Fig 1: 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 SAD?G-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
Fig 2: 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
Fig 3: 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 SAD?G-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
Fig 4: 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
Fig 5: 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
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