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. 2025 Nov 18;46(6):1501–1515. doi: 10.24272/j.issn.2095-8137.2025.068

Whole-brain mapping of monosynaptic afferents to GABAergic neurons in the sublaterodorsal tegmental nucleus of mice

Zhi-Gang Zhong 1,2, Shang-Qi Tang 1,2, Hui Ben 1,2, Jia-Lin Yang 1, Yong-Hua Chen 4, Wei-Min Qu 2, Zhi-Li Huang 2,3,*, Mei-Hong Qiu 1,2,*
PMCID: PMC12940752  PMID: 41320871

Abstract

The sublaterodorsal tegmental nucleus (SLD) is a critical hub for regulating rapid eye movement (REM) sleep and muscle atonia, with its dysfunction linked to various disorders such as REM sleep behavior disorder (RBD) and cataplexy. Despite its physiological significance, the presynaptic patterns influencing SLD γ-aminobutyric acid (GABA)ergic neurons—one of the primary neuronal subtypes within the SLD—remain poorly understood. This study applied a modified rabies virus tracing system combined with a Cre/loxP-based genetic approach to map and quantify the whole-brain monosynaptic afferents to SLD GABAergic neurons in mice. In total, 139 anatomically distinct nuclei were identified as sources of direct input, with predominant projections originating from the midbrain, pons, and medulla. Ipsilateral contributions accounted for 67.99% of all traced inputs, while 32.01% were contralateral. Prominent sources included the mesencephalic reticular nucleus, superior colliculus, oral part of the pontine reticular nucleus, gigantocellular reticular nucleus, lateral hypothalamic area, and zona incerta neurons. Several nuclei displayed contralateral projection biases. Immunofluorescence staining revealed molecular diversity among input neurons, suggesting that SLD GABAergic neurons integrate signals from anatomically and functionally distinct neuronal populations. These findings provide a comprehensive anatomical framework for understanding how SLD GABAergic neurons integrate multisource inputs and offer new perspectives for investigating their involvement in regulating complex physiological functions, including sleep and motor control.

Keywords: Sublaterodorsal tegmental nucleus, GABAergic neurons, Monosynaptic inputs, RBD, Cataplexy

INTRODUCTION

The sublaterodorsal tegmental nucleus (SLD), located rostral and ventral to the laterodorsal tegmental nucleus (LDTg) and ventromedial to the parabrachial nucleus (PB) (Clément et al., 2011; Krenzer et al., 2011; Shen et al., 2020), functions as a key integrative center involved in the regulation of diverse physiological and behavioral functions, including the modulation of anxiety-like behaviors, defensive aggression, processing of negative emotional memory, and the orchestration of rapid eye movement (REM) sleep and muscle atonia (Fan et al., 2023; Peever & Fuller, 2017; Saper et al., 2010; Vetrivelan & Bandaru, 2023; Wen et al., 2023). The SLD is composed of two predominant neuronal populations: glutamatergic and γ-aminobutyric acid (GABA)ergic neurons (Boissard et al., 2002; Clément et al., 2011; Kashiwagi et al., 2020, 2024; Wen et al., 2023). While glutamatergic neurons have been extensively implicated in REM sleep generation and muscle atonia, the functional contribution of GABAergic neurons remains less clear and somewhat controversial. Evidence supporting a REM-on role for SLD GABAergic neurons includes elevated c-Fos expression during REM sleep recovery and reciprocal inhibition with GABAergic neurons in the ventrolateral periaqueductal gray (VLPAG) (Lu et al., 2006). In contrast, juxtacellular recordings and calcium imaging indicate higher activity of many SLD GABAergic neurons during wakefulness than during REM sleep, suggesting functional heterogeneity (Boucetta et al., 2014; Cissé et al., 2020). Disruption of this neuronal subset has minimal influence on REM sleep architecture or atonia but alters circadian timing and REM onset dynamics, indicating a potential modulatory function in sleep regulation (Krenzer et al., 2011; Wen et al., 2023).

Previous investigations into the connectivity of SLD neurons have relied primarily on conventional tract-tracing techniques. Retrograde tracers such as Fluoro-Gold and cholera toxin subunit B (CTB), as well as anterograde tracers like Phaseolus vulgaris leucoagglutinin and adeno-associated viruses (AAV) expressing fluorescent protein, have revealed extensive afferent projections from regions such as the mesencephalic reticular nucleus (mRt), VLPAG, and lateral hypothalamic area (LH) (Boissard et al., 2003; Chen et al., 2022; Feng et al., 2020, 2024; Liang et al., 2014; Weber et al., 2018), and efferent targets such as the gigantocellular reticular nucleus (Gi), dorsal raphe nucleus (DR), and ventral spinal horn (Boissard et al., 2002; Krenzer et al., 2011; Lu et al., 2006). Although these studies have advanced understanding of SLD circuitry, they lack the specificity and resolution required to delineate the brain-wide presynaptic architecture of genetically defined neuronal populations. A comprehensive map of afferent projections targeting SLD GABAergic neurons across the entire brain has, until now, remained unavailable.

Recent advances in monosynaptic rabies trans-neuronal retrograde tracing, combined with the Cre/loxP genetic strategies, have revolutionized the mapping of whole-brain presynaptic connections to specific neuronal populations (Chen et al., 2019; Pollak Dorocic et al., 2014). In the present study, this approach was employed in vesicular GABA transporter (Vgat)-Cre mice to generate a whole-brain map of presynaptic inputs to SLD GABAergic neurons. Modified rabies virus tracing was used in combination with immunofluorescence staining to characterize the molecular phenotypes of input neurons originating from regions associated with behavioral state regulation. Furthermore, ipsilateral and contralateral projection patterns were analyzed to assess lateralization in afferent connectivity. These findings yield a high-resolution anatomical atlas of presynaptic inputs to SLD GABAergic neurons and establish a neuroanatomical framework for investigating their roles in sleep regulation and other physiological processes.

