Whole‐brain monosynaptic inputs to lateral periaqueductal gray glutamatergic neurons in mice

Abstract Objective The lateral periaqueductal gray (LPAG), which mainly contains glutamatergic neurons, plays an important role in social responses, pain, and offensive and defensive behaviors. Currently, the whole‐brain monosynaptic inputs to LPAG glutamatergic neurons are unknown. This study aims to explore the structural framework of the underlying neural mechanisms of LPAG glutamatergic neurons. Methods This study used retrograde tracing systems based on the rabies virus, Cre‐LoxP technology, and immunofluorescence analysis. Results We found that 59 nuclei projected monosynaptic inputs to the LPAG glutamatergic neurons. In addition, seven hypothalamic nuclei, namely the lateral hypothalamic area (LH), lateral preoptic area (LPO), substantia innominata (SI), medial preoptic area, ventral pallidum, posterior hypothalamic area, and lateral globus pallidus, projected most densely to the LPAG glutamatergic neurons. Notably, we discovered through further immunofluorescence analysis that the inputs to the LPAG glutamatergic neurons were colocalized with several markers related to important neurological functions associated with physiological behaviors. Conclusion The LPAG glutamatergic neurons received dense projections from the hypothalamus, especially nuclei such as LH, LPO, and SI. The input neurons were colocalized with several markers of physiological behaviors, which show the pivotal role of glutamatergic neurons in the physiological behaviors regulation by LPAG.


| INTRODUC TI ON
The periaqueductal gray (PAG) is located in the midbrain and regulates multiple functions, including defensive, 1 social, 2 antinociceptive, 3 itch-scratching, 4 and emotional behaviors. 5From the dorsal to ventral directions, the region is divided into four function-specific columns, namely the dorsomedial, dorsolateral, lateral (LPAG), and ventrolateral (VLPAG) columns. 5,6 the lateral part of the PAG, the LPAG comprises a heterogeneous nucleus in terms of neurotransmitter types and mainly contains glutamatergic and GABAergic neurons.1][12] There may be differences in the regulatory functions of different types of neurons in the LPAG.For example, the LPAG Vgat neurons support prey search, chase, and attack behaviors; however, LPAG Vglut2 neurons are involved in supporting the attack behavior only. 12In addition, LPAG is also involved in sleep-wake regulation.A previous study reported that LPAG neurotensinergic neurons promote non-rapid eye movement (NREM) sleep. 13Although the LPAG can regulate many physiological behaviors, it is unclear which specific excitatory or inhibitory signals directly control LPAG.Therefore, identification of the whole-brain inputs to LPAG can help to better understand the regulation of LPAG activity in different behavioral processes.
Rabies virus (RV)-based retrograde tracers allow us to identify neuronal presynaptic connections 14,15 and can be used to overcome the limitations of previous studies.In this study, we identified whole-brain monosynaptic inputs to LPAG glutamatergic neurons using transgenic mice that express Cre recombinase in glutamatergic neurons and combined a genetically modified RV with Cre-LoxP technology.Our results revealed that 59 afferent brain nuclei, and several nuclei with the highest input density (such as LH, LPO, and VP), are involved in the regulation of physiological behaviors.In conclusion, our findings provided substantial evidence for the structural framework of LPAG glutamatergic neurons and may guide neuronal pathway studies of glutamatergic neuron function in the LPAG.

| Animals
Adult transgenic mice (10-12-week-old, 25-28 g) with Cre recombinase expression in glutamatergic neurons (Vglut2-Cre mice) of the C57BL/6J strain and wild-type littermates without Cre expression were used for retrograde tracing experiments.The mice were housed under an automatically controlled 12/12-h light/dark cycle (lights on at 7 a.m.; intensity of 100 lux) 16 at a constant temperature of 22 ± 0.5°C and relative humidity of 60% ± 2%.The mice were provided free access to food and water.All animal experiments in this study were approved by Laboratory Animal Model Department, Shanghai Public Health Clinical Center, Fudan University (Permit No. 2023-A008-01).

