reduced in mice with upper-gut vagal afferents ablated by CCK-SAP, compared to those with intact vagal afferents (Fig. 1G, ****p<0.0001, one-way ANOVA). The ablation of upper gut vagal afferents was verified using Cholera Toxin Subunit B (CTB)-mediated retrograde labeling, a method previously employed to trace the gut-innervating vagal sensory neurons37. CTB was injected into the stomach and duodenal walls of mice that had received either SAP or SAP-CCK in the nodose ganglion (NG). The number of CTB-labeled cells was significantly reduced by approximately 68.6% in the SAP-CCK–injected group compared with the SAP group (Fig. 1H, ***p<0.001, t-test). These results together suggest that the feeding-induced activation of the NTS is predominantly driven by the gut vagal sensory neurons.

Chemogenetic activation of stomach/duodenum innervating vagal sensory neurons promotes sleep
Since feeding promoted sleep and activated gut vagal sensory neurons, we next investigated whether vagal sensory neurons innervating the stomach or intestines contribute to feeding-induced sleep. To examine the causal role of stomach/duodenum-innervating vagal sensory neurons in sleep modulation, we employed a dual-viral labeling strategy to selectively manipulate their activity10,13. Specifically, the stomach and duodenum of wild-type (WT) mice were injected with a retrogradely transported adeno-associated virus (rAAV-hSyn-CRE-EGFP-WPRE-hGHpolyA) under the control of the hSyn promoter. This approach enabled the expression of Cre recombinase in the soma of vagal sensory neurons that innervate stomach and duodenum (Vagalstom/duo neurons), located in the NG. A separate AAV virus carrying Cre-dependent designer receptors, hM3D(Gq), hM4D(Gi), or mCherry, was injected into the RNG to enable the selective expression of these receptors in the Vagalstom/duo neurons. The viral strategy and the expression of the receptors in the right NG are shown in Fig. 2A (upper panel). The EEG and EMG recordings were recorded for three hours, beginning 30 min after intraperitoneal injections of either saline or clozapine-N-oxide (CNO) at a dose of 1 mg/kg (Fig. 2A, lower panel).

In mice expressing hM3D(Gq)-mCherry in Vagalstom/duo neurons, CNO administration significantly increased both the proportion (Fig. 2B, C, upper panel; **p<0.01, ***p<0.001, two-way RMANOVA) and the total duration (Fig. 2C, lower panel; ***p<0.001, ****p<0.0001, t-test) of NREM sleep, while reducing wakefulness over the 3-h recording period compared with saline-treated controls. Because significant differences in wakefulness and NREM were observed during the first 30 min, we analyzed this interval in 2-min bins to rule out baseline effects. As shown in Supplementary Fig. 1A, both groups exhibited comparable wakefulness at recording onset, indicating the absence of baseline differences. Activation of Vagalstom/duo neurons quickly decreased the ratio of wakefulness within 30 min of recording (Supplementary Fig. 1A; *p<0.05, **p<0.01, ***p<0.001, two-way RMANOVA). It also extended NREM bout duration and reduced wake bout duration (Supplementary Fig. 1B; **p<0.01, t-test), without affecting the total number of bouts or the number of transitions between sleep states (Supplementary Fig. 1C, D).

Interestingly, CNO injections in mice expressing hM4D(Gi) in the Vagalstom/duo, did not affect sleep-wake states in terms of ratio and the total duration (Fig. 2D, E, supplementary Fig. 1E), however, it increased the bout duration of wakefulness and decreased the number of bouts of both wakefulness and NREM sleep (Supplementary Fig. 1F–H; *p<0.05, t-test). In control mice expressing mCherry alone, CNO injection elicited no significant changes in sleep-wake states (Fig. 2F–G, Supplementary Fig. 1I–L). We also conducted the caspase 3-mediated ablation of Vagalstom/duo neurons to further confirm the effects of stomach/duodenum vagal inhibition on sleep-wake states. The retrogradely transported virus rAAV-hSyn-CRE-mCherry-WPRE-hGH polyA was injected into the stomach and duodenal walls of WT mice, followed three weeks later by injection of either AAV-DIO-EGFP or AAV-DIO-taCaspase 3 into the NG. After four weeks of expression, the efficacy of taCaspase 3-mediated ablation was evaluated. As shown in Fig. 2H, the number of mCherry+ neurons was significantly reduced in the taCaspase 3 group (****p<0.0001, t-test). EEG and EMG recordings from these two groups revealed no significant differences in either the proportion or total duration of NREM sleep (Fig. 2I, J, Supplementary Fig. 1M–P). Together, these results indicate that activation of Vagalstom/duo neurons is sufficient to promote NREM sleep, but not necessary for its maintenance.

Using similar manipulation strategies, we next investigated whether small intestine-innervating vagal sensory neurons (VagalsInt neurons) contribute to sleep-wake modulation. The designer receptors hM3D(Gq) and hM4D(Gi) were selectively expressed in the VagalsInt neurons (Supplementary Fig. 2A). However, CNO administration produced no significant changes in sleep–wake states in mice expressing either hM3D(Gq) or hM4D(Gi) (Supplementary Fig. 2B–L) compared with their saline-injected controls, suggesting that VagalsInt neurons do not contribute to the regulation of sleep–wake states.

The stomach/duodenum innervating vagal sensory neurons mediate satiety-induced sleep
Given that stomach/duodenum-innervating vagal sensory neurons are activated during gastric distension, and our results indicate that their activation promotes NREM sleep, we next investigated whether these neurons underlie the sleep-wake activity changes induced by feeding, commonly known as postprandial somnolence. Using neuronal activation marker Fos, we first confirm the involvement of Vagalstom/duo neurons in the activation of the NTS in response to feeding. Chemogenetic inhibition of Vagalstom/duo neurons significantly reduced refeeding-induced NTS neuronal activation (Fig. 3A, **p<0.01, ****p<0.0001, one-way ANOVA), indicating that the Vagalstom/duo neurons partially contribute to the refeeding-induced activation of NTS neurons.

Next, we recorded and analyzed EEG and EMG in mice expressing hM3D(Gq) or hM4D(Gi) specifically in Vagalstom/duo neurons under both fasting and refeeding conditions. The experimental design was illustrated in Fig. 3B. In mice expressing hM4D(Gi)-mCherry in Vagalstom/duo neurons, nutritional state exerted a main effect on ratio and duration of wakefulness, NREM and REM sleep (Fig. 3C; *p<0.05). While CNO treatment did not significantly alter sleep. Post hoc analysis revealed that in saline injected control animals, refeeding significantly decreased wakefulness and increased NREM sleep, which were abolished by CNO administration (Fig. 3C; upper panel, **p<0.01, ***p<0.001 three-way RM ANOVA; lower panel, *p<0.05, two-way ANOVA), indicating that chemogenetic inhibition of Vagalstom/duo neurons prevents refeeding-induced decrease in wakefulness and increase in NREM sleep. The blockade of the reduction in wakefulness in mice with Vagalstom/duo neuron inhibition could result from the inability of refeeding to decrease the bout duration of wakefulness since the number of bouts was not significantly altered in the CNO-injected groups (Fig. 3D, E). The number of transitions between different sleep states were not affected (Fig. 3F).

In mice expressing hM3D(Gq)-mCherry in Vagalstom/duo neurons, both nutritional state and CNO treatment significantly affected the ratio and duration of wakefulness, NREM, and REM sleep (Fig. 3G; *p<0.05). Post hoc analysis revealed that refeeding in saline-injected controls reduced the ratio and total duration of wakefulness while increasing NREM sleep (Fig. 3G; upper panel, *p<0.05, #p<0.05, ##p<0.01 three-way RM ANOVA; lower panel, *p<0.05, ###p<0.001, two-way ANOVA). Notably, CNO injection under fasting conditions also decreased the duration of wakefulness and increased NREM sleep (Duration: ###p<0.001, two-way ANOVA). This effect was accompanied by a significant increase in NREM bout duration (Fig. 3H, #p<0.05, two-way ANOVA), without altering bout number or state transitions (Fig. 3I, J). These findings indicate that activation of Vagalstom/duo neurons under fasted condition reduces wakefulness and prolongs NREM sleep, further supporting their sleep-promoting role. Collectively, these results demonstrate that activation of Vagalstom/duo neurons facilitates sleep and mediates postprandial sleep.

The stomach/duodenum innervating vagal sensory neurons functionally regulates nts gabaergic neurons
To further dissect the circuit mechanisms underlying the regulation of sleep by feeding, we examined the responses of distinct neuronal populations in the NTS to fluctuations in energy status. Single-nucleus RNA sequencing reveal that the majority of neurons in the NTS express either the excitatory marker Slc17a6 (Vglut2) or the inhibitory marker Slc32a1 (Vgat)28. To label excitatory and inhibitory neurons in the NTS, we crossed Vglut2-Cre or Gad2-Cre mice with the Ai14 reporter strain, respectively. The expression of neuronal activity marker Fos was quantified in the NTS of mice in the fasted and refed groups. We found that in the NTS of Vglut2-Cre-Ai14 mice, the number of cells positive for Fos, as well as those positive for both tdTomato and Fos, was significantly elevated in the refed group compared to the fasted group (Fig. 4A, **p<0.01, ****p<0.0001, t-test), suggesting robust activation of excitatory neurons in the NTS by feeding. Notably, we observed a similar fold increase in GABAergic activation in the NTS of Gad2-Cre-Ai14 mice (Fig. 4B, ****p<0.0001, t-test), indicating that GABAergic inhibitory neurons in the NTS are also responsive to changes in energy status.

