Monosynaptic retrograde tracing of neurons expressing the G-protein coupled receptor Gpr151 in the mouse brain

Abstract

GPR151 is a G-protein coupled receptor for which the endogenous ligand remains unknown. In the nervous system of vertebrates, its expression is enriched in specific diencephalic structures, where the highest levels are observed in the habenular area. The habenula has been implicated in a range of different functions including behavioral flexibility, decision making, inhibitory control, and pain processing, which makes it a promising target for treating psychiatric and neurological disease. This study aimed to further characterize neurons expressing the Gpr151 gene, by tracing the afferent connectivity of this diencephalic cell population. Using pseudotyped rabies virus in a transgenic Gpr151-Cre mouse line, monosynaptic afferents of habenular and thalamic Gpr151-expressing neuronal populations could be visualized. The habenular and thalamic Gpr151 systems displayed both shared and distinct connectivity patterns. The habenular neurons primarily received input from basal forebrain structures, the bed nucleus of stria terminalis, the lateral preoptic area, the entopeduncular nucleus, and the lateral hypothalamic area. The Gpr151-expressing neurons in the paraventricular nucleus of the thalamus was primarily contacted by medial hypothalamic areas as well as the zona incerta and projected to specific forebrain areas such as the prelimbic cortex and the accumbens nucleus. Gpr151 mRNA was also detected at low levels in the lateral posterior thalamic nucleus which received input from areas associated with visual processing, including the superior colliculus, zona incerta, and the visual and retrosplenial cortices. Knowledge about the connectivity of Gpr151-expressing neurons will facilitate the interpretation of future functional studies of this receptor.

Keywords: RRID: AB_10743815; RRID: AB_2571870; RRID: IMSR_JAX:000664; RRID: IMSR_JAX:024109; RRID: SCR_003070; habenula; rabies; thalamus.

Figure 1

Distribution of fluorescent reporters in the Cre‐dependent rabies virus retrograde tracing system. Cre‐expressing neurons infected by AAV8‐Ef1a‐FLEX‐TVA‐mCherry express the sarcoma‐leukosis virus receptor (TVA) fused to the fluorescent protein mCherry (TVA‐mCherry; magenta). TVA enables entry of EnvA‐coated pseudotyped glycoprotein‐deleted rabies virus particles (SADΔG‐eGFP(EnvA)) into Cre‐positive neurons. Coinfection of these neurons with AAV8‐CA‐FLEX‐RG leads to expression of rabies glycoprotein (RG) which enables transsynaptic retrograde transport of the rabies virus particles. Both the Cre‐positive starter neurons and the primary afferent neurons will thus express the enhanced green fluorescent protein (eGFP; green) while TVA‐mCherry expression will be limited to Cre‐positive neurons

Figure 2

GPR151 protein expression in axons of Gpr151‐Cre neurons. Coronal sections of a Gpr151‐Cre mouse injected with AAV8‐hSyn1‐FLEX‐mCherry in the habenular region. Panels (g–i) represent magnifications of the insets in (d–f). Cell bodies positive for mCherry (magenta) were observed in the ventral medial habenula, the lateral habenula (excluding the oval subnucleus of the lateral division of lateral habenula), the lateroposterior, and paraventricular thalamic nuclei (b, c; −1.94 mm posterior to Bregma). Fibers expressing mCherry and GPR151 protein (green) exited the habenula through the fasciculus retroflexus (a–i; −2.30 mm posterior to Bregma), where colocalization between mCherry and GPR151 was observed (arrows; g–i). Scale bar (a–f) = 100 µm, (g–i) = 20 µm

Figure 3

Gpr151 mRNA expression in Gpr151‐Cre neurons. Coronal section of a Gpr151‐Cre mouse injected with AAV8‐hSyn1‐FLEX‐mCherry in the habenular region (−1.82 mm posterior to Bregma). Gpr151 mRNA expression (green) can be seen in the ventral medial habenula, lateral habenula (excluding the oval subnucleus of the lateral division), as well as in the lateral posterior, central lateral, and paraventricular thalamic nuclei (panels a, c, d, e). Expression of mCherry (magenta, panels b–e) show partial overlap with Gpr151 mRNA. Transduction efficiency and injection localization could explain the lack of mCherry expression in certain Gpr151‐positive subregions like the LHbMS. Examples of coexpression of mCherry and Gpr151 mRNA in the lateral posterior thalamic nucleus and lateral habenula are shown in panel (d) and (e) (magnifications of insets in panel c). Scale bar (a–c) = 100 µm, (d, e) = 20 µm

Figure 4

Expression of TVA‐mCherry and eGFP after injections of viral vectors at target sites. Coronal sections through the target areas of cases M80 (habenula; a–c), M18 (habenula; d–f), M118 (habenula, no RG; g–i), M77 (habenula, wild type; j–l), M119 (lateral posterior thalamic nucleus; m–o), and M113 (paraventricular thalamic nucleus; p–r) showing sarcoma‐leukosis virus receptor (TVA) fused to the fluorescent protein mCherry (TVA‐mCherry; magenta; b, e, h, k, n, q), eGFP expression (green; a, d, g, j, m, p), and an overlay between the two (c, f, i, l, o, r). Scale bar = 100 µm