| Surgery and viral injections
The surgical procedures were performed according to previous studies. 17Briefly, the mice were anesthetized by intraperitoneal injection of chloral hydrate (350 mg/kg) and were fixed in a stereotaxic apparatus.Then, the mice skulls were exposed and a small hole was drilled.The viral vectors were microinjected into the unilateral LPAG via a micropipette.The coordinate of LPAG was as follows: −4.10 mm from bregma, 0.35 mm lateral from midline, and 2.30 mm vertical from the pial surface.For retrograde tracing, helper viruses (AAV-EF1α-DIO-TVA-EGFP and AAV-EF1α-DIO-RvG were mixed at a 1:1 ratio; 100 nL) were injected into the LAPG and left for 15 min to allow diffusion away from the injection site.Three weeks later, RV-EnvA-ΔRG-DsRed (50 nL) was microinjected into the same location (n = 4 mice).At week 4, the mice were perfused.

| Histology and immunostaining
One week after injection of the RV, the mice were deeply anesthetized and perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4).
Following perfusion, the brains were removed and post-fixed in 4% PFA for 4-6 h at 4°C and then cryoprotected in 10%, 20%, and 30% sucrose at 4°C until they sank.Coronal brain sections (30μm-thick) were cut on a freezing cryostat (CM1950; Leica, Wetzlar, Germany) into four series and were collected in 0.1 M PBS (pH 7.4).

| Imaging and data analysis
Whole-brain sections were imaged under 10× or 20× magnification with the VS120 virtual microscopy slide scanning system (Olympus).
A confocal microscope was used to obtain 20× or 40× magnified images of the brain sections to obtain more details (Olympus Fluoview 1000, Tokyo, Japan).For cell mapping of neurons, the ImageJ software was used to semi-automatically quantify the neuronal bodies.
Based on a map of the mouse brain, the boundaries of specific brain regions were depicted using ImageJ. 18We also used ImageJ to distinguish between the cells that co-expressed DsRed and GFP for starter cell mapping.Then, we generated cell representation by applying the automatic wand (tracing) tool and bicubic interpolation to maximize neuronal fidelity.Next, we inverted the colorless regions to white and matched the contours of the cells to the corresponding brain regions based on the mouse brain atlas.Starter cells were binned at 0.12 mm along the anterior-posterior axis, with each coronal section image centered at the brain slice.For axonal varicosity counting, the images were captured using a 20× objective on the Olympus VS120 system.The axonal varicosity values of the whole brain were calculated semi-automatically using the particle analyzing plugin in ImageJ.If the transverse diameters of the axons were >0.5 mm, the varicosities were defined. 19ImageJ was further used to outline the brain regions based on the reference brain atlas.The strength and direction of the linear relationship between subregions and cells or varicosity proportion were measured using the Pearson product-moment correlation coefficient.

| Identification of monosynaptic inputs to LPAG glutamatergic neurons using an RV-based system
To identify monosynaptic inputs to the glutamatergic neurons in the LPAG, we used a cell type-specific, RG-deleted RV strategy 20 in transgenic mice line expressing Cre recombinase in glutamatergic neurons (Vglut2-Cre mice), which has been shown to label monosynaptic inputs of selected starter neurons with high specificity.On the first day, we injected two Cre-dependent helper viruses (AAV-EF1α-DIO-TVA-EGFP and AAV-EF1α-DIO-RvG) to express the enhanced green fluorescent protein (EGFP), avian-specific retroviral receptor (TVA), and the rabies glycoprotein G (RG) 15 in the unilateral LPAG in Vglut2-Cre mice or wild-type (WT) mice.Three weeks later, the modified RV (RV-EnvA-ΔRG-DsRed) that only infects neurons expressing TVA, and requires RG to spread retrogradely to presynaptic neurons was injected into the same area in the same Vglut2-Cre or WT mice.On the 28th day, the mice were perfused and their brains were processed (Figure 1A,B).To obtain a quantitative comparison between the brain samples, each sample was aligned with the Allen Mouse Brain Atlas after tissue sectioning and fluorescence imaging.After that, we manually identified and labeled the starter cells (expressing both DsRed and EGFP) and presynaptic neurons (expressing DsRed only), and registered their locations in the reference atlas.The starter cells were co-infected with AAV helper viruses and RV, and they were restricted to the LPAG ipsilateral to the injection area in the Vglut2-Cre mice (Figure 1C).The brains of the WT mice exhibited neither EGFP-positive nor DsRed-positive neurons in the LPAG (Figure 1D), showing that the expression of RG-deleted RV strategy was highly cell-type specific.