To further determine whether the activation of NTS neurons, particularly GABAergic neurons, following feeding is driven by vagal sensory input from the upper gut, we first combined herpes simplex virus (HSV)-mediated transsynaptic labeling and single-nucleus RNA sequencing to identify the cell types that receive direct inputs from the Vagalstom/duo neurons. We injected an anterogradely transported, polysynaptic HSV H129 strain expressing enhanced green fluorescent protein (HSV-EGFP) into the stomach and duodenum walls of WT mice and performed whole-brain HSV-EGFP tracing. EGFP signals were detected in the stomach, RNG, NTS, as well as in the PVH and the bed nucleus of the stria terminalis (BNST) (Supplementary Fig. 3A). Four days post-injection, the DVC was dissected for single-nucleus RNA sequencing (Fig. 4C). High-quality transcriptomes were obtained from 11,167 DVC cells. Neuronal population was re-clustered and 22 distinct neuronal populations were identified. After excluding area postrema (AP) and dorsal motor nucleus of the vagus (DMV) neurons based on established marker genes38, we identified eight excitatory (Glu_01-Glu_08) and eight inhibitory (GABA_01-GABA_08) clusters within the NTS (Fig. 4D, E). Among excitatory neurons in the NTS, Slc17a6+ (Vglut2+) neurons comprised the vast majority (96.66%) of the entire excitatory neuronal population. While the inhibitory neurons in the NTS were composed largely of Gad2+ neurons (68.71%), followed by Gad1+ (55.40%) and Slc32a1+ (Vgat+; 58.85%) populations, which exhibited varying degrees of reciprocal co-expression (Supplementary Fig. 3B). In addition, feature plots for co-expression of Gad2 and Slc17a6 showed that while 5.8% of these NTS neurons (124 out of 2132 cells) co-expressed both genes, the majority of clusters were clearly segregated (Fig. 4F–G). This segregation was further confirmed by in situ hybridization, which demonstrated a distinct separation between Slc17a6+ and Gad2+ populations, with an overlap rate of 8.47% (Fig. 4H).

To determine the proportions of GABAergic and glutamatergic neurons receiving input from Vagalstom/duo neurons, we isolated neurons infected by HSV-EGFP based on the presence of the EGFP-encoding sequence (717 bp). A total of 1,008 HSV-labeled neurons were re-clustered into seven distinct populations (Fig. 4I; Supplementary Fig. S3C and D). Of these, 55.16% were GABAergic (Gad1+/Gad2+/Slc32a1+), 40.18% were glutamatergic, and 4.66% were cholinergic (Chat+) (Fig. 4J). Among the GABAergic neurons, 282 were Gad2+, 193 were Gad1+, and 143 were Slc32a1+, indicating that Gad2+ GABAergic neurons receive the most projections from Vagalstom/duo neurons. Using Gad2-Cre-Ai14 mice with the HSV-EGFP transsynaptic labeling we confirmed these projections. Approximately 22.9 ± 1.9% of EGFP+ cells were also positive for tdTomato, indicating that NTS Gad2+ GABAergic neurons (NTSGABA) indeed receive direct projections from Vagalstom/duo neurons (Fig. 4K). These findings prompted us to investigate whether the GABAergic neurons in the NTS can be activated by Vagalstom/duo neurons and possibly contribute to the modulation of sleep by feeding. Chemogenetic activation of Vagalstom/duo neurons elicited a significant increase in neuronal activity in the NTS GABAergic neurons, as measured by the number of Fos+GAD67+ cells (Supplementary Fig. 3E, ****p<0.0001, t-test), demonstrating that NTSGABA neurons are functionally regulated by Vagalstom/duo neurons. Interestingly, chemogenetic inhibition of Vagalstom/duo neurons alleviated the feeding-induced activation of GABAergic neurons in the NTS, suggesting the involvement of NTSGABA neurons in transmitting satiety signals via Vagalstom/duo neurons (Supplementary Fig. 3F, **p<0.01, ***p<0.001, one-way ANOVA). Together, these results suggest that GABAergic neurons in the NTS are preferentially targeted and functionally regulated by projections from vagal sensory neurons innervating with the upper gut.

Activation of NTS GABAergic neurons reduces the duration of wakefulness
Given that Vagalstom/duo neurons activate NTSGABA neurons and promote sleep, we next assessed the functional role of NTSGABA neurons in sleep modulation. Gad2-Cre mice were microinjected with AAV-EF1a-DIO-ChR2-EYFP into the NTS to enable optogenetic activation of NTSGABA neurons (Fig. 5A). For acute optogenetic stimulation, a 30 Hz pulse train (10 ms pulse width) was delivered for 5 min at random intervals ranging from 15 to 25 min. For prolonged stimulation, optogenetic stimulation was applied using a 30 Hz pulse train (10 ms pulse width, 1 s duration with a 4 s inter-pulse interval) for 1 hour. Acute optogenetic stimulation (470 nm) suppressed wakefulness and increased NREM sleep (Fig. 5B, C, *p<0.05, t-test). Similarly, 1 hour of optogenetic stimulation resulted in a significant decrease in wakefulness and a corresponding increase in NREM sleep (Fig. 5D, *p<0.05, two-way RM ANOVA and t-test). Optic stimulation of NTS GABAergic neurons showed a significantly decreased bout duration of wakefulness and a trend toward increased bout duration of NREM sleep (Fig. 5E), while number of bouts appeared to increase across all three sleep stages (Fig. 5F, G). In contrast, optic stimulation in control mice expressing EYFP alone resulted in minimal changes in sleep-wake states, including total duration, bout duration, and number of bouts across different brain states (Supplementary Fig. 4).

We also confirmed the effects of NTSGABA activation on sleep using chemogenetic manipulation. The AAV-EF1a-DIO-hM3D(Gq)-mCherry was injected into the NTS of Gad2-Cre mice. EEG and EMG signals were recorded for 3 h, starting 30 min after CNO injection (Fig. 5H). Chemogenetic activation of NTSGABA neurons resulted in a significant reduction in the ratio and duration of wakefulness and an increase in the duration of NREM sleep (Fig. 5I, J, upper panel, ***p<0.001, two-way RMANOVA; lower panel **p<0.01, t-test). Surprisingly, the detailed analysis of the first 30 min recording (Fig. 5K) showed a main effect of CNO (**p<0.01), indicating that NTS GABAergic activation possibly elicited a rapid action on sleep within 30 min of CNO injection. Similar to the optic stimulation, CNO injection significantly decreased the bout duration of wakefulness without affecting the bout duration of NREM sleep (Fig. 5L, ***p<0.001, t-test). However, we observed an increase in the occurrence of both wakefulness and NREM sleep (Fig. 5M, *p<0.05, t-test), likely due to a greater number of transitions between wake-NREM and NREM-wake, suggesting a pattern of fragmented sleep (Fig. 5N, *p<0.05, t-test).

Because Vagalstom/duo neurons activate subpopulations of glutamatergic neurons in the NTS to suppress food intake after a meal18,39–41, we also sought to test whether glutamatergic neurons in the NTS are involved in the modulation of sleep. We injected the NTS of Vglut2-Cre mice with AAV-EF1a-DIO-ChR2-EYFP (Supplementary Fig. 5A). Surprisingly, optic stimulation (30 Hz pulse trains) of NTS glutamatergic neurons induced convulsive seizure-like behaviors in awake mice (Supplementary Fig. 5B). Taken together, these results suggest that activation of NTSGABA reduces wakefulness but leads to sleep fragmentation. Given that NTSGABAergic neurons are essential for regulating autonomic functions such as respiration, blood pressure, and cardiovascular activity, it is crucial to further investigate the circuitry mechanisms underlying their role in sleep regulation.

NTSGABA-PVH Projection Promotes NREM Sleep
Besides forming an inhibitory network that modulates autonomic functions, many NTS GABAergic neurons extend long-range projections to various brain regions33,42. To determine the projection area of NTSGABAergic neurons, we selectively labeled these neurons by local injecting AAV-EF1α-DIO-ChR2-EYFP into the NTS of GAD2-Cre mice. Four weeks post-injection, we observed clusters of axonal fibers in the PVH and BNST regions that were also identified through HSV-mediated trans-synaptic labeling following upper gut injection (Supplementary Fig. 6A). We further confirmed the NTSGABAergic projections to both brain regions using monosynaptic antegrade tracing. Gad2-cre mice were injected with AAV-DIO-mCh-2A-GK into the NTS followed by injection of H129-ΔGK-G4. Neurons labeled with GFP were observed in the regions of the injection sites and the downstream brain regions including PVH and BNST (Supplementary Fig 6B). Given that both the PVH and BNST are involved in the stress response and the maintenance of wakefulness, we next examined whether the NTSGABA-PVH or NTSGABA-BNST projection modulates sleep-wake states.

AAV-EF1α-DIO-ChR2-EYFP was injected into the NTS of GAD2-Cre mice, followed by optic fiber implantation at the PVH to selectively stimulate the axonal terminals of the NTSGABA-PVH projection. The expression of EYFP in both the NTS and PVH confirmed successful viral infection (Fig. 6A). Both the acute (Fig. 6B, C, *p<0.05, t-test) and prolonged (Fig. 6D, *p<0.05, two-way RM ANOVA, t-test) optic stimulation of NTSGABA-PVH projection showed a significantly decreased bout duration of wakefulness and elevated the NREM sleep, accompanied by a decrease in bout duration of wakefulness (Fig. 6E, *p<0.05, t-test). The number of bouts and the number of transitions between sleep states remained unchanged (Fig. 6F, G). Neither acute nor prolonged optic stimulation at the PVH in mice expressing the control virus induced significant changes in sleep-wake states (Supplementary Fig. 7).

The modulatory role of the NTSGABA-PVH projection in sleep was further confirmed through chemogenetic manipulation. AAV-EF1α-DIO-hM3D(Gq)-mCherry was injected into the NTS of GAD2-Cre mice, followed by the implantation of a guided cannula into the PVH. Successful viral infection was confirmed by mCherry fluorescence in both the NTS and PVH (Fig. 6H). EEG/EMG signals were recorded for 3 h, starting immediately after CNO infusion (0.5 μg) (Fig. 6H). Representative EEG and EMG recordings were shown in Fig. 6I. CNO infusion resulted in a marked decrease in ration and duration of wakefulness and an increase in NREM sleep (Fig. 6J, upper panel *p<0.05, two-way RM ANOVA, lower panel, *p<0.05, **p<0.01, t-test), which was not attributable to baseline differences among the groups as shown in Fig. 6K. Interestingly, in contrast to the phenotype observed with NTSGABA stimulation, changes in sleep-wake states were primarily driven by a reduction in the bout duration of wakefulness and extension of bout duration of NREM sleep (Fig. 6L, *p<0.05, t-test). The number of bouts as well as the number of transitions between sleep states remained unchanged (Fig. 6M, N). Since activation of PVH neurons is essential for maintaining wakefulness, these results suggest that NTSGABA neurons reduce wakefulness and promote sleep by suppressing PVH neurons via the NTSGABA-PVH projection.