MATERIALS AND METHODS

Animals and ethics statement

Adult male Vgat-Cre mice (12–16 weeks, 25–30 g) (Jackson Laboratory, Stock No. 017535), derived from the C57BL/6J background and optimized for specific targeting of whole-brain GABAergic neurons, were used for viral retrograde tracing experiments. Animals were housed under a controlled 12:12 h light/dark cycle (lights on at 0700h; light intensity 100 lux), with ambient temperature maintained at 22°C±1°C and relative humidity at 60%±5%. Food and water were provided ad libitum. All procedures were approved by the Committee on the Ethics of Animal Experiments at the School of Basic Medical Science at Fudan University (Permit number: 20190221-097) and were conducted in compliance with relevant guidelines and regulations. Every effort was made to minimize animal pain and discomfort.

Viral vectors and stereotaxic surgery

Three viral vectors were used in this study, including AAV2/9-EF1α-DIO-RVG, AAV2/9-EF1α-DIO-H2B-TVA-EGFP, and RV-EnVA-∆G-dsRed (BrainVTA, China). Viral titers were 5×1012 vg/mL for both AAV vectors and 2×108 vg/mL for the EnvA-pseudotyped glycoprotein (RG)-deleted and DsRed-expressing rabies virus (RV-EnVA-∆G-dsRed). Prior to injection, AAV2/9-EF1α-DIO-RVG and AAV2/9-EF1α-DIO-H2B-TVA-EGFP were mixed in a 1:1 ratio, yielding a final concentration of 2.5×1012 vg/mL.

Vgat-Cre mice were anesthetized with 1.5%–2% isoflurane and secured in a stereotaxic apparatus (RWD Instruments, China) following confirmation of eyelid reflex loss. Stereotaxic coordinates for the SLD were determined based on the fourth edition of Paxinos’s Mouse Brain Atlas (Paxinos, 2013). After aseptic preparation, a midline scalp incision was made to expose the skull, and overlying connective tissue was carefully removed. A micro-craniotomy was performed above the SLD, and 20 nL of the AAV-helper virus mixture was injected unilaterally at a rate of 4 nL/min using a micropipette (coordinates: anteroposterior –5.2 mm, mediolateral +0.75 mm, dorsoventral –4.0 mm). The micropipette was left in place for another 5 min to ensure diffusion before being slowly retracted. Two weeks later, 40 nL of RV-ENVA-∆G-dsRed was injected into the same coordinates using the same procedures. Mice were allowed to survive for 7 days following rabies virus injection prior to perfusion.

Histology and immunofluorescence staining

Mice were deeply anesthetized with 1.5%–2% isoflurane and perfused transcardially with 40 mL of ice-cold saline, followed by 60 mL of 4% paraformaldehyde (PFA; P804536; Macklin, China) in 0.1 mol/L phosphate buffer (PB, pH 7.4). After perfusion, brains were extracted, post-fixed in 4% PFA for 6–8 h at 4°C, and cryoprotected in 30% sucrose until fully saturated. Tissue was sectioned coronally at 30 µm using a cryostat (CM1950; Leica, Germany) into four serial sets and stored at –20°C in cryoprotectant solution.

For immunofluorescence staining, free-floating sections were washed three times in 0.01 mol/L phosphate-buffered saline (PBS) and incubated overnight at 4°C with primary antibodies diluted in PBS containing 2.5% Triton X-100 (T9284; Sigma-Aldrich, USA). Primary antibodies included rabbit anti-GABA (A2052; 1:1 000; Sigma-Aldrich, USA), mouse anti-glutamate decarboxylase 1 (GAD67; 1:500; Millipore, USA), rabbit anti-glutamate (G6642; 1:1 000; Sigma-Aldrich, USA), goat anti-choline acetyltransferase (ChAT; AB144P; 1:2 000; Millipore, USA), rabbit anti-serotonin (S5545; 1:1 000; Sigma-Aldrich, USA), goat anti-parvalbumin (PV; PVG213; 1:1 000; Swant, USA), and rabbit anti-orexin A (ab6214; 1:2 000; Abcam, UK). The sections were then washed and incubated for 1.5 h at room temperature with appropriate secondary antibodies (1:1 000; Life Technologies, USA), including Alexa Fluor 488 donkey antibody against rabbit IgG, Alexa Fluor 647 donkey antibody against mouse IgG, Alexa Fluor 488 donkey antibody against goat IgG, and Alexa Fluor 647 donkey antibody against goat IgG. Sections were then rinsed, mounted on glass slides, and cover-slipped with DAPI Fluoromount-GTM (0100-20; Southern Biotech, USA).

Imaging and quantification of retrograde labeling

Only mice with verified and efficient infection of starter neurons in the SLD (n=6) were selected for whole-brain imaging and input quantification. Coronal sections were scanned using a high-resolution virtual microscopy slide scanning system (VS120; Olympus, Japan) with a 10× objective, and images were saved in TIFF format for analysis. To determine the distribution and abundance of DsRed-labeled neurons, each image was manually aligned to the corresponding coronal layer of the Mouse Brain Atlas (Paxinos, 2013) via Adobe Illustrator. Brain subregion boundaries were delineated, and DsRed-positive neurons were manually identified based on somatic morphology and fluorescence intensity. Serial sections were sampled every 90 µm, spanning from bregma +1.42 mm to –7.20 mm (75.5±1.96 sections per animal). Labeled neurons were bilaterally counted within each anatomically defined brain region. Counts for each region were summed across sections and expressed as mean±standard error of the mean (SEM). The proportion of total brain-wide inputs contributed by each region was calculated as a percentage of the total number of DsRed-positive neurons (Supplementary Data S1). Input regions were categorized based on input density: numerous (>4%), substantial (3%–4%), moderate (2%–3%), or sparse (1%–2%). Regions contributing <1% input were reported in the Supplementary Data but excluded from density-based classification. Full quantification data, including raw counts and regional percentages, are provided in Supplementary Data S1.