| Whole-brain input patterns to LPAG glutamatergic neurons
To determine the inputs to LPAG glutamatergic neurons, the brains of these injected mice were cut serially in coronal sections after adequate infection time (Figure 2).Sections from a representative Vglut2-Cre mouse (Figure 2) revealed that DsRed-labeled presynaptic neurons were observed in many brain nuclei.DsRed-labeled afferent neurons were largely located in the hypothalamus, while some were also found in the thalamus, midbrain, and medulla.
Moreover, a few input neurons were found in the cortex, superior colliculus and pons (Figure 2).Next, to display the LPAG presynaptic neurons in greater detail (Figure 3), representative images of inputs from typical subregions were enlarged and selected, such as the lateral hypothalamic area (LH), lateral preoptic area (LPO), substantia

| Immunofluorescence of DsRed-labeled neurons and several markers of physiological behaviors
Next, we determined the colocalization of inputs to the LPAG glutamatergic neurons using several neuronal markers associated with important physiological functions via immunofluorescence.
In the LH, the inputs to LPAG glutamatergic neurons were found to be partly colocalized with orexin (also known as hypocretin) (8.41% ± 1.86% in Figure 4A), which is a central hub for the integration of a wide range of inputs from the brain regions that regulate physiological homeostasis and complex behaviors. 213][24] Deficiency of orexin or its receptor leads to narcolepsy in animals. 25In addition, the LH orexin system is also involved in appetitive, stress response, and other behaviors necessary for survival. 26,27The dense monosynaptic projections in the LH to the LPAG glutamatergic neurons may be to excitatory glutamatergic or inhibitory GABAergic neurons, because a large number of cell populations in the LH are glutamatergic and GABAergic neurons. 28The input neurons from the LPO to LPAG glutamatergic neurons were mostly colocalized with GABA neurons (44.10% ± 4.86% in Figure 4B), where a center for the induction of NREM and REM sleep is located. 29In the LPO, GABAergic cell populations are vital for arousal and sleep homeostasis.Optogenetic stimulation of GABAergic neurons in the LPO of mice during sleep leads to rapid wake induction, and the awake state produced is characterized by increased EEG theta activity.These findings suggest a role of LPO GABAergic neurons in linking arousal to sleep homeostasis. 30[33] Notably, in our study, a large number (32.41% ± 3.92%) of dsRedlabeled neurons in the VP were GABAergic populations (Figure 4C).
In addition, we also found that 36.45% of the input neurons from the MPA and 41.67% from the SI to LPAG glutamatergic neurons were also GABAergic (Figure 4D,E).[36][37][38]

| Analysis of input neurons innervating LPAG glutamatergic neurons
Next, in order to better identify the distribution of these DsRedlabeled neurons, we performed statistical analysis after identifying the brain regions with monosynaptic inputs to LPAG glutamatergic neurons based on standard mouse brain atlases.We divided the whole brain into seven brain structures, namely the cortex, thalamus, hypothalamus, superior colliculus, midbrain, pons, and medulla.
Then, we calculated the proportion of DsRed-labeled afferent neurons of each brain region in the whole brain.We defined the brain regions in which DSRed-labeled neurons accounted for >0.05% of the total labeled neurons as the nuclei with monosynaptic connections with LPAG glutamatergic neurons and generated a list of whole-brain inputs to LPAG glutamatergic neurons (Figure 5).We found that the total afferent neurons, including 59 nuclei, originated in the seven brain structures: the cortex, thalamus, hypothalamus, superior colliculus, midbrain, pons, and medulla (n = 4 mice).We found that the highest numbers of inputs to LPAG were localized in the hypothalamus.The anatomical and functional connectivity of the hypothalamic-PAG circuit, especially the ventromedial hypothalamus and ventrolateral area (VMHvl), to the LPAG has been reported previously, 10 which is highly consistent with our findings.
Finally, to compare the broad distribution of the input neurons more intuitively in the whole brain, we showed the sagittal sections for schematic illustrations of the proportion of input neurons within each nucleus of the whole brain monosynaptic inputs to LPAG glutamatergic neurons, which clearly showed that the numerous afferent neurons of the total whole brain were located in the hypothalamus (Figure 6).However, a technical limitation of our study is that the olfactory bulb was often damaged when the mice brain was cut to create coronal sections, which led to a significant underestimation of labeling in the olfactory bulb.In conclusion, our results provide insights into input distribution to LPAG glutamatergic neurons in the whole brain, except for the olfactory bulb.

