Extended Data Fig. 1: Neuroanatomical characterization of LPB → SNR and LPB → VTA neurons.
From: Pain modulates dopamine neurons via a spinal–parabrachial–mesencephalic circuit
(a) eYFP-expressing LPBVGLUT2 neurons. (b-g) LPBVGLUT2 terminals expressing eYFP in different brain regions. Bregma: 0.14 mm (b), −1.22 mm (c), −1.46 mm (d), −1.46 mm (e), −3.40 mm (f), −3.40 mm (g). Note fibers of passage in VTA (red arrow) (scale bar 50 μm). (h) Mean eYFP fluorescence intensity in different brain areas (n = 5 mice; Data represent mean ± SEM). (i) Synaptophysin-mCherry expression in LPB (left) and ventral midbrain (right) from VGLUT2-Cre mice (scale bars 50 μm). (j) High resolution images of synaptophysin-expressing LPBVGLUT2 terminals close to lateral VTA (lVTA) TH-immunopositive and SNR GABA-immunopositive neurons (scale bar 20 μm). (k) Mean synaptophysin intensity (quantified as number of particles in a defined region) for different ventral midbrain subregions (n = 3 mice). (l) Experimental design. (m) Sample of a recorded and neurobiotin (NB)-filled, mCherry-positive SNR cell (scale bar 50 μm). (n) Light-evoked EPSC recorded in mCherry-positive SNR cell (black) in response to stimulation of LPB inputs (red trace: 20 µM CNQX). (o) Mean EPSC amplitude before (ACSF) and after CNQX application. (p) eYFP-expressing LPBGAD2 neurons at bregma −5.30 mm. (q-s) LPBGAD2 terminals expressing eYFP in (q) DLPAG (Bregma: −4.60 mm), but not in (r) ventral midbrain (Bregma: −3.30 mm) or (s) amygdala (Bregma: −1.50 mm; scale bar 200 μm). (t) Left: Retrobead injection site in NAcLat of a VGLUT2-Cre mouse for experiment in Fig. 1g (scale bar 200 µm). Middle/Right: lVTA cell that was recorded for experiment in Fig. 1g and filled with neurobiotin (NB). It is retrogradely labeled (that is, projects to NAcLat) and TH-immunopositive (scale bar 50 µm). (u) Same as in (t) but cell is in SNR and TH-immunonegative (scale bar 50 µm). (v) Recorded VTA cell from a GAD2tdTomato mouse and filled with NB (Refers to Fig. 1k; scale bar 50 µm). Data represent mean ± SEM. Significance was calculated by means of one-way RM ANOVA with Tukey’s post-hoc test (k) or paired t-test (o). * p < 0.05, ** p < 0.01, *** p < 0.001.