RESULTS

Monosynaptic input mapping of GABAergic neurons in the SLD

A cell type-specific retrograde tracing strategy was applied in transgenic Vgat-Cre mice using an RG-deleted rabies virus to map the brain-wide monosynaptic inputs to GABAergic neurons in the SLD. This approach enabled precise identification of direct monosynaptic input neurons with high specificity. Notably, no starter cells or retrogradely labeled neurons were observed in wild-type control mice (Chen et al., 2019; Taniguchi et al., 2011). Neurons expressing the TVA receptor were labeled with the enhanced green fluorescent protein (EGFP), while those infected by the rabies virus were labeled with DsRed. Dual-labeled starter neurons (EGFP/DsRed) were strictly confined to the SLD. Their anatomical localization was verified by immunostaining for choline acetyltransferase (ChAT) to delineate the adjacent LDTg (Figure 1). Only samples in which starter neurons were confined to the SLD (n=6) were aligned to the Mouse Brain Atlas for subsequent brain-wide and region-specific quantification of presynaptic neurons (expressing DsRed only). DsRed single-labeled neurons were distributed across multiple distant brain regions and also detected within the SLD itself, indicating that local GABAergic neurons provide direct recurrent input, consistent with intra-SLD connectivity (Figure 1E). To verify the neurochemical identity of starter cells, immunofluorescence staining for GAD67 was performed, confirming the specificity of the viral labeling strategy for GABAergic neurons (Supplementary Figure S1). Additionally, no EGFP signal colocalized with ChAT-positive neurons, further confirming the specificity of the labeling strategy for GABAergic populations.

Figure 1.

Figure 1

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Trans-synaptic rabies retrograde tracing of monosynaptic inputs to SLD GABAergic neurons

A: Schematic overview of experimental strategy. Adeno-associated viruses (AAV)-helper viruses and rabies glycoprotein deleted rabies virus (RV) were sequentially injected into the sublaterodorsal tegmental nucleus (SLD) of Vgat-Cre mice to trace monosynaptic inputs to GABAergic neurons. B: Schematic coronal section illustrating viral infection in the SLD. Starter neurons (co-expressing EGFP and DsRed) are shown in yellow, AAV helper virus-infected neurons are shown in green, and RV-infected neurons are shown in red. C: Representative immunofluorescence image of the SLD region from a Vgat-Cre mouse, showing starter neurons, presynaptic afferent neurons (DsRed, red), and cholinergic neurons (ChAT, pink) in the laterodorsal tegmental nucleus (LDTg). Nuclei are labeled with DAPI (blue). White box indicates region enlarged in (E). D: Representative immunofluorescence image from a wild-type (non-Cre control mouse, demonstrating absence of both starter cells and retrogradely labeled neurons, confirming Cre-dependence and specificity of the tracing system. E: High-magnification photomicrographs of white rectangular region in C. White arrowhead marks a DsRed single-labeled neuron and yellow arrow points to a starter neuron. 4V, fourth ventricle; 5N, motor trigeminal nucleus; DTg, dorsal tegmental nucleus; scp, superior cerebellar peduncle. Scale bars: 100 μm (C), 25 μm (E).

Whole-brain distribution of monosynaptic inputs to SLD GABAergic neurons

A comprehensive brain-wide analysis was performed using serial coronal sections from six mice with confirmed restriction of starter neurons to the SLD. A total of 139 anatomically defined input regions projecting directly to SLD GABAergic neurons were identified, spanning the telencephalon, diencephalon, midbrain, pons, medulla, and cerebellum (Figures 2, 3A; Supplementary Data S1). Input distributions were highly conserved across individuals, as indicated by strong spatial correlations (ipsilateral, 0.907±0.008; contralateral, 0.933±0.007, n=6) (Figure 3A, B), underscoring the reproducibility of the input architecture. The brainstem constituted the dominant source of input (82.24%±0.92%), with the midbrain contributing the largest fraction (46.80%±0.85%), followed by the medulla (20.37%±1.37%) and pons (15.07%±1.26%). Additional projections arose from the diencephalon (11.50%±0.69%) and telencephalon (5.25%±0.36%), with the cerebellum providing sparse input (1.01%±0.21%) (Figure 3C).

Figure 2.

Figure 2

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Monosynaptic inputs to SLD GABAergic neurons across the whole brain

Representative coronal sections (+1.42 mm to –7.20 mm relative to bregma) from a single mouse, illustrating the distribution of monosynaptic input neurons to SLD GABAergic neurons. Red dots indicate injection site in the SLD. Brain regions housing these input neurons were denoted in accordance with the Mouse Brain Atlas for precise localization and identification. M2, secondary motor cortex; M1, primary motor cortex; Cg1, cingulate cortex, area 1; LPO, lateral preoptic; MPA, medial preoptic area; LHb, lateral habenular nucleus; ZI, zona incerta; LH, lateral hypothalamus; CeA, central amygdaloid nucleus; PF, fascicular nucleus; PSTh, parasubthalamic nucleus; p1Rt, p1 reticular formation; APT, anterior pretectal nucleus; ml, medial lemniscus; SC, superior colliculus; mRt, mesencephalic reticular nucleus; SNr, substantia nigra, reticular part; SNc, substantia nigra, compact part; DK, nucleus of Darkschewitsch; VTA, ventral tegmental area; Su3, supraoculomotor; PnO, pontine reticular nucleus; MnR, median raphe nucleus; DR, dorsal raphe nucleus; PAG, periaqueductal gray; MPL, medial paralemniscal nucleus; RtTg, reticulotegmental nucleus of the pons; LDTg, laterodorsal tegmental nucleus; SPTg, subpeduncular tegmental nucleus; VLPAG, ventrolateral periaqueductal gray; Su5, supratrigeminal nucleus; SubCV, subcoeruleus nucleus, ventral part; PB, parabrachial nucleus; PnC, pontine reticular nucleus, caudal part; SLD, sublaterodorsal tegmental nucleus; RIP, raphe interpositus nucleus; LC, locus coeruleus; Bar, Barrington’s nucleus; Ve, vestibular nucleus; IRt, intermediate reticular nucleus; RMg, raphe magnus nucleus; Gi, gigantocellular reticular nucleus; LPGi, lateral paragigantocellular nucleus; Lat, lateral cerebellar nucleus; Med, medial cerebellar nucleus; DPGi, dorsal paragigantocellular nucleus; GiA, gigantocellular reticular nucleus, alpha part; PCRt, parvicellular reticular nucleus; Pr, prepositus nucleus; PMn, paramedian reticular nucleus. Scale bar: 1 mm.

Figure 3.