| DISCUSS ION
The LPAG is involved in the regulation of a variety of brain functions, including defensive, offensive, 1 and social behaviors 2,7 and pain. 8In addition, our recent study revealed an important role of LPAG glutamatergic neurons in the sleep-wake neural circuit.It is crucial to explore the afferent inputs to LPAG glutamatergic neurons for understanding their regulation in brain functions.In this study, we used a cell type-specific, RG-deleted RV strategy and

| Advantages of specific trans-synaptic tracing compared to traditional retrograde tracing
In recent years, the neural connectivity of the LPAG has been investigated because it has pivotal roles in multiple brain functions.Most previous studies on the inputs to the LPAG have focused on specific regions connected to the LPAG, for example, the auditory cortex, 11 anterior cingulate cortex, 39 lateral parabrachial nucleus, 40 LH, CeA, and ZI. 12 These findings provide some evidence for brain-wide neuronal inputs to the LPAG.In addition, previous investigations used conventional tracing techniques, such as cholera-toxin subunit B (CTB) and wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP).Keay et al. 41 injected the retrograde tracer CTB into the LPAG of rats and revealed the spinal afferents to the LPAG.The reciprocal connections between the medial preoptic area and the midbrain PAG in rats were also found via exploiting the WGA-HRP. 42ese studies had several limitations because the input of specific brain regions to the LPAG is difficult to assess based on whole-brain mapping.Traditional retrograde tracers, such as CTB and HRP, cannot identify the inputs to the LPAG-specific cell types (Table 1).
In this study, we used a RV-mediated retrograde tracing system in Vglut2-Cre mice, which allowed specific labeling of whole-brain monosynaptic inputs to the LPAG glutamatergic neurons (Table 1).
We found that the majority of these inputs originate from the LH, LPO, and VP of the hypothalamus, which is also consistent with previous findings. 43In addition, the SI, LGP, and MPA of the hypothalamus contained a large number of DsRed-labeled cells.Moreover, relatively dense inputs to the LPAG also originated from the CPu in the thalamus and the Gi in the medulla.In conclusion, our findings provide a comprehensive map of the presynaptic patterns that control LPAG glutamatergic neurons.

| Implications of LPAG activity in offensive and defensive behaviors
Studies conducted in recent decades have firmly established the pivotal role of LPAG in the defensive and offensive neural circuit.
LPAG receives the densest projections from the VMHvl, 43 which is a critical hub for the attack behavior. 44 LGP, lateral globus pallidus; LH, lateral hypothalamic area; LPGi, lateral paragigantocellular nucleus; LPO, lateral preoptic area; Lve, lateral vestibular nucleus; M1, primary motor cortex; M2, secondary motor cortex; MPA, medial preoptic area; MPOM, medial preoptic nucleus, medial part; MVeMC, medial vestibular nucleus, magnocellular part; PAG, periaqueductal gray; PH, posterior hypothalamic area; PnC, pontine reticular nucleus, caudal part; RMg, raphe magnus nucleus; RPF, retroparafascicular nucleus; RSG, retrosplenial granular cortex; Rt, reticular thalamic nucleus; S1HL, primary somatosensory cortex, hindlimb region; SC, superior colliculus; SI, substantia innominata; SNC, substantia nigra, compact part; SNR, substantia nigra, reticular part; Su3C, supraoculomotor cap; SubI, subincertal nucleus; SuVe, superior vestibular nucleus; V1, primary visual cortex; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VP, ventral pallidum; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus; VTA, ventral tegmental area; ZI, zona incerta; ZID, zona incerta, dorsal part; 12N, hypoglossal nucleus; 7N, facial nucleus.can induce defensive behaviors characterized by alertness, freezing, and escape. 45The different neural populations of LPAG may play different roles in offensive and defensive behaviors.LPAG GABAergic neurons are required for prey detection, chase, and attack, while LPAG glutamatergic neurons are selectively required for attack. 12In addition, LPAG glutamatergic neurons, rather than GABAergic neurons, receive direct input from auditory cortical centers and mediate sound-driven defensive behavior. 11A recent study revealed the importance of the hypothalamic-midbrain circuit in coordinating aggressive action.The LPAG glutamatergic cells receive preferential projection from the VMHvl glutamatergic cells, and chemogenetic inactivation of the LPAG glutamatergic neurons results in aggression-specific deficits. 10We mapped the monosynaptic inputs to the LPAG glutamatergic cells and found that the projections from the LPAG glutamatergic populations were mainly distributed in the hypothalamus.Furthermore, the top nuclei projections to the LPAG were also involved in offensive and defensive behaviors, for example, the LH 46 and SI, 47     Activation of neurotensinergic neurons in the LPAG could promote NREM sleep. 13 this study, we found that the LPAG glutamatergic populations receive strong inputs from several brain regions associated with sleep-wake regulation.MCH-expressing neurons and orexin/ hypocretin neurons in the LH are important for sleep-wake regulation. 22,50The LH GABAergic and glutamatergic neurons also mediate the sleep-wake cycle. 51,52Our results revealed that LPAG glutamatergic neurons received the densest projections from the LH; thus, the LH-LPAG neural circuit may be important for sleepwake regulation.4][55] The direct inputs from several sleep-wake related nuclei located in the hypothalamus to the LPAG glutamatergic neurons suggested that the LPAG may function via integrating sleep-wake regulatory signals from the hypothalamus.