NTSGABA-BNST Projection Reduces Wakefulness
We next examined whether the NTSGABA-BNST projection also regulates sleep-wake states. Using a similar approach as for testing the NTSGABA-PVH projection, AAV-EF1α-DIO-ChR2-EYFP was injected into the NTS of GAD2-Cre mice, followed by implantation of optic fibers at the BNST to selectively stimulate the axonal terminals of the NTSGABA-BNST projection. The EYFP expression at both the NTS and BNST confirmed successful viral infection (Fig. 7A). Neither the acute (Fig. 7B, C) nor prolonged (Fig. 7D) optical stimulation of NTSGABA-BNST projection elicited changes in sleep-wake states. Although prolonged stimulation resulted in a reduction in the bout duration of wakefulness (Fig. 7E *p<0.05), the total duration of wakefulness remained unaffected. Neither the number of bouts nor the number of transitions between sleep states were affected by optic stimulation either (Fig. 7F, G). Optogenetic stimulation of the BNST in control virus-expressing mice was conducted to exclude the effects of stimulation itself on sleep-wake activity (Supplementary Fig. 8).

Chemogenetic manipulation of the NTSGABA-BNST projection was employed to confirm its role in modulating sleep-wake states. AAV-EF1α-DIO-hM3D(Gq)-mCherry was injected into the NTS of GAD2-Cre mice, and a guide cannula was subsequently implanted into the BNST. The fluorescent expression of mcherry in both the NTS and BNST indicated successful viral infection. EEG/EMG signals were recorded for 3 h, starting immediately after CNO infusion (0.5 μg) (Fig. 7H). Representative EEG and EMG recordings are shown in Fig. 7I. CNO infusion produced a modest but significant reduction in ratio and duration wakefulness and a corresponding increase in NREM sleep (Fig. 7J; upper panel, *p<0.05, ***p<0.001; two-way ANOVA; lower panel, *p<0.05, **p<0.01, t-test). Analysis of the first 30 min revealed that these changes emerged rapidly, were evident within the first 20 min, and dissipated thereafter, indicating an acute effect (Fig. 7K, *p<0.05, two-way RMANOVA). These alterations were accompanied by a decrease in wakefulness bout duration, with NREM bout duration remaining unchanged (Fig. 7L; **p<0.05, t-test). Number of bouts and the number of transitions between sleep states were unaffected (Fig. 7M, N), consistent with the effects observed during optogenetic stimulation. These results together suggest that activation of the NTSGABA-BNST projection acutely reduces wakefulness without affecting NREM sleep.

Methods
Animals
Animal care and all experimental protocols were approved by the Scientific Research Ethics Committee of Qilu Hospital, Shandong University. Animals were housed in groups of five under a 12-hour light–dark cycle (light on at 07:00 and off at 19:00) with ad libitum access to food and water. For the fasting condition, mice were deprived of food for 16 h starting at 16:00 on the previous day, and food was reintroduced at 08:00 the following morning. The mouse strains used in this study are as follows: C57BL/6J (Charles River stock number: 219), Gad2-ires-cre (Jackson Laboratory stock number: 028867), Vglut2-ires-cre (Jackson Laboratory stock number: 028863) and Ai14 (Jackson Laboratory stock number: 007914). Detailed information on the mice used in this study was listed in Supplementary Table 1. All experiments were conducted in male adult mice in normal health conditions. Following surgical procedures for electrode or fiber optic implantation, mice were singly housed and allowed at least 1 week of rest prior to the subsequent experiments. All mice were under 16 weeks of age at the time of the experiments.

Surgical procedures
Peripheral organs viral injections. Mice were anesthetized using isoflurane (4% for induction and 1%-2% for maintenance). A midline abdominal incision was made to expose the target organs (stomach/duodenum or small intestine). The viruses, including AAV2/R rAAV-hSyn-CRE-EGFP-WPRE-hGH polyA, AAV2/R rAAV-hSyn-CRE-mCherry-WPRE-hGH polyA, or HSV-EGFP (co-administered with Bortezomib, 0.1 mg/mL, I.P.) and CTB555 were injected into the target organ using a Nanofil™ 36G beveled needle (World Precision Instruments, Sarasota, FL) attached to a Nanofil™ 5 μL syringe (World Precision Instruments, Sarasota, FL), mounted on a Pump 11 Elite Nanomite (Harvard Apparatus, Holliston, MA). For the stomach and duodenum injection, 3 μl of viral particles (titer: 1.08E+13 vg/ml) were injected into the submucosal layer at 10-12 different sites. For the small intestine injection, 3 μl of viral particles were injected into the proximal jejunum/ileum at a minimum of 10 injection sites over a total length of 10 cm.

Nodose ganglia injections. Adult male mice were anesthetized with isoflurane (4% for induction and 1%-2% for maintenance) and the right nodose ganglia were exposed and injected with the virus following the procedure described previously66. Briefly, a midline incision was made to expose the submandibular glands, which were retracted to reveal the trachea. The carotid artery was carefully separated and retracted toward the skull base to uncover the vagus trunk. The hypoglossal nerve was then pulled caudally to provide a clear view of the nodose ganglia. A total volume of 0.5 μl of viral aliquots was loaded into a Nanofil™ 36G beveled needle (World Precision Instruments, Sarasota, FL) and injected using the Pump 11 Elite Nanomite at a rate of 0.1 μl/min. The following viruses and chemicals were used for the different experiments as indicated: AAV2/5 rAAV-hSyn-DIO-hM3D(Gq)-mCherry-WPRE-hGH polyA, AAV2/5 rAAV-hSyn-DIO-hM4D(Gi)-mCherry-WPRE-hGH polyA, AAV2/5 rAAV-hSyn-DIO-mCherry-WPRE-hGH polyA, AAV2/5 rAAV-EF1a-DIO-EGFP-WPRE-hGH polyA, AAV2/5 rAAV-EF1a-DIO-taCasp3-TEVp-P2A-EGFP-WPRE-hGH polyA, CCK-SAP (250 ng/ml; IT-31), and SAP (250 ng/ml; IT-21). For each nodose ganglion, injections were made at two sites: rostral and caudal to the laryngeal nerve branch.

Stereotaxic viral injections and optic fiber implantation. Stereotaxic surgery was performed as previously described67,68. Briefly, mice were anesthetized with isoflurane (4% for induction and 1%-2% for maintenance) and secured in a stereotaxic frame. For virus injection, a total volume of 1.0 µl of AAV vectors (0.5 µl per side) was bilaterally infused into the NTS at the following coordinates: AP, −7.5 mm; ML, ±0.3 mm; DV, −5.4 mm. The following viruses were used for the different experiments as indicated: AAV2/9 AAV-hSyn-DIO-hM3D(Gq)-mCherry-WPRE-hGH polyA, AAV2/9 AAV-hSyn-DIO-mCherry-WPRE-hGH polyA, AAV2/9 rAAV-EF1a-DIO-hChR2(H134R)-EYFP-WPRE-hGH polyA, AAV2/9 AAV-EF1a-DIO-EYFP-WPRE-hGH polyA, HSV129-dgK-G4, AAV-EF1a-DIO-mCh-2A-gK-WPRE-hGH polyA. For cannula implantation, inserts were placed into the PVH and BNST at the following coordinates: PVH (AP, −0.7 mm; ML, ±0.2 mm; DV, −4.7 mm) and BNST (+0.26 mm; ML, ±0.8 mm; DV, −4.5 mm). Intra-nucleus microinjections of CNO were performed in conscious, unrestrained, and freely moving mice in their home cages. A 33-G stainless-steel injector connected to a 5-μl syringe was inserted into the guide cannula and extended 0.5 mm beyond the tip. A total volume of 1 μl of CNO (0.5 μg/μl) or artificial cerebrospinal fluid (aCSF) was infused bilaterally into the PVH or BNST at a rate of 0.125 μl/min, followed by a 5-minute waiting period to prevent backflow during withdrawal. For optic fiber implantation, a single optical fiber cannula (Doric lens) was surgically implanted in the PVH (AP, −0.7 mm; ML, ±0.2 mm; DV, −4.4 mm) or BNST (+0.26 mm; ML, ±0.8 mm; DV, −4.2 mm). EEG/EMG recordings and behavioral tests were conducted at least 21 days after virus injection and 7 days following cannula/optic fiber implantation. Detailed information on the viruses and chemicals used in this study is provided in Supplementary Table 1.

EEG/EMG implantation. Mice were anesthetized with isoflurane (4% for induction and 1–2% for maintenance) and placed in a stereotactic apparatus (RWD, Shenzhen, China). The coordinates for EEG electrode placement were based on previously published studies69. EEG signals were recorded from the frontal cortex (AP, +2 mm; ML, +2 mm) and the occipital cortex (AP, −3.5 mm; ML, +2.5 mm). A reference screw was positioned in the cerebellum, 1.5 mm posterior to Lambda, with a lateral (ML) position of 0. Two EMG electrodes were then inserted into the neck muscle tissue. The EEG/EMG implant and cannula were securely fixed to the skull using 3M resin and dental cement. After surgery, mice were singly housed and allowed at least 7 days for surgical recovery before EEG data acquisition.