Figure 3

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Distribution of presynaptic inputs to SLD GABAergic neurons across the whole brain

A: Line graph showing percentage distribution of DsRed-positive presynaptic neurons across different brain regions along the rostrocaudal axis. Inputs are plotted separately for ipsilateral (top) and contralateral (bottom) hemispheres, with distinct colors representing different cases (n=6). Brain regions are color-coded: telencephalon (green), diencephalon (light purple), midbrain (blue), pons (yellow), medulla (light pink), and cerebellum (purple). B: Heatmaps depicting spatial correlation of input distributions across different animals for ipsilateral (left) and contralateral (right) hemispheres. Color scale represents degree of correlation, with warmer colors indicating higher correlations. C: Percentage of total DsRed-positive neurons distributed across major brain regions in the whole brain (left), ipsilateral hemisphere (middle), and contralateral hemisphere (right). Each dot represents an individual animal. Data are presented as mean±SEM. (n=6). Source data are provided as Supplementary Data S1.

To provide a more detailed depiction of the whole-brain monosynaptic input regions to SLD GABAergic neurons, representative images from several subregions were selected (Figure 4), including cingulate cortex, area 1 (Cg1), secondary motor cortex (M2), primary motor cortex (M1), medial preoptic area (MPA), and lateral preoptic area (LPO) in the telencephalon; lateral habenular nucleus (LHb), LH, ZI, and parasubthalamic nucleus (PSTh) in the diencephalon; p1 reticular formation (p1Rt), mRt, superior colliculus (SC), nucleus of Darkschewitsch (DK), substantia nigra, compact part (SNc), substantia nigra, reticular part (SNr), ventral tegmental area (VTA), supraoculomotor (Su3), pontine reticular nucleus, oral part (PnO), paramedian raphe nucleus (PMnR), DR, VLPAG, LPAG, and medial paralemniscal nucleus (MPL) in the midbrain; LDTg, pontine reticular nucleus, caudal part (PnC), raphe interpositus nucleus (RIP), locus coeruleus (LC), and Barrington’s nucleus (Bar) in the pons; lateral paragigantocellular nucleus (LPGi), Gi, raphe magnus nucleus (RMg), gigantocellular reticular nucleus, alpha part (GiA), prepositus nucleus (Pr), dorsal paragigantocellular nucleus (DPGi), gigantocellular reticular nucleus, ventral part (GiV), intermediate reticular nucleus (IRt), and parvicellular reticular nucleus (PCRt) in the medulla; and lateral cerebellar nucleus (Lat) in the cerebellum.

Figure 4.

Figure 4

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Typical images of presynaptic inputs to SLD GABAergic neurons across different brain regions

Fluorescent micrographs showing DsRed-positive neurons (red) across various brain regions, indicating presynaptic input to SLD GABAergic neurons. Brain regions are outlined with dashed lines. Cg1, cingulate cortex, area 1; M2, secondary motor cortex; M1, primary motor cortex; MPA, medial preoptic area; LPO, lateral preoptic; LHb, lateral habenular nucleus; LH, lateral hypothalamus; ZI, zona incerta; PSTh, parasubthalamic nucleus; p1Rt, p1 reticular formation; mRt, mesencephalic reticular nucleus; SC, superior colliculus; InG, intermediate layer of the superior colliculus; InWh, intermediate white layer of the superior colliculus; DpG, deep gray layer of the superior colliculus; DK, nucleus of Darkschewitsch; SNc, substantia nigra, compact part; SNr, substantia nigra, reticular part; VTA, ventral tegmental area; Su3, supraoculomotor; Su3C, supraoculomotor cap; PnO, pontine reticular nucleus, oral part; PMnR, paramedian raphe nucleus; PAG, periaqueductal gray; Aq, aqueduct; DR, dorsal raphe nucleus; LPAG, lateral periaqueductal gray; VLPAG, ventrolateral periaqueductal gray; MPL, medial paralemniscal nucleus; LDTg, laterodorsal tegmental nucleus; PnC, pontine reticular nucleus, caudal part; RIP, raphe interpositus nucleus; Bar, Barrington's nucleus; LC, locus coeruleus; LPGi, lateral paragigantocellular nucleus; Gi, gigantocellular reticular nucleus; RMg, raphe magnus nucleus; GiA, gigantocellular reticular nucleus, alpha part; DPGi, dorsal paragigantocellular nucleus; Pr, prepositus nucleus; 4V, 4th ventricle; Lat, lateral cerebellar nucleus; GiV, gigantocellular reticular nucleus, ventral part; IRt, intermediate reticular nucleus; PCRt, parvicellular reticular nucleus. Scale bar: 200 μm.

To further elucidate the distribution and relative contributions of input sources to SLD GABAergic neurons, 73 brain nuclei were identified in which either ipsilateral or contralateral projections exceeded 0.2% of total input across six major brain regions (Figure 5). Afferent neurons were predominantly distributed within the midbrain (33 nuclei), followed by the pons (14 nuclei), medulla (10 nuclei), diencephalon (7 nuclei), and telencephalon (7 nuclei), with sparse input also detected from two cerebellar nuclei. Among these monosynaptic inputs, 67.99%±0.86% of DsRed-labeled neurons were located ipsilaterally, while 32.01%±0.86% were located contralaterally (Figures 3A, B, 6C).

Figure 5.

Figure 5

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Statistical analysis of ipsilateral and contralateral inputs to SLD GABAergic neurons