| Neural circuitry underlying modulation of LPAG in pain responses
The LPAG plays a vital role in pain-related behavioral responses.
Several clinical studies have shown that the functional connectivity of LPAG is disrupted in patients with pain. 56In addition, LPAG mediates pain avoidance behaviors.For example, increased signals from the anterior cingulate cortex to the DL/LPAG are critical for fear avoidance in chronic pain disability. 39The hypothalamus, especially the LH, is also involved in pain perception and promotion. 57,58The lateral septum-LH circuit is critical for pain modulation, 57 and activation of the orexin system facilitates pain control. 59In addition, Siemian et al. 60 demonstrated that the LH parvalbumin-positive (LH PV ) glutamatergic neurons have a potential role as a target for analgesia, in which the LH PV neurons-VLPAG axonal projections play a vital role.More interestingly, an increasing number of studies have suggested bidirectional regulation of sleep-wake and pain behavior. 61,62In our research, we found that numerous hypothalamic subregions associated with sleep-wake regulation, especially LH, send dense projections to the LPAG, which may provide several new directions for future research.For example, future studies should explore whether the LPAG can achieve bidirectional control of sleep-wake regulation and pain processing and whether this process is related to the integration of signals from the hypothalamus.

| The functional implications of LPAG activity for heart rate and respiration
LPAG is involved in fear, defensive, sleep-wake, pain and other behavioral functions.These behaviors are accompanied by changes in heart rate (HR) and respiration, which demonstrates the significant potential implications of LPAG activity for HR and respiratory control.For HR, manipulating the activity of LPAG neurons can effectively alter cardiovascular effects, including bradycardia or increases in HR. [63][64][65][66] The different effects may result from the differential activation of excitatory or inhibitory neurons in the LPAG.
In addition, the cardiovascular response controlled by hypothalamic neurons is largely dependent on the activity of PAG neurons, [63][64][65][66] TA B L E 2 The proportion (>2%) and involved physiological behaviors of nuclei that input to the LPAG glutamatergic neurons.demonstrating the vital role of the hypothalamus-PAG circuit in HR control.For respiration, the PAG-substructures of different animals play differential roles in respiration control. 67For example, in humans, the VLPAG is involved in anticipation of breathing difficulty, whereas difficult breathing is associated with activity in the LPAG. 68 addition, Subramanian et al. 69 stimulated the LPAG in cats and found three types of respiratory responses, including tachypnea, inspiratory apneusis, and respiratory changes in the context of vocalization.In brief, as a control center for behavioral regulation, the PAG may be involved in the integration of sensory signals from the periphery, including HR and respiration.
In conclusion, we mapped the monosynaptic afferents to the LPAG glutamatergic neurons and found that they received projections from other brain areas, especially the nuclei localized in the hypothalamus.This suggests a vital role of LPAG glutamatergic neurons in a wide range of physiological and pathological functions, including sleep-wake regulation, offensive-defensive behaviors, and pain response.We also summarized the involved physiological behaviors of nuclei that input to LPAG glutamatergic neurons (Table 2), which may help us better understand the implications of LPAG in a variety of physiological and behavioral functions.Therefore, our neuroanatomical data could be useful for future explorations of the LPAG and provide a structural framework for the underlying neural mechanisms related to certain physiological functions.

