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.

Keywords: glutamatergic neurons; lateral periaqueductal gray; monosynaptic inputs; rabies virus.

FIGURE 1

Experimental strategy for RV‐based retrograde tracing in LPAG glutamatergic neurons. (A) Design of viral vectors for RV‐based 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).

FIGURE 2

Representative images of monosynaptic inputs to LPAG glutamatergic neurons from the whole brain. Regions are labeled according to the mouse brain atlas. Scale bar: 1 mm. AI, agranular insular cortex; AcbSh, accumbens nucleus, shell; AHP, anterior hypothalamic area, posterior part; BSTL, bed nucleus of the stria terminalis, lateral division; CeMAD, central amygdaloid nucleus, medial division, anterodorsal part; DR, dorsal raphe nucleus; DMTg, dorsomedial tegmental area; ECIC, external cortex of the inferior colliculus; 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; I, intercalated nuclei of the amygdala; LA, lateroanterior hypothalamic nucleus; LC, locus coeruleus; LPGi, lateral paragigantocellular nucleus; LPAG, lateral periaqueductal gray; LDTg, laterodorsal tegmental nucleus; LH, lateral hypothalamic area; LPO, lateral preoptic area; LSI, lateral septal nucleus, intermediate part; LO, lateral orbital cortex; MPA, medial preoptic area; MdD, medullary reticular nucleus, dorsal part; MHb, medial habenular nucleus; MPOL, medial preoptic nucleus, lateral part; Pir, piriform cortex; PAG, periaqueductal gray; PnV, pontine reticular nucleus, ventral part; Pr, prepositus nucleus; PMD, premammillary nucleus, dorsal part; SI, substantia innominata; SN, substantia nigra; TC, tuber cinereum area; VP, ventral pallidum; VMH, ventromedial hypothalamic nucleus; VTA, ventral tegmental area; VLPAG, ventrolateral periaqueductal gray.

FIGURE 3

Schematic representation of the typical regions with monosynaptic inputs to LPAG glutamatergic neurons. (A–I) Primary inputs originated from regions involved in physiological behaviors (e.g., VP, LH, MPB, LC, LDT, DPGi, LPGi, and Giv). Scale bar: 200 μm. 12N, hypoglossal nucleus; 3V, 3rd ventricle; ADP, anterodorsal preoptic nucleus; AHA, anterior hypothalamic area, anterior part; APTV, anterior pretectal nucleus, ventral part; Bar, Barrington's nucleus; BSTLV, bed nucleus of the stria terminalis, lateral division, ventral part; BSTMA, bed nucleus of the stria terminalis, medial division, anterior part; BSTMPI, bed nucleus of the stria terminalis, medial division, posterointermediate part; BSTMPM, bed nucleus of the stria terminalis, medial division, posteromedial part; CGA, central gray, alpha part; CI, caudal interstitial nucleus of the medial longitudinal fasciculus; CPu, caudate putamen; DLG, dorsal lateral geniculate nucleus; DM, dorsomedial hypothalamic nucleus; DPGi, dorsal paragigantocellular nucleus; f, fornix; F, nucleus of the fields of Forel; FF, fields of Forel; fr, fasciculus retroflexus; Gem, gemini hypothalamic nucleus; Gi, gigantocellular reticular nucleus; GiV, gigantocellular reticular nucleus, ventral part; HDB, nucleus of the horizontal limb of the diagonal band; 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; LA, lateroanterior hypothalamic nucleus; LC, locus coeruleus; LDTg, laterodorsal tegmental nucleus; LH, lateral hypothalamic area; LPBC, lateral parabrachial nucleus, central part; LPBD, lateral parabrachial nucleus, dorsal part; LPBI, lateral parabrachial nucleus, internal part; LPGi, lateral paragigantocellular nucleus; LPO, lateral preoptic area; MCPO, magnocellular preoptic nucleus; MeAD, medial amygdaloid nucleus, anterodorsal; ml, medial lemniscus; MPO, medial preoptic nucleus; mt, mamillothalamic tract; ns, nigrostriatal bundle; PMV, premamillary nucleus, ventral part; PS, parastrial nucleus; PSTh, parasubthalamic nucleus; Ro, nucleus of Roller; scp, superior cerebellar peduncle; SI, substantia innominata; SLEAC, sublenticular extended amygdala, central part; SLEAM, sublenticular extended amygdala, medial part; SPF, subparafascicular thalamic nucleus; Subl, subincertal nucleus; Te, terete hypothalamic nucleus; VMHC, ventromedial hypothalamic nucleus, central part; VMHDM, ventromedial hypothalamic nucleus, dorsomedial part; VMHVL, ventromedial hypothalamic nucleus, ventrolateral part; VP, ventral pallidum; ZID, zona incerta, dorsal part; ZIV, zona incerta, ventral part.

FIGURE 4

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.

FIGURE 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; 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.

FIGURE 6

Schematic diagrams showing the distribution of the monosynaptic inputs innervating the LPAG glutamatergic neurons. Sagittal sections for a schematic illustration of whole‐brain inputs to LPAG glutamatergic neurons in Vglut2‐Cre mice. BSTLI, bed nucleus of the stria terminalis, lateral division, intermediate part; CPu, caudate putamen; DM, dorsomedial hypothalamic nucleus; DpMe, deep mesencephalic nucleus; Gi, gigantocellular reticular nucleus; LGP, lateral globus pallidus; LH, lateral hypothalamic area; LPAG, lateral periaqueductal gray; LPO, lateral preoptic area; MPA, medial preoptic area; MPOM, medial preoptic nucleus, medial part; PH, posterior hypothalamic area; SI, substantia innominata; SNC, substantia nigra, compact part; SNR, substantia nigra, reticular part; SubI, subincertal nucleus; VM, ventromedial thalamic nucleus; VP, ventral pallidum; VTA, ventral tegmental area; ZI, zona incerta; ZID, zona incerta, dorsal part.