Proportions of presynaptic inputs to SLD GABAergic neurons from various brain regions, expressed as a percentage of total inputs. Red bars indicate ipsilateral inputs, orange bars indicate contralateral inputs. Brain regions exhibiting a significant contralateral bias (>30% more than ipsilateral inputs) are marked with an asterisk. Data are presented as mean±SEM, n=6. Brain regions are color-coded: telencephalon (green), diencephalon (light purple), midbrain (blue), pons (yellow), medulla (pink), and cerebellum (purple). M2, secondary motor cortex; CeA, central amygdaloid nucleus; M1, primary motor cortex; LPO, lateral preoptic; BNST, bed nucleus of the stria terminalis; MPA, medial preoptic area; Cg1, cingulate cortex, area 1; ZI, zona incerta; LH, lateral hypothalamus; LHb, lateral habenular nucleus; PH, posterior hypothalamic nucleus; PF, parafascicular thalamic nucleus; PSTh, parasubthalamic nucleus; PeF, perifornical nucleus; DM, dorsomedial hypothalamic nucleus; VM, ventromedial thalamic nucleus; mRt, mesencephalic reticular nucleus; SC, superior colliculus; LPAG, lateral periaqueductal gray; PnO, pontine reticular nucleus, oral part; VLPAG, ventrolateral periaqueductal gray; DR, dorsal raphe nucleus; p1Rt, p1 reticular formation; SNr, substantia nigra, reticular part; VTA, ventral tegmental area; MPL, medial paralemniscal nucleus; APT, anterior pretectal nucleus; SNc, substantia nigra, compact part; PTg, pedunculotegmental nucleus; PrCnF, precuneiform area; RPC, red nucleus, parvicellular part; PMnR, paramedian raphe nucleus; DMPAG, dorsomedial periaqueductal gray; PR, prerubral field; xscp, decussation of the superior cerebellar peduncle; DK, nucleus of Darkschewitsch; InC, interstitial nucleus of Cajal; PAG, periaqueductal gray; Su3C, supraoculomotor cap; Su3, supraoculomotor; RRF, retrorubral field; MN, mammillary nucleus; mlf, medial longitudinal fasciculus; PaR, prerubral nucleus; SPTg, subpeduncular tegmental nucleus; Pa4, paratrochlear nucleus; CnF, cuneiform nucleus; MnR, median raphe nucleus; PnC, pontine reticular nucleus, caudal part; LDTg, laterodorsal tegmental nucleus; PB, parabrachial nucleus; LC, locus coeruleus; Pr5, principal sensory trigeminal nucleus; SLD, sublaterodorsal tegmental nucleus; SubCV, subcoeruleus nucleus, ventral part; Bar, Barrington’s nucleus; Su5, supratrigeminal nucleus; DMTg, dorsomedial tegmental area; SubCD, subcoeruleus nucleus, dorsal part; CG, central gray; RtTg, reticulotegmental nucleus of the pons; RIP, raphe interpositus nucleus; Gi, gigantocellular reticular nucleus; PCRt, parvicellular reticular nucleus; IRt, intermediate reticular nucleus; Ve, vestibular nucleus; LPGi, lateral paragigantocellular nucleus; Sp5, spinal trigeminal nucleus; GiA, gigantocellular reticular nucleus, alpha part; DPGi, dorsal paragigantocellular nucleus; Sol, solitary nucleus; Pr, prepositus nucleus; Lat, lateral cerebellar nucleus; Med, medial cerebellar nucleus. Source data are provided as Supplementary Data S2.

The midbrain represented the dominant input source. Of the 33 contributing midbrain nuclei with monosynaptic connections to SLD GABAergic neurons, nine had a proportion exceeding 1% of total labeled neurons (Figures 5, 6A). The mRt provided the strongest ipsilateral projections (4.73%±0.28%), followed by the SC (3.12%±0.34%). For contralateral inputs, the most substantial projections were from the SC (3.64%±0.39%) and PnO (3.33%±0.36%). In the pons, the PnC exhibited substantial bilateral input, with a contralateral predominance (ipsilateral 1.59%±0.24%, contralateral 2.06%±0.23%). In the medulla, the most prominent input to SLD GABAergic neurons originated from the Gi (ipsilateral 3.49%±0.23%, contralateral 2.12%±0.06%). In contrast to the brainstem, projections from the diencephalon and telencephalon were less abundant. The most prominent diencephalic inputs originated from the ipsilateral LH (3.47%±0.21%) and ZI (3.19%±0.35%), while all seven telencephalic and both cerebellar nuclei each contributed less than 1% of total input (Figure 5).

Figure 6.

Figure 6

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Summary of ipsilateral and contralateral monosynaptic inputs to SLD GABAergic neurons

A, B: Schematic sagittal brain maps depicting distribution of ipsilateral (A) and contralateral (B) monosynaptic inputs to SLD GABAergic neurons. Strength of input from each region is categorized as numerous (>4%, dark red), substantial (3%–4%, red), moderate (2%–3%, light red), or sparse (1%–2%, pale red). Green dot represents SLD injection site. C: Pie chart showing proportion of ipsilateral (67.99%±0.87%) and contralateral (32.01%±0.87%) inputs across the brain. Data are presented as mean±SEM, n=6. DPGi, dorsal paragigantocellular nucleus; DR, dorsal raphe nucleus; Gi, gigantocellular reticular nucleus; IRt, intermediate reticular nucleus; LDTg, laterodorsal tegmental nucleus; LH, lateral hypothalamus; LPAG, lateral periaqueductal gray; mRt, mesencephalic reticular nucleus; p1Rt, p1 reticular formation; PCRt, parvicellular reticular nucleus; PMnR, paramedian raphe nucleus; PnC, pontine reticular nucleus, caudal part; PnO, pontine reticular nucleus, oral part; SC, superior colliculus; SLD, sublaterodorsal tegmental nucleus; SNr, substantia nigra, reticular part; VLPAG, ventrolateral periaqueductal gray; VTA, ventral tegmental area; ZI, zona incerta. Source data are provided as Supplementary Data S1, S2.

Several nuclei exhibited marked contralateral dominance (Figure 5). Within the midbrain, the PnO and PMnR projected more densely to the contralateral SLD, with contralateral-to-ipsilateral ratios of 1.56 and 1.83, respectively. In the pons, the PnC showed a similar bias (1.30). Although projections from the DPGi and Pr in the medulla were relatively sparse, they displayed pronounced contralateral lateralization (DPGi=3.11; Pr=2.25). Notably, the two cerebellar nuclei innervating SLD GABAergic neurons were also dominated by contralateral innervation, with the proportion of DsRed-positive neurons in the contralateral Lat being 42.86% higher than that on the ipsilateral side.

To visualize the spatial architecture of afferent inputs, all contributing nuclei were grouped into four density categories and color-coded on a sagittal brain map: numerous (>4%), substantial (3%–4%), moderate (2%–3%), and sparse (1%–2%). This visualization highlighted that the most robust projections to SLD GABAergic neurons were concentrated in the midbrain, medulla, and pons (Figure 6).