Figure 5

Omission of RG limits eGFP expression to the injected area. Coronal brain sections from 1.34 to −4.84 mm relative to Bregma of a Gpr151‐Cre mouse (case M118) in which AAV8‐Ef1a‐FLEX‐TVA‐mCherry and SADΔG‐eGFP(EnvA) was injected unilaterally in the habenula. Since AAV8‐CA‐FLEX‐RG was omitted, transsynaptic transport of the rabies vector is inhibited. Although many eGFP positive (green) neurons could be seen in various nuclei in the targeted area (medial and lateral habenula, lateral posterior thalamic nucleus, parafascicular thalamic nucleus, paraventricular thalamic nucleus, and precommissural nucleus), no eGFP expressing neurons were detected elsewhere in the brain. Scale bar = 1000 µm

Figure 6

Afferents of habenular Gpr151‐Cre neurons. Coronal brain sections from 1.98 to −4.96 mm relative to Bregma of a Gpr151‐Cre mouse (case M80) where AAV8‐Ef1a‐FLEX‐TVA‐mCherry, AAV8‐CA‐FLEX‐RG and SADΔG‐eGFP(EnvA) was injected unilaterally in the habenula. Neurons coexpressing eGFP and mCherry (starter neurons; magenta outline) were almost exlusively located in the medial and lateral habenula. EGFP positive neurons (green) were found throughout the brain, most notably in the medial septal nucleus, nucleus of the diagonal band, lateral preoptic area, and the lateral hypothalamus (peduncular part). Scale bar = 1000 µm

Figure 7

Main afferent neuronal populations of the habenular Gpr151‐Cre starter neurons. Coronal sections showing eGFP‐expressing afferent neurons (green cell bodies and fibers) in the medial septal nucleus and the nucleus of the diagonal band (a; case M17), the lateral and medial preoptic area (b; case M18), the bed nucleus of stria terminalis and the paraventricular thalamic nucleus (c; case M17), the triangular nucleus of the septum and the bed nucleus of the anterior commissure (d; case M17), the entopeduncular nucleus and the lateral hypothalamus (e, f; case M18). Scale bar = 100 µm

Figure 8

Afferents of Gpr151‐Cre neurons in the paraventricular thalamic nucleus. Coronal brain sections from 1.98 to −4.84 mm relative to Bregma of a Gpr151‐Cre mouse (case M113) where AAV8‐Ef1a‐FLEX‐TVA‐mCherry, AAV8‐CA‐FLEX‐RG, and SADΔG‐eGFP(EnvA) was injected into the anterior part of the paraventricular thalamic nucleus. Afferent eGFP expressing neurons (green) were found in great numbers in the zona incerta, medial preoptic area, medial and lateral hypothalamus and to a minor extent in the prelimbic cortex, accumbens nucleus, lateral preoptic area, bed nucleus of stria terminalis, and periaqueductal gray. Neurons coexpressing eGFP and mCherry (starter neurons; magenta outline) were located in the paraventricular thalamic nucleus. Scale bar = 1000 µm

Figure 9

Main afferent neuronal populations of Gpr151‐Cre starter neurons in the paraventricular thalamic nucleus. Coronal sections (case M113) showing eGFP‐expressing afferent neurons (green cell bodies and fibers) in the prelimbic cortex (a, left hemisphere), the septohypothalamic nucleus (b), the median preoptic nucleus (b), the periventricular hypothalamic nucleus (b, d), the medial preoptic area (b), the zona incerta (c, right hemisphere), the anterior hypothalamic area (d), the retrochiasmatic area (d), the peduncular part of lateral hypothalamus (e, right hemisphere), the ventromedial hypothalamic nucleus (e, right hemisphere), and the arcuate hypothalamic nucleus (f). Scale bar = 100 µm

Figure 10

Efferent projections of Gpr151‐Cre neurons in the paraventricular thalamic nucleus. Coronal sections of a Gpr151‐Cre mouse injected with AAV8‐hSyn1‐FLEX‐mCherry in the paraventricular nucleus of the thalamus (case M74) showing 3,3'‐diaminobenzidine enhanced mCherry immunostaining in cell bodies in the injected area and in efferent projections to the zona incerta (a), the prelimbic area (b), the shell and core of accumbens nucleus (c), and the basolateral amygdala (d). Scale bar = 1000 µm

Figure 11

Afferents of Gpr151‐Cre neurons in lateral thalamic nuclei. Coronal brain sections from 1.98 to −5.02 mm relative to Bregma of a Gpr151‐Cre mouse (case M119) where AAV8‐Ef1a‐FLEX‐TVA‐mCherry, AAV8‐CA‐FLEX‐RG, and SADΔG‐eGFP(EnvA) was injected into the lateral posterior thalamic nucleus. Starter neurons expressing both mCherry and eGFP (outlined in magenta) were not restricted to the target area, but were also found in the laterodorsal thalamic nucleus, central lateral thalamic nucleus, parafascicular thalamic nucleus, posterior thalamic group, lateral habenula, precommissural nucleus, and medial pretectal nucleus. Afferent eGFP expressing neurons (green) were detected in many areas throughout the brain including the cingulate and prelimbic cortices, bed nucleus of stria terminalis, ventral pallidum, reticular thalamic nucleus, lateral preoptic area, lateral hypothalamic area, zona incerta, and superior colliculus. Scale bar = 1000 µm