Polysomnography recording and data analysis
Before EEG/EMG recording, each mouse was individually placed in the recording environment and allowed to acclimate to the recording cable for 2 days. Electroencephalogram (EEG) and electromyogram (EMG) signals were recorded using the Plexon multi-channel acquisition system (Plexon, Hong Kong). The length of the recording cable was selected to ensure that the mouse’s movement was not restricted. Signals were amplified using a Plexon high-gain preamplifier (X192 gain, filter set to 0.1–7500 Hz, sampling rate: 2000 Hz). EEG/EMG signals were recorded between 10:00 and 13:00, and the data were analyzed after converting the raw signals into the European data format. Bandpass filters were applied to the EEG (0.3–35 Hz) and EMG (50–200 Hz) signals. Data were analyzed in 4-second epochs, and brain states were classified as NREM, REM, or wakefulness. Wakefulness was characterized by desynchronized EEG and high EMG activity; NREM sleep was defined by high-amplitude, low-frequency (0.5–4 Hz) EEG signals and low EMG activity; REM sleep was identified by predominant theta-like (4–9 Hz) EEG signals and low EMG activity. All brain states were scored using AI-driven software, Lunion Stage, developed by Lunion Data (Shanghai, China)70.

Chemogenetic manipulation
The CNO was dissolved in the saline. For the chemogenetic activation of gastric vagal sensory neurons and GABAergic neurons in the NTS, the CNO (1 mg/kg) or saline was given intraperitoneally (i.p) 30 min before EEG recording. For chemogenetic activation of GABA neuron axons locally, a total amount of 0.5 μg CNO (0.5 ug/ul, 1 ul) was infused into the PVH or BNST through a guide cannula. EEG/EMG recordings were performed either 0 or 30 min after intra-nucleus infusion or intraperitoneal injection of CNO, respectively, and continued for 3 h.

Optogenetic manipulation
Mice were given 2 days to acclimate to the fiber optic cables (100 cm long, 200 μm diameter; Inper, China) before testing. A pulse generator was used to delivered 465 nm laser stimulation (Inper, China). The laser power was adjusted to achieve an intensity of 5–10 mW/mm2 at the tip of optic fiber. For acute optogenetic stimulation, 30 Hz pulse train (10 ms pulse width) was delivered for 5 min at random intervals ranging from 15-25 min. For prolonged stimulation, the light was delivered at 30 Hz pulse train (10 ms pulse width, 1 s duration with 4 s inter-pulse interval) for 1 h. EEG/EMG recordings were performed both before and after optic stimulation, as indicated for each experiment.

Single nucleus RNA sequencing
Nuclei isolation from the dorsal vagal complex (DVC)
Mice were anesthetized with 2-4% isoflurane and decapitated. The dorsal vagal complex (DVC) were dissected from the brain stem immediately and stored at –80°C. The nuclei were isolated using GEXSCOPE® Nucleus Separation Solution (Singleron Biotechnologies, Nanjing, China) in strict accordance with the manufacturer’s instructions. Isolated nuclei were resuspended in PBSE (PBS buffer containing 2 mM EGTA) to 106 nuclei per 400 μl, filtered through a 40 μm cell strainer, and counted with Trypan blue. Nuclei enriched in PBSE were stained with DAPI (1:1,000) (TermoFisher Scientifc, D1306). Nuclei were defined as DAPI-positive singlets.

Library preparation for single nucleus RNA-sequencing
The concentration of the single-nucleus suspension was adjusted to 3–4×105 nuclei/mL in PBS before loading onto a microfluidic chip (Singleron Biotechnologies). SnRNA-seq libraries were then constructed using the GEXSCOPE® Single-Nucleus RNA-seq Kit (Singleron Biotechnologies) according to the manufacturer’s instructions. The libraries were sequenced on an Illumina NovaSeq 6000 platform with 150 bp paired-end reads.

Single-nucleus RNA-sequencing data analysis
Raw reads were processed to generate gene expression profiles using CeleScope v2.0.7 (Singleron Biotechnologies). The sequencing data were aligned to the Mus musculus (mm10) genome using STAR version 2.7.11a with default parameters. Briefly, Barcodes and UMIs were extracted from R1 reads and corrected. Adapter sequences and polyA tails were trimmed from R2 reads and the trimmed R2 reads were aligned against the GRCm38 (mm10) transcriptome using STAR Solo in STAR (2.7.11a). Successfully assigned reads with the same cell barcode, UMI and gene were grouped together to generate the gene expression matrix for further analysis. The exogenous gene sequence (EGFP), was integrated into the mouse genome for subsequent snRNA sequencing analysis. Initially, the exogenous gene (EGFP) was prepared in FASTA format, accompanied by a GTF file detailing gene annotations. These files were then appended to the ends of the mouse reference genome FASTA and GTF files. The modified reference genome was constructed using the Celescope mkref command and the raw sequencing data were processed using Celescope version 2.0.7, resulting in the generation of a gene expression matrix that included the EGFP sequence.

RNA in situ hybridization
Mice were anesthetized with 2–4% isoflurane and decapitated. Brains were immediately removed and cryosectioned at a thickness of 10 μm using a cryostat (CM1950, Leica, Germany). RNA amplification-based single molecule fluorescent in situ hybridization (asmFISH) was performed as previously described71, using SEERNA® FISH RNA Fluorescence in Situ Hybridization Kit (Dynamic Biosystems, Suzhou, China). The label probe was co-njugated to Cy7 (Gad2) and Cy5 (Slc17a6) (Life Technologies, Thermo Fisher Scientific, Shanghai, China). Probe Genomic Targeted Sequences. Briefly, tissue sections in 10 μm thickness were fixed in 4% (w/v) paraformaldehyde (Sigma, Shanghai, China) for 5 min, and washed twice with diethyl pyrocarbonate (DEPC)-treated PBS, followed by dehydration in an ethanol series. After three washes with Wash Buffer (Dynamic Biosystems, Suzhou, China), sections were permeabilized with 0.1 M HCl for 5 min in an incubator at 42°C. The sections were then incubated in target probes in hybridization mix at 42°C for 4 h, ligation mix at 42°C for 30 min, splint primers in the circularization mix at 42°C for 30 min, and amplification mix for rolling circle amplification (RCA) at 42°C for 2 h. After washing three times with Wash Buffer, the sections were then incubated in hybridization buffer containing the label probes in a 42°C incubator for 30 min. The slides were mounted with SlowFade Gold Antifade Mountant (Thermo Fisher Scientific, Shanghai, China) medium containing 0.5 μg/mL DAPI (Sigma, Shanghai, China). Images were acquired using the 3D-Hitech Pannoramic® MIDI II equipped with a pco.edge 4.2 bi camera under 20x objective lens.

Histology and immunohistochemistry
Mice were anesthetized with avertin (250 mg/kg, Sigma-Aldrich, Shanghai, China) and intracardially perfused with 30 mL phosphate-buffered saline (PBS), followed by 30 mL of 4% paraformaldehyde (PFA) to verify the correct injection site after completing the relevant experiments. Their brains were then removed and fixed overnight in 4% PFA. After fixation, the brains were dehydrated in 30% sucrose at 4°C until it was fully submerged. Coronal sections (30 μm) were made using a cryostat (Leica), and fluorescence at the site of injection as well as at the projection area were examined directly under a microscope (Fluoview 1200, Olympus, Japan). For immunohistochemistry, floating sections were rinsed in PBS, then incubated in PBS containing 0.3% Triton X-100 (PBST) at 4°C with the following primary antibodies: rabbit anti-Fos or mouse anti-GAD67 (1:400, 12 h). After primary antibody incubation, sections were rinsed three times in PBS, and secondary antibodies (Alexa 488 or 594, 1:400, Invitrogen) were applied for 4 h at room temperature. Detailed information on the antibodies used in this study was listed in Supplementary Table 1. Finally, sections were mounted on glass slides, dried, dehydrated, and coverslipped. Fluorescence images were captured using a confocal microscope (Leica, Germany) and analyzed with Fiji ImageJ software.