Immunofluorescence characterization of DsRed-labeled neurons using markers associated with behavioral state regulation

Immunofluorescence staining revealed that monosynaptic inputs to SLD GABAergic neurons colocalized with multiple neurochemical markers associated with diverse physiological functions, including REM muscle atonia, cataplexy, sleep-wake cycle, and motor control. In the medulla, input neurons from the Gi—a region involved in modulating motor activity—exhibited substantial colocalization with GABA (47.78%±1.94%; Figure 7A) and glutamate (34.55%±4.24%; Figure 7D) (Capelli et al., 2017; Lemieux & Bretzner, 2019). Additional analysis revealed that 37.36%±3.28% of input neurons from the GiA (Figure 7B) and 40.42%±4.10% from the GiV (Figure 7C) were identified as GABAergic, consistent with previous findings implicating these subregions in REM sleep, cataplexy, and muscle atonia (Uchida et al., 2021; Valencia Garcia et al., 2018; Zhong et al., 2019).

Figure 7.

Figure 7

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Neurochemical characterization of monosynaptic inputs to SLD GABAergic neurons

A–G: Representative immunofluorescence images showing colocalization of DsRed-labeled presynaptic neurons (red) with various neurochemical markers (green), including GABA (A–C), glutamate (Glu, D), serotonin (E), orexin (F), and parvalbumin (PV, G) in different brain regions. Nuclei are stained with DAPI (blue). Three right panels in each row show magnified views of white-boxed region in the leftmost image. Colocalized neurons are indicated by arrows in the rightmost images. Bar graphs on the right quantify percentage of colocalized neurons within each region. Data are presented as mean±SEM (n=4). DR, dorsal raphe nucleus; Gi, gigantocellular reticular nucleus; GiA, gigantocellular reticular nucleus, alpha part; GiV, gigantocellular reticular nucleus, ventral part; LH, lateral hypothalamus; SNr, substantia nigra, reticular part. Scale bar: 100 μm (leftmost images), 50 μm (three right panel images). Source data are provided as Supplementary Data S3.

Notably, a minor subset (22.71%±2.20%; Figure 7E) of DsRed-labeled neurons in the DR was identified as serotonergic, which have been shown to promote sleep in mice and zebrafish (Oikonomou et al., 2019). Within the LH, 12.03%±2.01% of input neurons were immunoreactive for orexin (Figure 7F). The LH operates as a central hub for behavioral state regulation, coordinating homeostatic and instinctive functions through widespread projections (Dawson et al., 2023; Gao & Horvath, 2014; Jennings et al., 2015; Viskaitis et al., 2024). Although orexin neurons are classically associated with arousal promotion (Adamantidis et al., 2007; De Luca et al., 2022), recent research indicates that a specific orexinergic subset projecting to the SLD may facilitate REM sleep initiation and alleviate REM sleep pressure (Feng et al., 2020, 2024). Dysregulation or loss of orexin neurons, peptides, or receptors has been directly linked to narcolepsy in both human and animal models (Chemelli et al., 1999; Chen et al., 2022; España et al., 2007; Hara et al., 2001; Uchida et al., 2021; Willie et al., 2003).

Unexpectedly, 23.77%±4.69% of DsRed-labeled neurons colocalized with parvalbumin in the SNr (Figure 7G), a region predominantly composed of GABAergic neurons and implicated in movement suppression and sleep regulation (Liu et al., 2020).

DISCUSSION

The SLD plays a pivotal role in regulating a broad spectrum of physiological functions, including REM sleep generation, muscle atonia, emotional processing, and motor control (Fan et al., 2023; Peever & Fuller, 2017; Vetrivelan & Bandaru, 2023; Wen et al., 2023). Elucidating its afferent architecture is essential for understanding the neural mechanisms underlying these functions and their dysregulation in neuropsychiatric and sleep disorders. Although earlier studies employing conventional tracers have outlined general connectivity patterns, the specific presynaptic organization targeting SLD GABAergic neurons remains poorly resolved. In this study, a genetically restricted rabies virus-based transsynaptic tracing strategy in Vgat-Cre mice enabled comprehensive identification of direct inputs to SLD GABAergic neurons. Afferent neurons were mapped across 139 distinct brain regions, encompassing the telencephalon, diencephalon, midbrain, pons, medulla, and cerebellum. Many of these input sources are involved in core behavioral and physiological domains, including arousal regulation, motor control, emotional processing, and muscle atonia. The input landscape was dominated by projections from the brainstem, particularly the midbrain, medulla, and pons, underscoring the strong integration of SLD GABAergic circuits within lower brain networks essential for state transitions and reflexive control. In addition, a prominent ipsilateral bias was observed, with a subset of nuclei—particularly within the midbrain and medulla—exhibiting marked contralateral projection asymmetry.

Comparison with previous retrograde tracing studies

To the best of our knowledge, only two prior investigations have systematically examined the afferent organization of the SLD (Boissard et al., 2003; Chen et al., 2022). In a foundational study, Boissard et al. (2003) employed CTB, a classical nonspecific retrograde tracer, and identified 38 distinct input nuclei projecting to the SLD, with prominent labeling from the mRt, PnC, VLPAG, PCRt, and LH. Although this study provided essential anatomical groundwork, the use of a non-cell-type-specific tracer limited resolution and introduced ambiguity due to potential labeling of fibers of passage. In contrast, the present study utilized a Cre-dependent monosynaptic rabies tracing approach in Vgat-Cre mice, enabling selective identification of direct inputs to GABAergic neurons within the SLD (Pollak Dorocic et al., 2014). This strategy not only corroborated previously reported projections but also uncovered a significantly expanded input network encompassing 139 anatomically defined nuclei, including novel afferents from cerebellar regions. Notably, contralateral projections were robustly represented in the current dataset, particularly from the PnO, PnC, PMnR, DPGi, Pr, and Lat. These findings highlight hemispheric asymmetries in the afferent architecture of SLD GABAergic neurons and suggest lateralized integration of sensorimotor signals.

A more recent study by Chen et al. (2022) employed an analogous rabies-based approach in Vglut2-Cre mice to selectively trace monosynaptic inputs to glutamatergic neurons in the SLD. Although substantial overlap in upstream regions was observed, notable distinctions emerged between the two neuronal populations. Chen et al. (2022) identified 50 nuclei exceeding a 0.2% input threshold, whereas the current analysis revealed a far more extensive input repertoire to GABAergic neurons (139 nuclei total), suggesting that GABAergic neurons may receive a broader array of modulatory and sensory inputs. Moreover, the pronounced contralateral projection bias evident in the GABAergic dataset—especially from brainstem and cerebellar structures—was absent in the glutamatergic dataset, suggesting functional divergence between these two neuronal subtypes. These results imply that GABAergic neurons may function as bilateral integrators of diverse behavioral-state and sensorimotor signals, while glutamatergic neurons may operate within more localized and lateralized circuits. Future studies combining transsynaptic tracing with functional labeling strategies or single-cell resolution analyses will be essential for clarifying whether these afferent pathways converge onto overlapping or segregated microcircuits within the SLD.