F I G U R E 1
Experimental strategy for RV-based retrograde tracing in LPAG glutamatergic neurons.(A) Design of viral vectors for RVbased trans-synaptic retrograde tracing, including helper viruses with Cre-dependent expression of TVA receptor (AAV EF1α-DIO-TVA-EGFP) and RvG (AAV-EF1α-DIO-RvG).The RV was genetically modified by pseudotyping with EnvA (RV-EnvA-△RG-DsRed). (B) Schematic of the injection procedure and experimental timeline for helper viruses and RV in the Vglut2-Cre mouse.(C, D) Fluorescence images showing GFP-and DsRed-expressing neurons in the LPAG after helper virus and RV injected in Vglut2-Cre mouse and wild type mouse.The lower panels are the enlarged view of the white boxed region in the upper panels.Scale bar: 200 μm (upper panels and right panels), 50 μm (lower panels).

| 4153 MAF I G U R E 4
et al. accurately determined the whole-brain monosynaptic inputs to glutamatergic neurons in the LPAG.We found that LPAG glutamatergic neurons received extensive direct inputs from the whole brain.Moreover, these input neurons preferentially originated from a wide range of nuclei in the hypothalamus, such as the LH, LPO, SI, VP, LGP, and MPA.Taken together, our results revealed a comprehensive map of the presynaptic patterns that may control LPAG glutamatergic neuron activity, which contributes to a better understanding of the role of LPAG in multiple functions and behaviors.Immunofluorescence of DsRed and several markers of physiological behaviors.(A-E) Images showing DsRed-labeled afferent neurons colocalized with several markers of physiological behavior regulation in typical brain regions.Enlarged views of the white boxed regions in the left-most images are shown in the three right images.Colocalized neurons are indicated by arrows in the rightmost images.Images showing that some DsRed-labeled neurons were colocalized with orexin + neurons in the LH, GABAergic neurons in the LPO, VP, MPA, or SI.The quantification of DsRed + cells that are positive for special biomarkers is presented in the rightmost columns.n = 4, each data point represents one experimental animal.Scale bar: 100 μm (left-most images), 20 μm (three right images).GABA, γ-aminobutyric acid; LH, lateral hypothalamic area; LPO, lateral preoptic area; MPA, medial preoptic area; SI, substantia innominate; VP, ventral pallidum.
Excitation of the LPAG F I G U R E 5 Statistical analysis of the whole-brain distribution of monosynaptic inputs to LPAG glutamatergic neurons.Average proportion of DsRed-labeled neurons in brain regions with more than 0.05% average input proportions from LPAG glutamatergic neurons.Error bars represent the standard error of mean.n = 4, each data point represents one experimental animal.Brain regions are grouped into seven general structures listed at the top, and specific brain regions are listed at the bottom.AI, agranular insular cortex; APT, anterior pretectal nucleus; BSTLI, bed nucleus of the stria terminalis, lateral division, intermediate part; CGPn, central gray of the pons; cp, cerebral peduncle, basal part; CPu, caudate putamen; DCIC, dorsal cortex of the inferior colliculus; Dk, nucleus of Darkschewitsch; DM, dorsomedial hypothalamic nucleus; DpG, deep gray layer of the superior colliculus; DPGi, dorsal paragigantocellular nucleus; DpMe, deep mesencephalic nucleus; ECIC, external cortex of the inferior colliculus; Eve, nucleus of origin of efferents of the vestibular nerve; Gi, gigantocellular reticular nucleus; GiA, gigantocellular reticular nucleus, alpha part; GiV, gigantocellular reticular nucleus, ventral part; HDB, nucleus of the horizontal limb of the diagonal band; ic, internal capsule; InG, intermediate gray layer of the superior colliculus; InWh, intermediate white layer of the superior colliculus; IPACL, interstitial nucleus of the posterior limb of the anterior commissure, lateral part; IPACM, interstitial nucleus of the posterior limb of the anterior commissure, medial part; IRt, intermediate reticular nucleus; which suggests that LPAG may play an important role in defensive-offensive circuits by integrating complex signals from the hypothalamus.10

4. 3 |
Neuroanatomical evidence for the potential role of the LPAG in sleep-wake regulationThe functional columns of the PAG play important roles in the sleepwake cycle.Zhong et al.48 showed that NTS-expressing glutamatergic neurons in the VLPAG are preferentially active during NREM sleep, and their activation strongly promotes NREM sleep.The activation of GABAergic neurons in the VLPAG suppresses the initiation and maintenance of REM sleep and consolidates NREM sleep.49 figure drawing.LXK, HZ, and PCY contributed to acquisition of the data and statistical analysis.All authors have read and agreed to the published version of the manuscript.