Statistics and reproducibility
All data are presented as means ± standard error. Statistical data were analyzed using the GraphPad Prism software package (GraphPad Software, Inc.). One-way ANOVA, two-way (RM) ANOVA, three-way ANOVA, and Student’s t-test (two-tailed) were performed when appropriate. For all statistical measures, a p-value < 0.05 was considered to be statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Chen et al. used optogenetics and chemogenetics neural manipulation and claimed that increased NREM sleep in post-fasting refeeding is caused by decreasing activity in the PVH through the GABAergic innervation from NTS and input from the nodose ganglion to NTS. The manuscript is well written, and the control groups and statistics are sound. Although the finding is interesting and advances our knowledge in this field of research, it could be improved by considering the following points.\nComments\n1) It is not clear what kind of signals by refeeding, activate the nodose ganglion. It would be interesting to clarify what signals are involved, such as physical signals from the stomach wall, blood glucose level changes, etc. Please also indicate whether this pathway is also functional during normal feeding behavior.\n2) In p11, the reason to focus on the analysis of the GABAergic innervation of the NTS is relatively weak, not to study the glutamatergic innervation enough since c-Fos is clearly expressed in the glutamatergic neurons in Figure 4A. Also, the authors are using Gad2-cre mice, but they need to confirm whether these are indeed GABAergic neurons or not. Recent studies have shown that some neurons express Gad 1 or 2 but do not express vGAT and express vGlut instead; these neurons act as glutamatergic neurons. This means gad2-cre might not be suitable to label GABAergic neurons in this brain region. For confirmation, please use vGAT-Cre, stain with vGAT antibody, or show these neurons are not co-expressed with vGlut.\n3) In p12, please show the physiological validity of using 30Hz stimulation. It is stated that activation of glutamatergic neurons with optogenetics causes epilepsy, simply because the firing rate of baselines is different for glutamatergic neurons and GABA neurons, and 30Hz may be too strong for glutamatergic neurons.\n4) In p9, please clearly indicate what time in the light/dark cycle the 16-hour fasting experiment.\n5) In Discussion, There is not much discussion of the effect on REM sleep, but the reason why the effect on REM sleep are not significant may simply be due to the small sample size or the fact that the experiment was conducted at a time of day with a small percentage of REM, so at least discuss the possibility a little more.", "reviewer_2": "This study describes a gut-brain pathway that regulates sleep, potentially providing a mechanism to account for the sleep-inducing effects of food intake. The topic is important and the paper presents some interesting data. However, there are basic problems with several of the experiments that must be addressed before this would be suitable for publication in any journal.\nMajor points.\n1. There are several problems with the chemogenetic experiments in Fig. 2 and elsewhere. (1) They all require genotype controls (mice injected with fluorophore AAV, treated with CNO). This is particularly important because CNO (rather than DCZ) was used in these experiments, which is less specific and could modulate sleep. (2) The hM4D-CNO experiments must be compared to hM4D-saline in the same animals - it is not appropriate to compare them solely to hM3D-saline or CNO in different animals. (3) It is not clear exactly how the experiment was performed in Fig. 2, but the logical test is to measure whether activation with hM3D rescues sleep in fasted mice, and whether silencing with hM4D blocks increased sleep in fed mice, as in Fig. 3. (4) The statistical analysis of the chemogenetic data in Fig. 3 is unclear. First, the hM3D and hM4d experiments should be analyzed separately, and compared to their own both drug and genotype controls. Second, you cannot use a two-way ANOVA when you are simultaneously varying three parameters (genotype, drug treatment, nutritional state). Further, for the all of the chemogenetic and optogenetic data, the significance of differences in means between any two specific experiments has be tested using a test that is corrected for multiple comparisons, not just a t-test.\n2. Existing evidence suggests that GABAergic projections from NTS to forebrain are extremely sparse, so it is surprising that the authors claim strong projections to the PVH and BNST. This is a particular concern because (1) The tracing data in Fig. 7 is not convincing -- the images are very zoomed in and the axonal fibers appear to actually label surrounding structures more strongly than PVH, (2) the use of GAD2-Cre is problematic, because in other brain regions (e.g. hypothalamus) it has been shown that GAD2 is expressed in many neurons that are glutamatergic, and (3) there is a lack of convincing data showing that the viral injections are confined to NTS. It is very possible to label GABAergic neurons in structures adjacent to the NTS to give the appearance of spurious projections. For example, the histology in Fig. 5a is very zoomed in, whereas Fig. 5I appears to show infection primarily in DMV, not NTS, whereas Fig. 6I clearly labels AP as well. The authors should (1) confirm the specificity of the GAD2 driver by showing there is no overlap with Vglut2 in NTS, (2) confirm the projections to PVH and BNST exist using tracing with Vgat-Cre, with comprehensive histological data showing the injections are confined to the NTS and that the projections specifically label the downstream structures claimed (i.e. with more zoomed out images and replication). (3) change the nomenclature throughout the text to make it clear that the functional manipulations in the brain target the dorsal vagal complex (DVC), not the NTS specifically, and refrain from making claims (in the title, abstract or elsewhere) about the function of GABAergic NTS neurons specifically, since the injections are not restricted to the NTS. (4) Provide histological documentation of the spread of viral infection in every mouse used for manipulations. This can included in the supplement.\n3. For Fig. 2A, there needs to be direct immunohistochemical validation of the efficiency of vagal afferent ablation with CCK-SAP. Showing changes in Fos in NTS is not sufficient.\nMinor points\n1. Many of the manipulations target innervation of both stomach and intestine, and therefore the text should make this clear (as opposed to referring solely to \"gastric\" afferents). CCK is expressed in many intestinal innervating vagal afferents.\n2. There is no such thing as \"c-fos\", although the term is erroneously used throughout the scientific literature. The gene in the mouse is Fos (first letter capitalized, italicized) and the mouse protein is FOS. Please use one or the other.", "reviewer_3": "The authors investigated the role of GABAergic NTS neurons modulating sleep-wake states and explored the mechanisms underlying satiety related sleep. They show that GI signals sensed by nodose ganglion (NG) cells which then pass this information to the hindbrain NTS regions in the control of sleep-wake states. While it’s interesting to understand hunger and sleep control by the central nervous system interacting with gut, the hypothesis and strategies and organization of the manuscript are not yet in a good shape.\nMany issues arise as the authors firstly need to enhance relevant research progress and background of the studies with apparent important literature to be cited, and secondly the organization and the presentation of the figures are in a mass making them very hard to follow and understand, there is a lack of logic. Finally, the verification of the injections and ablation of cells are not robust enough.\n1. Discussion for the feeding and sleeping research related to GABAergic neurons in the NTS is necessary in the introduction section.\n2. In figure 1 (and other figures, too), many panels are not labeled, therefore it’s hard to logically understand the data. For instance, it could be difficult to understand that panel B may contain Fasted and also Refed recordings, in panel C it could be hard to follow that it does include WAKE, NREM and REM three separate conditions that are meant to be in one panel. Also by the way, in this C panel the Y axis of REM condition could have been adjusted so that readers to understand the difference better between fasted and refed conditions.\n3. In figure 2, the best would be NG cells innervating stomach/intestine are ablated, so it needs to demonstrate first whether CCK NG cells fall into this category, if yes then maybe SAP-CCK would be appropriated to use, and second the authors need to verify the efficacy of the ablation of CCK NG cells, the same stays true for the AAV-DIO-Caspase3 strategy for which that representatives the authors provided (in the supplement) are very hard to determine the NG cells were ablated.\n4. In figure 3 panel B, it’s very hard to understand if there is a difference or not with the Wake, NREM and REM ratios. It would be great to separate activating DREADD and inhibitory DREADD data in this figure and other figures too, so that it’s easier to read and understand.\n5. Figure 4, because many refed activated NTS neurons are not GABAergic as shown in the panels A and B, therefore it has to be cautious to call that those GABAergic neurons are more important in control of the wake-sleep conditions in the satiety mice. Also, it’d be great to label what panels represent vGlut2 AI14 and which is for vGat AI14. It again shows in the panel C that only around 20% of the HSV positive cells in NTS are GABAergic, the result informs that most of the HSV positive cells are not GABAergic in NTS, which could weaken the authors argument that NTS GABAergic neurons are important."}, "author_response": "Dear Reviewers,\nWe sincerely appreciate your thoughtful and constructive comments, which have been invaluable in improving the quality and clarity of our manuscript. In response, we have carefully revised the manuscript following your suggestions in a point-by-point manner. The introduction, methods, results, and discussion sections were revised according to the suggestions, and some of the figures were rearranged to facilitate understanding, with the corresponding figure legends updated accordingly. All textual changes in the revised manuscript are highlighted in yellow for ease of reference.\nReviewer #1 (Remarks to the Author):\nChen et al. used optogenetics and chemogenetics neural manipulation and claimed that increased NREM sleep in post-fasting refeeding is caused by decreasing activity in the PVH through the GABAergic innervation from NTS and input from the nodose ganglion to NTS. The manuscript is well written, and the control groups and statistics are sound. Although the finding is interesting and advances our knowledge in this field of research, it could be improved by considering the following points.\nComments\n1. It is not clear what kind of signals by refeeding, activate the nodose ganglion. It would be interesting to clarify what signals are involved, such as physical signals from the stomach wall, blood glucose level changes, etc. Please also indicate whether this pathway is also functional during normal feeding behavior.\nResponse: Thank you very much for your suggestions. During feeding, gastric vagal sensory neurons projecting to the nodose ganglion are activated by multiple types of signals. Physical cues from the stomach wall, such as tension and tactile stimuli as the stomach accommodates food, stimulate vagal sensory neurons via mechanosensors, particularly subtypes of IGLEs within the myenteric plexus that express GLP1R or OXTR. In parallel, chemical cues contribute as nutrients entering the intestine—including glucose, fatty acids, and amino acids—and activate vagal sensory neurons either through direct sensing at vagal terminals or via enteroendocrine cell-mediated pathways. Hormonal signals further modulate this activity: the presence of food in the gut induces release of CCK, PYY, and GLP1, which act on their respective receptors expressed by vagal terminals in the submucosal layer of the intestinal wall1, 2.