Circuit-level modulation of SLD GABAergic neurons in REM sleep behavior disorder (RBD)

RBD is a parasomnia marked by the loss of REM-associated muscle atonia and is widely recognized as a prodromal feature of neurodegenerative conditions (Antelmi et al., 2021; Claassen et al., 2010; Dauvilliers et al., 2018; Iranzo et al., 2006; Peever & Fuller, 2017; Saper et al., 2010; Sulaman et al., 2023; Vetrivelan & Bandaru, 2023). Glutamatergic neurons in the SLD have been shown to induce muscle atonia through activation of ventromedial medullary (vM) inhibitory neurons or spinal interneurons (Lu et al., 2006; Valencia Garcia et al., 2018). The present study revealed that GABAergic neurons in the SLD received dense monosynaptic inputs from local SLD neurons—likely glutamatergic—as well as from GABAergic neurons in the Gi, GiA, and GiV (Figure 1, Figure 6A–C). This anatomical arrangement suggests their involvement in REM atonia circuitry and provides new structural evidence implicating SLD GABAergic neurons in RBD-related dysfunction.

Neuroanatomical substrates linking SLD GABAergic neurons in cataplexy

Cataplexy, a defining feature of narcolepsy type 1, manifests as abrupt, emotionally triggered muscle atonia during wakefulness and arises from the degenerative loss or dysfunction of the orexin system, including its neurons, neuropeptides, or receptors (Chemelli et al., 1999; Chen et al., 2022; España et al., 2007; Hara et al., 2001; Peever & Fuller, 2017; Scammell, 2015; Uchida et al., 2021; Willie et al., 2003). The resemblance between cataplexy-related atonia and REM sleep paralysis has led to the hypothesis that cataplexy reflects aberrant recruitment of REM atonia pathways during waking states. Experimental activation of SLD glutamatergic neurons or vM GABA/glycine neurons elicits cataplexy-like episodes, while their inhibition attenuates these events in narcoleptic mice (Torontali et al., 2019; Uchida et al., 2021). Additional structures—including GABAergic neurons in the mRt and projections from the CeA—also modulate cataplexy occurrence (Chen et al., 2022; Li & Sheets, 2018; Mahoney et al., 2017; Snow et al., 2017; Torontali et al., 2019; Uchida et al., 2021). In the current study, SLD GABAergic neurons were shown to receive direct monosynaptic input from these cataplexy-related regions. Notably, approximately 12% of inputs from the LH co-expressed orexin, and nearly 40% of projections from the Gi, GiA, and GiV were GABAergic (Figure 7A–C). These data suggest that SLD GABAergic neurons integrate convergent emotional and arousal-related signals from orexinergic and inhibitory brainstem systems, and are thus strategically positioned to modulate cataplexy through aberrant engagement of REM-atonia circuitry during wakefulness.

Functional integration of SLD GABAergic neurons in sleep-wake regulation

Our findings revealed that SLD GABAergic neurons receive extensive inputs from multiple brain regions involved in sleep-wake regulation. These include: in the midbrain, GABAergic neurons in the mRt and VLPAG have been shown to suppress REM sleep and promote NREM sleep (Chen et al., 2022; Weber et al., 2018). While glutamatergic neurons in the adjacent PAG regulate NREM sleep through projections to the caudal medulla (Zhong et al., 2019). DR also provided prominent input, with serotonergic neurons promoting sleep and dopaminergic neurons enhancing wakefulness (Cho et al., 2017; Oikonomou et al., 2019). In the diencephalon, the LH contributed heterogeneous inputs. Melanin-concentrating hormone (MCH) neurons facilitate REM sleep, whereas orexinergic, glutamatergic, and GABAergic neurons promote arousal (Adamantidis et al., 2007; Arrigoni et al., 2019; De Luca et al., 2022; Yamashita & Yamanaka, 2017).The ZI also sent multiple projections to SLD GABAergic neurons. A subpopulation of Lhx6-expressing ZI GABAergic neurons enhance both NREM and REM sleep (Liu et al., 2017), suggesting a potential role of the ZI-SLD pathway in sleep regulation. Similarly, the vM, including GiA, GiV, and LPGi, contains GABAergic neurons that could induce NREM or REM sleep upon activation (Weber et al., 2015; Zhong et al., 2019). Given our findings that SLD GABAergic neurons received dense input from the vM, this circuit may be crucial for sleep-wake transitions.

Other key inputs included the LC, PB, LDTg, SNr, VTA, LPO, DPGi, and Pr, all of which support sleep-wake transitions and arousal regulation (Eban-Rothschild et al., 2016; Kashiwagi et al., 2024a; Kaur et al., 2013; Liang et al., 2021; Liu et al., 2020; Miracca et al., 2022; Qiu et al., 2019; Tossell et al., 2023; Van Dort et al., 2015; Yu et al., 2019). Additionally, the presence of DsRed single-labeled neurons within the SLD indicated local glutamatergic input to SLD GABAergic neurons. Since SLD glutamatergic neurons are preferentially active during REM sleep and vigorously promote its occurrence (Boucetta et al., 2014; Clément et al., 2011; Cox et al., 2016; Erickson et al., 2019; Torontali et al., 2019; Wen et al., 2023), this finding suggests a local circuit regulating REM sleep within the SLD. Altogether, these diverse inputs underscore the role of SLD GABAergic neurons in integrating signals related to arousal and sleep, potentially modulating the transition between sleep and wake states.

Potential role of SLD GABAergic neurons in motor control

SLD GABAergic neurons also received extensive monosynaptic inputs from motor-related regions, including the basal ganglia, midbrain, and caudal brainstem, implicating this population in the coordination of locomotion and behavioral state transitions. Prominent afferents arose from SNr, SNc, VTA, DR, SC, and caudal brainstem motor circuits.

