Fig 2: Decrease in the synaptic localization of inhibitory synaptic markers in Tg-FDD mice. (A) Immunofluorescence for GABAA receptor (green) and GAD (red) on cortex and hippocampus. (B) Quantification of the number of merge puncta for both markers on A. (C) Double staining for GABAB receptor (green) and GAD (red). (D) Quantification of the number of merged puncta for both markers on C. p < 0.05 indicated on each graph, Mann–Whitney test, n = 9 photographs from 3 different animals per genotype. Data represented as mean ± SEM. Scale bar 10 µm.

Fig 3: Pain induced local VTA cell-type phenotype alterations. Representative 2.5× images showing an upregulation in (a) GAD65/67 marker of GABAergic inhibitory neurons, and a downregulation in (b) dopamine transporter (DAT) and (c) dopamine D2 receptors (D2r) in the VTA of Sham- and CFA-treated rats. While for (d) tyrosine hydroxylase (TH, red), no significant change was observed between experimental groups. Illustrative immunofluorescent images of Sham- (subpanels: a.1, b.1, c.1, and d.1; respectively) and CFA-treated rats (subpanels: a.3, b.3, c.3, and d.3; respectively). DAPI labeling (blue) was used to nuclear counterstain (Sham group subpanels: a.2, b.2, c.2, and d.2; and CFA group subpanels: a.4, b.4, c.4, and d.4; respectively). Scale bars, 500 µm. All sections are taken 5.80–6.00 mm posterior to the bregma. White polygon indicates the coronal slice area used to measure the mean relative fluorescence intensity. Comparisons between experimental groups are based on parametric unpaired t-test or non-parametric Mann-Whitney (MW) test. n = 5–9 per group (right panels). Values are presented as mean ± S. D. Significant results are indicated by * when p < 0.05 and by ** when p < 0.01. Supplementary material are provided in Source Data S2.

Fig 4: Tissue validation of LHb eNpHR3.0-expressing neurons targeting into the VTA. Immunofluorescent images showing eNpHR3.0-expressing axonal terminals of LHb neurons targeting into the VTA GABAergic cells. (a) Colocalization of LHb terminals with VTA GAD67-positive neurons; the arrow indicates a green inhibitory GABAergic neuron co-labeled with a mCherry LHb terminal in the VTA. DAPI labeling (blue) was used to nuclear counterstain. (b) From the left to the right, LHb terminals in VTA (b.1; mCherry, red), VTA GABAergic inhibitory (b.2; GAD65/67-marker)-positive neurons (pink), VTA tyrosine hydroxylase (TH)-positive neurons (b.3; green), and finally the respective merged image of all channels (b.4). The arrows show some examples of colocalization of LHb terminals with VTA inhibitory neurons. Scale bars, 20 µm. All sections are taken 5.40–6.00 mm posterior to the Bregma. Supplementary material are provided in Source Data S1.