\nRefeeding after acute food restriction is expected to engage the same pathways, although the magnitude of activation may be altered. Short-term fasting has been shown to reduce the mechanosensitivity of gastric tension receptors, resulting in delayed satiety signaling and compensatory increases in food intake3. In addition, fasting decreases circulating levels of hormones that promote satiety and maintain metabolic homeostasis, including leptin, insulin, CCK, GLP1, and PYY4-7, which predominantly activate or sensitize vagal sensory neurons8, 9-12. While levels of ghrelin increase, which inhibits gastric vagal sensory neuron activity13. These could lead to a potential reduction in the baseline activity of these neurons. Fasting also alters the expression of peptide receptors located at the terminals of gastric vagal sensory neurons, particularly G-protein-coupled receptors14-17, providing an additional layer of modulation of vagal sensory neuron activity under fasting conditions. Evidence indicates that refeeding can rapidly restore circulating hormones (eg. Leptin，insulin, CCK, GLP1 6, 18, 19, etc) and the expression of certain peptide receptors20, implying that vagal sensory neuron activation may differ between normal feeding and post-fasting refeeding. Further electrophysiological studies are needed to demonstrate the differences in vagal sensory neuron responses between normal feeding and refeeding after acute fasting.\nWe have revised our description in the introduction according to your suggestions, please see lines 56-64.\n2. In p11, the reason to focus on the analysis of the GABAergic innervation of the NTS is relatively weak, not to study the glutamatergic innervation enough since c-Fos is clearly expressed in the glutamatergic neurons in Figure 4A. Also, the authors are using Gad2-cre mice, but they need to confirm whether these are indeed GABAergic neurons or not. Recent studies have shown that some neurons express Gad 1 or 2 but do not express Vgat and express vGlut instead; these neurons act as glutamatergic neurons. This means gad2-cre might not be suitable to label GABAergic neurons in this brain region. For confirmation, please use Vgat-Cre, stain with Vgat antibody, or show these neurons are not co-expressed with vGlut.\nResponse: We greatly appreciate your thoughtful suggestions regarding the rationale for investigating GABAergic neurons in the NTS and clarifying their identity.\nIndeed, in the NTS both glutamatergic and GABAergic neurons are activated during normal feeding as well as refeeding after acute fasting21, 22. With respect to glutamatergic neurons in the NTS, extensive work has examined their role in regulating energy balance, including Lepr+, Calcr+, Glp-1r+ and CCK+ subpopulations23-26. Interestingly, a recent study reported that CART⁺ glutamatergic neurons in the NTS are activated by elevations in blood pressure and promote NREM sleep27. Collectively, these observations indicate that glutamatergic neuron subpopulations in the NTS are primarily involved in energy homeostasis, with a subpopulation also participating in sleep modulation. In contrast, the functional role of GABAergic neurons in the NTS has been less well studied. Given that a substantial proportion of GABAergic neurons is also activated by refeeding, we initially focused on investigating their role.\nTo more precisely examine whether Gad2+ neurons in the NTS co-express vglut2 and to determine the identities and proportion of GABAergic neurons receiving direct input from gastric vagal sensory neurons, we combined HSV-mediated transsynaptic labeling with single-nucleus RNA sequencing. After mice were injected with HSV-EGPF into the upper gut, we dissected the dorsal vagal complex (DVC) and sent it for single nucleus RNA sequencing. We captured a total of 11,167 DVC cells, and after excluding area postrema (AP) and dorsal motor nucleus of the vagus (DMV) neurons based on established marker genes28. A clear segregation of Slc17a6⁺ (vglut2⁺) excitatory neurons and Gad2⁺ inhibitory neurons within the NTS was illustrated by co-expression of feature plot. We further validated such segregation by in situ hybridization. Using similar strategies, we also analyzed the co-expression of Slc32a1(Vgat) and vglut2. Both snRNA-seq data and in situ hybridization results showed that among neurons expressing Vgat and Vglut2, less than 10% co-express both (Please see ppt file named” additional figures for reviewer 1” for the results). These results indicate that in the NTS, the vast majority of GABAergic neurons (Gad2+ and Vgat+) do not co-release glutamate. In addition, our data showed that the NTS inhibitory neurons consisted largely of Gad2⁺ neurons (68.7%), of which 41.1% (443 of 1079 cells) co-expressed Vgat. Conversely, 54.1% (443 of 818 cells) of Vgat⁺ neurons co-expressed Gad2. These results indicate that in the NTS, Gad2⁺ and Vgat⁺ inhibitory populations partially overlap. Notably, previous studies have reported that, in the mid brain dorsal raphe nucleus, the Gad2⁺ and Vgat⁺ GABAergic subpopulations could exert opposing effects on sleep29-31. Therefore, while our study focused on Gad2⁺ GABAergic neurons, future work could examine the role of NTS Vgat⁺ GABAergic neurons in sleep modulation.\nFurthermore, we determined the identities and proportions of GABAergic and glutamatergic neurons receiving input from gastric vagal input, we isolated neurons infected by HSV-EGFP based on the presence of the EGFP-encoding sequence (717 bp) from the single-nucleus RNA sequencing data. Of these, 55.16% were GABAergic (Gad1⁺/Gad2⁺/Slc32a1⁺), 40.18% were glutamatergic, and 4.66% were cholinergic (Chat⁺) (Figure 4J). Among the GABAergic neurons, 282 were Gad2⁺, 193 were Gad1⁺, and 143 were Vgat⁺, indicating that Gad2⁺ GABAergic neurons receive the most projections from vagal sensory input innervating the upper gut. We confirmed the projection using Gad2-Cre-Ai14 mice with the HSV-EGFP transsynaptic labeling (Figure 4K). These results together provided us strong rational to investigate the role of Gad2+ GABAergic neurons in regulation of postprandial sleep.\nWe have revised the manuscript to include single-nucleus RNA sequencing data and in situ hybridization results, along with the corresponding results and discussion. Please refer to lines 454–489 and 688–702.\n3. In p12, please show the physiological validity of using 30Hz stimulation. It is stated that activation of glutamatergic neurons with optogenetics causes epilepsy, simply because the firing rate of baselines is different for glutamatergic neurons and GABA neurons, and 30Hz may be too strong for glutamatergic neurons\nResponse: Thank you for bringing up this point. We also considered this possibility during the experimental design and tested the responses under both 3 Hz and 10 Hz optogenetic stimulation. The animals exhibited varying levels of seizure-like behaviors, including neck jerks, forelimb trembling, and seizures while lying on their belly (please see the attached file named “additional figures for reviewer 1”). These observations suggest that the effects are more likely due to the involvement of NTS glutamatergic neurons in modulating seizure-like behaviors, rather than overstimulation per se. Moreover, previous studies have also applied 30 Hz optogenetic stimulation to a subset of NTS glutamatergic neurons and demonstrated their role in blood pressure regulation and sleep27. Further experiments are warranted to delineate the specific role and circuit mechanisms of NTS glutamatergic neurons in mediating seizure-like behaviors.\n4. In p9, please clearly indicate what time in the light/dark cycle the 16-hour fasting experiment.\nResponse: Thank you for your suggestion. Animals were housed in groups of five under a 12-hour light–dark cycle (lights on at 07:00 and off at 19:00) with ad libitum access to food and water. For the fasting condition, mice were deprived of food for 16 hours starting at 16:00 on the previous day, and food was reintroduced at 08:00 the following morning. The fasting mainly occurred during the dark cycle.\nWe have added the description for the fasting condition in the Materials and Methods section. Please see lines 121-122.\n5. In Discussion, There is not much discussion of the effect on REM sleep, but the reason why the effect on REM sleep are not significant may simply be due to the small sample size or the fact that the experiment was conducted at a time of day with a small percentage of REM, so at least discuss the possibility a little more。\nResponse: Thank you for your precious suggestion. The main reason that we did not observe significant changes in REM sleep was due to the time and duration of recording. Studies have shown that the proportion of REM in bursts was lowest at the first 2 hours of the light period (ZT2) and higher at the end of the dark period (ZT20)32. Most of our EEG data were collected at 9:00-10:00 (ZT2-3) for three hours. This could be the main reason why we did not observe significant changes in REM sleep.\nWe have modified the discussion according to your suggestions. Please see lines 648-652.\nReviewer #2 (Remarks to the Author):\nThis study describes a gut-brain pathway that regulates sleep, potentially providing a mechanism to account for the sleep-inducing effects of food intake. The topic is important and the paper presents some interesting data. However, there are basic problems with several of the experiments that must be addressed before this would be suitable for publication in any journal.\nMajor points\n1. There are several problems with the chemogenetic experiments in Fig. 2 and elsewhere. (1) They all require genotype controls (mice injected with fluorophore AAV, treated with CNO). This is particularly important because CNO (rather than DCZ) was used in these experiments, which is less specific and could modulate sleep. (2) The hM4D-CNO experiments must be compared to hM4D-saline in the same animals - it is not appropriate to compare them solely to hM3D-saline or CNO in different animals. (3) It is not clear exactly how the experiment was performed in Fig. 2, but the logical test is to measure whether activation with hM3D rescues sleep in fasted mice, and whether silencing with hM4D blocks increased sleep in fed mice, as in Fig. 3. (4) The statistical analysis of the chemogenetic data in Fig. 3 is unclear. First, the hM3D and hM4d experiments should be analyzed separately, and compared to their own both drug and genotype controls. Second, you cannot use a two-way ANOVA when you are simultaneously varying three parameters (genotype, drug treatment, nutritional state). Further, for the all of the chemogenetic and optogenetic data, the significance of differences in means between any two specific experiments has be tested using a test that is corrected for multiple comparisons, not just a t-test.\nResponse: We thank the reviewer for raising very constructive suggestions to help improve the manuscript.\n(1) Suggestions about proper controls\nRegarding genotype controls, we sincerely appreciate your suggestion. We have now included control animals for hM4D (AAV-DIO-hM4D–injected mice treated with saline) in Figures 2 and 3, along with additional mice in the hM4D group in parallel to minimize potential batch effects. We agree that comparing CNO and saline treatments within the same animals would be ideal. However, in our study we used different animals for the following reasons: (1) the experiments were designed in parallel so that saline- and CNO-injected groups were tested under identical conditions, including the same ZT time at the onset of recording and the same environment, thereby minimizing potential variations across different testing sessions; and (2) the parallel design helped avoid possible carryover effects from repeated injections or recording sessions in the same animals, which could otherwise confound the interpretation of CNO-specific effects. This parallel approach has also been adopted in previous sleep studies using chemogenetic manipulation33, 34. We will take your suggestion into careful consideration for future experimental design.\n(2) Concerns about the effects of CNO on sleep\nIndeed, CNO itself has been reported to modulate sleep, including suppression of REM sleep and changes in sleep architecture, such as prolonged maximum NREM episode duration and reduced NREM occurrence. However, these effects were observed only at doses of 5 mg/kg or higher. Even at 5 mg/kg, the ratio and total duration of Wake and NREM sleep during the 2 hours post-injection were not significantly affected35, 36. At the dosage used in our study (1 mg/kg), CNO does not seem to elicit significant changes in sleep, as we have tested in AAV-DIO-mCherry–injected mice (revised Figure 2F and G ). In addition, this dosage has been used in sleep studies using chemogenetic manipulations and consistently shown to have no effects on Wake/NREM/REM ratio or duration, number of bouts, or the number of state transitions33, 37, 38.