As a principal output structure of the basal ganglia, the SNr consists predominantly of GABAergic neurons that exert powerful inhibitory control over motor execution and oculomotor behavior (Basso & Sommer, 2011; Hikosaka & Wurtz, 1985; Kravitz et al., 2010; Liu et al., 2020). Recent studies have identified distinct neuronal subpopulations within the SNr that play specialized roles in motor and sleep regulation. Specifically, PV-positive neurons are preferentially active during voluntary movement and facilitate its termination, whereas GAD2-positive neurons maintain tonic activity during sleep states and are suppressed during wake-related locomotion (Liu et al., 2020). In the present study, SLD GABAergic neurons received direct synaptic input from PV-positive SNr neurons, delineating a pathway through which basal ganglia circuits may influence motor suppression via the SLD. In parallel, monosynaptic inputs from the motor cortex to SLD GABAergic neurons further support the notion that the SLD plays an integrative role in motor control.

Dopaminergic neurons in the SNc and VTA also provided inputs to SLD GABAergic neurons. Given that dopaminergic degeneration in the midbrain is a hallmark of Parkinson’s disease and contributes to motor dysfunction (Carmichael et al., 2021; Gaertner et al., 2022), these projections raise the possibility that the SLD participates in dopaminergic regulation of motor circuits. Similarly, serotonergic neurons in the DR, which suppress locomotion through downstream projections to the orbitofrontal cortex and CeA (Correia et al., 2017; Ren et al., 2018), innervated SLD GABAergic neurons, indicating a serotonergic route for inhibition of motor activity. The midbrain SC, involved in visual processing and the initiation of rapid eye movements (saccades), also sent dense projections to SLD GABAergic neurons, suggesting that visual cues may influence motor output via this circuit.

Within the caudal brainstem, both glutamatergic and GABAergic neurons in the Gi, GiA, GiV, and LPGi projected to SLD GABAergic neurons. These caudal brainstem nuclei contain functionally heterogeneous neuronal subtypes that differentially regulate locomotion (Arber & Costa, 2022; Leiras et al., 2022). Inhibitory neurons in the Gi, GiA, or GiV suppress movement, whereas LPGi glutamatergic neurons sustain movement (Capelli et al., 2017; Lemieux & Bretzner, 2019).

Altogether, the input architecture of SLD GABAergic neurons supports a functional role in integrating motor commands from basal ganglia, midbrain dopaminergic and serotonergic systems, and medullary locomotor circuits. This integrative capacity may enable dynamic modulation of motor output in accordance with behavioral state and sensory context.

Potential interplay between GABAergic and glutamatergic neurons in the SLD

Although the present study focused on afferent inputs to SLD GABAergic neurons, emerging evidence suggests that functional crosstalk between GABAergic and glutamatergic neurons within the SLD may play a critical role in regulating REM sleep, atonia, and motor transitions. Glutamatergic neurons within the SLD constitute a well-established REM-on population that projects to spinal cord circuits to enforce muscle atonia during REM sleep (Lu et al., 2006; Peever & Fuller, 2017; Valencia Garcia et al., 2018). However, the timing and patterning of their activity may be shaped by intranuclear inhibitory mechanisms. Local GABAergic neurons are positioned to regulate the excitability or recruitment thresholds of glutamatergic neurons, thereby refining REM onset, offset, or motor suppression during state transitions.

Histological and electrophysiological studies support the presence of intranuclear inhibitory connections within the SLD (Boissard et al., 2003), and the detection of DsRed single-labeled neurons in the present study—likely reflecting local glutamatergic input to GABAergic neurons—further indicates the existence of reciprocal signaling between these two cell populations. Additionally, several afferent regions identified here, including the VLPAG, ZI, and GiA, are known to innervate both glutamatergic and GABAergic neurons within the SLD, suggesting coordinated or opposing regulatory influences. Chen et al. (2022) similarly reported abundant intranuclear DsRed-labeled neurons projecting to the SLD itself. These findings suggest that glutamatergic neurons, like GABAergic ones, receive substantial local input from neighboring neurons, supporting the notion that local microcircuit regulation is a general feature of the SLD neural architecture. Functionally, such cross-inhibition may help regulate transitions between REM and wake states, prevent premature motor output during REM, and coordinate emotional and locomotor information. Future experiments using cell-type-specific calcium imaging or optogenetics will be essential to dissect the dynamic interactions between these two SLD neuronal subtypes in behaving animals.

CONCLUSIONS

This study generated a comprehensive, brain-wide map of monosynaptic inputs to SLD GABAergic neurons, revealing extensive integration of signals from circuits governing sleep regulation, motor control, and emotional processing. The breadth of this connectivity highlights the pivotal role of SLD GABAergic neurons in orchestrating complex behaviors and physiological states. Future research should focus on elucidating the specific mechanisms through which these afferents influence SLD function and exploring their potential contributions to neurological conditions such as RBD, cataplexy, and Parkinson’s disease. A more complete understanding of SLD GABAergic circuit function will also require systematic characterization of their efferent projections. Anterograde tracing and cell-type-specific projection mapping will be critical for identifying downstream targets and clarifying how these outputs contribute to REM sleep atonia, motor inhibition, and state transitions.

SUPPLEMENTARY DATA

Supplementary data to this article can be found online.

zr-46-6-1501-S1.zip (2.1MB, zip)

Acknowledgments

COMPETING INTERESTS

The authors declare that they have no competing interests.

AUTHORS’ CONTRIBUTIONS

Z.G.Z., M.H.Q., S.Q.T., H.B., J.L.Y., and Y.H.C. performed the experiments and analyzed the data. M.H.Q and Z.G.Z. drafted the manuscript. M.H.Q. and Z.L.H. designed the experimental protocols. M.H.Q., W.M.Q., and Z.L.H. secured funds for the study and revised the manuscript. All authors read and approved the final version of the manuscript.

Funding Statement

This work was supported by the National Key Research and Development Program of China (2023YFC2306500), National Nature Science Foundation of China (81971239, 81771430), and National Major Project of China Science and Technology Innovation 2030 for Brain Science and Brain-Inspired Technology (2021ZD0203400)

Contributor Information

Zhi-Li Huang, Email: huangzl@fudan.edu.cn.

Mei-Hong Qiu, Email: mhqiu@shmu.edu.cn.

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