\n(3) Concerns about experimental design clarity (Figure 2 and 3)\nThank you for this insightful suggestion. In the original submission (Figure 3), we examined hM3D activation in both fasted and refed mice. We found that in fasted mice, activation of Vagalstom/duo neurons also reduced wake duration and increased NREM sleep, further confirming the promotive role of gastric vagal afferents in sleep. In the revised Figure 3, we have included the genotype controls for hM4D and reorganized the data presentation to separately show the effects of hM3D and hM4D under both fasted and refed conditions. Please see the revised Figure 3. We also revised the description in the results accordingly. Please see lines358-383 and lines 408-436.\n(4) Concerns about statistical analysis\nThank you for your valuable advice. We agree that the original presentation of Figure 3 may have caused confusion. In the revised version, we reorganized the figure and analyzed the hM3D and hM4D experiments separately, each compared to their respective treatment and genotype controls, as suggested. We also included additional hM4D mice in parallel to minimize potential batch effects. All statistical analyses in Figure 3 have been updated accordingly. Specifically, for the chemogenetic experiments: (1) The ratios of different sleep stages (Wake, NREM, and REM) were analyzed using Three-way repeated-measure ANOVA, with CNO treatment as independent variable 1, nutritional state as independent variable 2, and testing time as independent variable 3 (repeated measure). (2) The total durations of different sleep stages were presented as bar graphs and analyzed using two-way ANOVA, with CNO treatment as independent variable 1 and nutritional state as independent variable 2. Statistical significance for all ANOVA tests was determined using Bonferroni post hoc correction. Please see the revised Figure 3. We have revised the results and figure legend accordingly. Please see lines 408-436 and lines 879-922.\nFor the optogenetic and chemogenetic data presented in other figures, the statistical analysis was performed as follows: (1) Line charts (e.g., ratios of different sleep stages, Figure 2C upper panel): analyzed with two-way RM ANOVA, followed by Bonferroni post hoc correction. (2) Single bar graphs (e.g., total sleep stage duration, Figure 2C lower panel): analyzed using two-tailed t-tests. (3) Combined bar graphs (e.g., bout duration/bout occurrence, Supplementary Figure 1B–C): Although the bout duration and the number of bouts of different sleep stages were displayed within the same bar graph, comparisons were not made between the different sleep stages. Therefore, t-tests were used to assess the treatment effects of CNO. This approach to data presentation and analysis has also been applied in previous studies39-41.\n2. Existing evidence suggests that GABAergic projections from NTS to forebrain are extremely sparse, so it is surprising that the authors claim strong projections to the PVH and BNST. This is a particular concern because (1) The tracing data in Fig. 7 is not convincing -- the images are very zoomed in and the axonal fibers appear to actually label surrounding structures more strongly than PVH, (2) the use of GAD2-Cre is problematic, because in other brain regions (e.g. hypothalamus) it has been shown that GAD2 is expressed in many neurons that are glutamatergic, and (3) there is a lack of convincing data showing that the viral injections are confined to NTS. It is very possible to label GABAergic neurons in structures adjacent to the NTS to give the appearance of spurious projections. For example, the histology in Fig. 5a is very zoomed in, whereas Fig. 5I appears to show infection primarily in DMV, not NTS, whereas Fig. 6I clearly labels AP as well. The authors should (1) confirm the specificity of the GAD2 driver by showing there is no overlap with Vglut2 in NTS, (2) confirm the projections to PVH and BNST exist using tracing with Vgat-Cre, with comprehensive histological data showing the injections are confined to the NTS and that the projections specifically label the downstream structures claimed (i.e. with more zoomed out images and replication). (3) change the nomenclature throughout the text to make it clear that the functional manipulations in the brain target the dorsal vagal complex (DVC), not the NTS specifically, and refrain from making claims (in the title, abstract or elsewhere) about the function of GABAergic NTS neurons specifically, since the injections are not restricted to the NTS. (4) Provide histological documentation of the spread of viral infection in every mouse used for manipulations. This can included in the supplement.\nResponse: We sincerely appreciate your constructive suggestions.\n(1) Concerns about tracing data (Supplementary Fig 7) and projections from NTS GABAergic neurons to the PVH and BNST:\nWe thank the reviewer for pointing out the limitations in our original tracing data. While NTS GABAergic neurons have been primarily studied for their local inhibitory networks that regulate autonomic functions such as cardiovascular and respiratory control42-44, recent studies indicate that they also form long-range projections. For example, using Gad2-Cre-Ai14 mice, study revealed their projections to PVH and BNST, further confirmed by CTB-mediated antegrade tracing45. Additionally, whole-brain mapping using Vgat-Cre mice demonstrated that PVH is indeed a target of NTS GABAergic neurons46.\nTo strengthen our evidence and directly address the reviewer’s concern, we employed HSV-mediated monosynaptic antegrade tracing in both Gad2-Cre and Vgat-Cre mice47. Cre-dependent AAV helper expressing glycoprotein gK (AAV2/9-DIO-mCh-gK) was injected into the NTS, followed three weeks later by H129-dgK-G4. Because gK expression was restricted to the NTS, the tracer selectively labeled only the monosynaptic targets of NTS GABAergic neurons. GFP-labeled neurons were consistently observed in both PVH and BNST in Gad2-Cre and Vgat-Cre mice (see Supplementary Fig. 6B and file named “additional figures for reviewer 2”), confirming monosynaptic projections to these regions.\nIn response to the reviewer’s request for more convincing histology, we have now replaced the original Supplementary Fig. 7A with a less zoomed-in image that clearly shows the location of NTS GABAergic axonal fibers in PVH. Together, these data provide stronger support that NTS GABAergic neurons form direct projections to PVH and BNST.\n(2) Concerns about the specificity of using Gad2-cre mice (co-expression of Gad2 and vglut2):\nWe fully understand the reviewer’s concern, as several studies have reported that Gad2 can be expressed in vglut2⁺ glutamatergic neurons in other brain regions48. To directly assess this issue in the NTS, we combined HSV-mediated transsynaptic labeling with single-nucleus RNA sequencing to determine the identities and proportions of GABAergic neurons, and to confirm whether they receive direct input from gastric vagal sensory neurons. Following HSV-EGFP injection into the upper gut, we dissected the dorsal vagal complex (DVC) for single-nucleus RNA sequencing. From a total of 11,167 captured DVC cells, we excluded AP and DMV neurons based on established marker genes28. Within the NTS population, Slc17a6⁺ (vglut2⁺) excitatory neurons and Gad2⁺ inhibitory neurons formed clearly segregated clusters. Feature plots revealed that only 5.8% of NTS neurons (124 of 2132 cells) co-expressed both genes, while the vast majority showed distinct segregation. This segregation was independently validated by in situ hybridization, which demonstrated a clear separation of Slc17a6⁺ and Gad2⁺ populations, with an overlap rate of 8.47%. These results are consistent with previous single-nucleus RNA sequencing studies showing that glutamatergic and GABAergic NTS neurons segregate into distinct clusters22, 28, 49, 50. These findings support that in the NTS, Gad2 and vglut2 co-expression is minimal. Furthermore, the Gad2-Cre line used in this study has been shown to drive reporter expression almost exclusively in GABAergic neurons across the brain, including brainstem regions51. Based on these data, we are confident that our Gad2-Cre–based experiments in the NTS reliably target GABAergic neurons.\nWe have incorporated the new single-nucleus RNA sequencing and in situ hybridization results into Figure 4, and revised the Results and Discussion sections accordingly (lines 454-489 and 688-702).\n(3) Concerns about spread expression of virus and suggestions about changing the nomenclature from NTS to DVC.\nWe thank the reviewer for these thoughtful comments. In response, we have provided histological images of viral expression for mouse used in the manipulations related to NTS viral injections ( please see file named “Additional figures for reviewer 2”). The NTS, AP, and DMV are in very close anatomical proximity, making some spread of viral expression into neighboring regions unavoidable.\nIn the DMV, the majority of neurons are cholinergic, and GABAergic neurons are very sparse to absent in this region52, 53. Functional inhibition of DMV neurons is therefore mediated largely by GABAergic inputs arising outside the DMV, most prominently from NTS GABAergic neurons54, 55. This could explain the presence of mCherry/EGFP fluorescence observed in the DMV.\nWe did notice viral expression in the area postrema (AP) in some cases. Because AP contains a substantial GABAergic population, we complemented the chemogenetic experiments with optogenetic manipulations in which optic fibers were restricted to the NTS, thereby minimizing potential contributions from AP GABAergic neurons. While chemogenetic and optogenetic activation did not produce identical outcomes, these differences can be explained in part by variable spread of viral expression into AP. In addition, AP area has not been reported to project to PVH or BNST45, 56, 57. At last, projection-specific optogenetic manipulations yielded consistent results, supporting the interpretation that NTS GABAergic neurons and their downstream targets are primarily responsible for the observed effects on sleep.\nTaken together, these data strengthen our confidence that the observed effects reflect the activity of NTS GABAergic neurons and their downstream projections, rather than GABAergic neurons in the broader DVC.\n3. For Fig. 2A, there needs to be direct immunohistochemical validation of the efficiency of vagal afferent ablation with CCK-SAP. Showing changes in Fos in NTS is not sufficient.\nResponse: We sincerely appreciate your critical suggestions. We now include results verifying the efficiency of ablation anatomically as previously reported58. We injected the retrograde tracer CTB into the stomach wall of SAP- or SAP-CCK–injected mice. The number of CTB-labeled neurons in the nodose ganglion was significantly reduced by 68.6 % in SAP-CCK–treated animals compared with SAP controls (t-test, p < 0.001). The results are now presented in Figure 1H, and the Results section has been updated accordingly (Please see lines 325-334).\nMinor points\n1. Many of the manipulations target innervation of both stomach and intestine, and therefore the text should make this clear (as opposed to referring solely to \"gastric\" afferents). CCK is expressed in many intestinal innervating vagal afferents.\nResponse: Thank you for this helpful suggestion. We would like to clarify that SAP-CCK was used only once at the beginning, to verify that the effects of NTS activation after refeeding were mediated by vagal inputs from the gastrointestinal tract. This provided the rationale to further investigate the role of gut-innervating vagal inputs in sleep regulation. We then separately examined vagal sensory neurons innervating the stomach and duodenum (Vagalstom/duo neurons) versus those innervating the more distal small intestine (VagalsInt neurons). Our results demonstrated that the sleep-modulating effects were primarily mediated by activation of Vagalstom/duo neurons. For this reason, we initially used the term “gastric input.” In light of the reviewer’s comment, we have revised the wording throughout the manuscript and replaced “gastric afferents” with “upper gut–innervating vagal sensory input.”\n2. There is no such thing as \"c-fos\", although the term is erroneously used throughout the scientific literature. The gene in the mouse is Fos (first letter capitalized, italicized) and the mouse protein is FOS. Please use one or the other.\nResponse: We sincerely appreciate the reviewer’s suggestion. We have revised the manuscript to consistently use the correct nomenclature, referring to the it as Fos.\nReviewer #3 (Remarks to the Author):\nThe authors investigated the role of GABAergic NTS neurons modulating sleep-wake states and explored the mechanisms underlying satiety related sleep. They show that GI signals sensed by nodose ganglion (NG) cells which then pass this information to the hindbrain NTS regions in the control of sleep-wake states. While it’s interesting to understand hunger and sleep control by the central nervous system interacting with gut, the hypothesis and strategies and organization of the manuscript are not yet in a good shape.\nMany issues arise as the authors firstly need to enhance relevant research progress and background of the studies with apparent important literature to be cited, and secondly the organization and the presentation of the figures are in a mass making them very hard to follow and understand, there is a lack of logic. Finally, the verification of the injections and ablation of cells are not robust enough.\n1. Discussion for the feeding and sleeping research related to GABAergic neurons in the NTS is necessary in the introduction section.\nResponse: Thank you for your valuable suggestions; we fully agree with your points. However, we did not find any literature reporting the role of NTS GABAergic neurons in sleep regulation, which represents one of the novel findings of our study. We did identify studies demonstrating the important role of GABAergic neurons in regulating feeding behaviors, including postprandial feeding inhibition21 and the mediation of liraglutide-induced suppression of feeding59. In line with your advice, we have revised the introduction accordingly (see lines 95-98).\n2. In figure 1 (and other figures, too), many panels are not labeled, therefore it’s hard to logically understand the data. For instance, it could be difficult to understand that panel B may contain Fasted and also Refed recordings, in panel C it could be hard to follow that it does include WAKE, NREM and REM three separate conditions that are meant to be in one panel. Also by the way, in this C panel the Y axis of REM condition could have been adjusted so that readers to understand the difference better between fasted and refed conditions.\nResponse: We greatly appreciate your thoughtful suggestions. To improve the clarity and interpretability of the figures, we have made the following changes across all figures:\n(1) Panel labeling: We have added proper labels to all panels to clearly indicate the conditions and measurements. Please see the revised Figures\n(2) Data grouping: We have combined the ratio of WAKE/NREM/REM sleep and the total duration of these brain states into a single group. In each figure, the upper panels now show the curve of the ratio across three-hour recordings, while the lower panels display the corresponding bar graph of the total duration.\n(3) Y-axis adjustment: The Y-axis for REM has been adjusted to match the maximum range of REM values, allowing for better comparison between conditions (e.g., Fasted vs. Refed).\n3. In figure 2, the best would be NG cells innervating stomach/intestine are ablated, so it needs to demonstrate first whether CCK NG cells fall into this category, if yes then maybe SAP-CCK would be appropriated to use, and second the authors need to verify the efficacy of the ablation of CCK NG cells, the same stays true for the AAV-DIO-Caspase3 strategy for which that representatives the authors provided (in the supplement) are very hard to determine the NG cells were ablated.\nResponse:\nThank you for raising this important point. Several studies have demonstrated that CCK-expressing NG neurons innervate the stomach and intestine, and SAP-CCK injection directly into the nodose ganglion has been established as an effective approach for selectively ablating this population. This method achieves up to ~80% reduction in CCK NG neurons58, 60. A key advantage of SAP-CCK ablation is that it selectively eliminates vagal afferents from the upper gut while sparing vagal efferent neurons. Accordingly, SAP-CCK has been widely applied in studies investigating vagal sensory neuron function10, 61-63.\nIn our study, we verified the efficacy of SAP-CCK ablation anatomically as previously reported58. First, we injected the retrograde tracer CTB into the stomach wall of SAP- or SAP-CCK–injected mice. The number of CTB-labeled neurons in the nodose ganglion was significantly reduced by ~68.6% in SAP-CCK–treated animals compared with SAP controls (t-test, p < 0.01). These results are now presented in Figure 1G, and the Results section has been updated accordingly (Please see lines 325-334).\nRegarding the AAV-DIO-Caspase3 strategy, we quantified the ablation efficacy and found that approximately 83.68% of NG sensory neurons were eliminated. These quantifications have been incorporated into Figure 2H, and the Results section has been revised accordingly (Please see lines 358-369).\n4. In figure 3 panel B, it’s very hard to understand if there is a difference or not with the Wake, NREM and REM ratios. It would be great to separate activating DREADD and inhibitory DREADD data in this figure and other figures too, so that it’s easier to read and understand.\nResponse: We sincerely appreciate your valuable advice and fully agree with your suggestion. In Figure 3, we have now separated the activating (hM3Dq, Figure 3B-E) and inhibitory (hM4Di, Figure 3F-I) DREADD data, which makes the differences in Wake, NREM, and REM easier to interpret. We have included control animals for hM4D (AAV-DIO-hM4D–injected mice treated with saline) in Figures 3, along with additional mice in the hM4Di group in parallel to minimize potential batch effects. We have also applied the same strategy to the other relevant figures by presenting the effects of chemogenetic activation and inhibition separately, along with their corresponding control groups. The Results section has also been revised accordingly to the updated figures (Please see lines 408-436).\n5. Figure 4, because many refed activated NTS neurons are not GABAergic as shown in the panels A and B, therefore it has to be cautious to call that those GABAergic neurons are more important in control of the wake-sleep conditions in the satiety mice. Also, it’d be great to label what panels represent vGlut2 AI14 and which is for Vgat AI14. It again shows in the panel C that only around 20% of the HSV positive cells in NTS are GABAergic, the result informs that most of the HSV positive cells are not GABAergic in NTS, which could weaken the authors argument that NTS GABAergic neurons are important.\nResponse: We greatly appreciate your thoughtful suggestions regarding the importance of studying GABAergic neurons in the NTS.\nIndeed, in the NTS both glutamatergic and GABAergic neurons are activated during normal feeding as well as refeeding after acute fasting21, 22. With respect to glutamatergic neurons in the NTS, extensive work has examined their role in regulating energy balance including Lepr+, Calcr+, Glp-1r+ and CCK+ subpopulations23, 24, 25, 26. To date, these studies have primarily characterized the functions of NTS glutamatergic neurons in energy homeostasis.\nUsing HSV-mediated transsynaptic labeling combined with single-nucleus RNA sequencing, we observed a clear segregation of Slc17a6⁺ (vglut2⁺) excitatory neurons and Gad2⁺ inhibitory neurons within the NTS. Further analysis revealed that NTS GABAergic neurons receive projections from gastric vagal sensory inputs that are comparable to, if not greater than, those received by NTS glutamatergic neurons (revised Figure 4), underscoring their potential role in regulating postprandial behaviors. Using both chemogenetic and optogenetic manipulations, we provided evidence that NTS GABAergic neurons could participate in the modulation of NREM sleep. However, we do not exclude the role for glutamatergic subpopulations in modulating postprandial sleep, as a recent study reported that CART⁺ glutamatergic neurons in the NTS are activated by increases in blood pressure and promote NREM sleep27. Further experiments are needed to identify the specific glutamatergic neuron subpopulations involved in this modulation.\nThe observation that only ~20% of HSV-labeled neurons were co-localized with tdTomato⁺ cells in Gad2-Ai14 mice may be due to the limited efficiency of Cre-mediated recombination. Previous characterizations of GABAergic Cre lines demonstrated that, although Cre activity is highly specific, recombination efficiency can vary considerably across brain regions51, 64. In addition, tdTomato fluorescence may not always be detectable when expression levels are low, even if the mRNA is present, which could further contribute to the underestimation of co-localization.\nWe have updated Figure 4 with proper labeling and incorporated single-nucleus RNA sequencing data along with in situ hybridization results to strengthen our rational on investigating role of NTS GABAergic neurons in modulating postprandial sleep. The Results and Discussion sections have been revised accordingly to align with the updated figures. Please see lines 454–489 and 688–702."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This is a revised manuscript by Chen et al. They adequately addressed and responded to this reviewer’s concerns. Now the manuscript is improved. I appreciate snRNA-sequencing data. The authors mentioned that “In addition, our data showed that the NTS inhibitory neurons consisted largely of Gad2⁺ neurons (68.7%), of which 41.1% (443 of 1079 cells) co-expressed Vgat. Conversely, 54.1% (443 of 818 cells) of Vgat⁺ neurons co-expressed Gad2.” This means there is 58.8% of Gad2+ cells do not express Vgat. What exactly do these neurons release? And can they be called GABAergic neurons?", "reviewer_2": "I have no further comments. The manuscript can be accepted.", "reviewer_3": "The authors have now made significant changes to satisfy the previous questions."}, "author_response": "Dear Reviewers,\nWe sincerely appreciate your thoughtful and constructive comments, which have greatly improved the clarity and overall quality of our manuscript. We have carefully addressed Reviewer 1’s comment.\nReviewer #1 (Remarks to the Author):\nThis is a revised manuscript by Chen et al. They adequately addressed and responded to this reviewer’s concerns. Now the manuscript is improved. I appreciate snRNA-sequencing data. The authors mentioned that “In addition, our data showed that the NTS inhibitory neurons consisted largely of Gad2⁺ neurons (68.7%), of which 41.1% (443 of 1079 cells) co-expressed Vgat. Conversely, 54.1% (443 of 818 cells) of Vgat⁺ neurons co-expressed Gad2.” This means there is 58.8% of Gad2+ cells do not express Vgat. What exactly do these neurons release? And can they be called GABAergic neurons?\nResponse: We thank the reviewer for raising such an insightful yet highly important question. We had the same concerns when we got the single-nucleus data. Therefore, we actually performed a triple in situ hybridization for Gad2, Vglut2 and Vgat to confirm the identity of these neurons, during the revision process (please see the following images). We did observe neurons co-expressing Gad2 and Vglut2 but lacking Vgat, which appeared to cluster in the ventromedial region of the NTS, near the DMV. Quantitative analysis showed that approximately 69.1 ± 5.91% of Gad2⁺ cells expressed Vgat, while 21.1 ± 3.71% of Gad2⁺ cells co-expressed Vglut2. These results indicate that the majority of Gad2⁺ neurons in the NTS can be classified as GABAergic. Since we noticed that the Gad2⁺/Vglut2⁺ neurons were spatially condensed, suggesting they may possess distinct projections and functions, which we plan to investigate in future studies.\nReviewer #2 (Remarks to the Author):\nI have no further comments. The manuscript can be accepted.\nResponse: We sincerely appreciate your time and constructive comments, which have greatly helped us improve the manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors have now made significant changes to satisfy the previous questions.\nResponse: We sincerely appreciate your time and constructive comments, which have greatly helped us improve the manuscript."}]

Decision letter