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. 2023 Apr 17;14:2198. doi: 10.1038/s41467-023-37878-y

Fig. 1. Neuropathic pain-induced depression (NPID) triggers hyperactivity in BLA neurons projecting to the ACC and increases functional connectivity between the ACC and BLA.

Fig. 1

a Retrograde tracing strategy, with the injection of the cholera toxin B subunit (CTB) into the anterior cingulate cortex (ACC). b Representative image of retrogradely labeled cell bodies in the anterior part of the basolateral nucleus of the amygdala (BLA). Scale bar = 100 µm. c Experimental design for quantifying the activity of BLA neurons projecting to the ACC, during NPID. d–f Peripheral nerve injury induced an ipsilateral mechanical hypersensitivity (d; F(21,224) = 2.710; P < 0.0001; post hoc 1–7 weeks P < 0.05), decreased grooming in the splash test (ST) (e; sham: 125.90 ± 10.80; NPID: 80.67 ± 8.22; P = 0.0042) and increased latency to feed in novelty-suppressed feeding (NSF) (f; sham: 120.7 ± 10.49; NPID: 173.7 ± 14.38; P = 0.0089) (n = 9 mice/group). g Representative images showing positive cells for fluorogold (FG + , upper panel), c-Fos (c-Fos + , middle), or co-labeled (bottom), in the contralateral (sham: right column; NPID: middle right) or ipsilateral BLA (sham: middle left; NPID: left). Scale bar = 100 µm. h–m Quantification of FG + , c-Fos+ cells and their co-localization revealed that, at 8 weeks postoperative (PO), the number of FG + cells was not altered (h; contralateral BLA: sham: 135.8 ± 31.52; NPID: 267.2 ± 72.58; P = 0.21; i; ipsilateral BLA: sham: 163.5 ± 28.85; NPID: 170.6 ± 25.43; P = 0.3651), as well as c-Fos + (j; sham: 309.3 ± 47.60; NPID: 378.2 ± 49.10; P = 0.14) and % of FG + /c-Fos + (l; sham 30.50 ± 4.20; NPID: 28.33 ± 4.72; P = 0.45) cells in the contralateral BLA. In contrast, the number of c-Fos + (k; sham: 245.5 ± 34.37; NPID: 362.4 ± 21.62; P = 0.0238) and % of FG + /c-Fos+ cells (m sham 22.06%±3.55; NPID: 32.32%±2.81; P = 0.0159) were increased in the ipsilateral BLA (sham: n = 4 mice; NPID: n = 5 mice). n Close-up image showing the co-localization of c-Fos+ and FG + cells. Scale bar = 20 µm. o Inter-group statistical comparisons showed increased functional connectivity between ACC and amygdala (AMY) at 8 (right image) but not 2 (left) PO weeks. FWER corrected at cluster level for P < 0.05. Data are mean ± SEM. *P < 0.05; **P < 0.01, ***P < 0.001. Two-way ANOVA repeated measures (Time × Surgery; VF); two-sided unpaired t test (ST and NSF); one-sided Mann–Whitney test (FG, c-Fos quantification). Contra contralateral, ipsi ipsilateral, BLP posterior part of the BLA, LA lateral amygdala. Sagittal mouse brain cartoons (a, c) were created with Biorender.com. Source data are provided as a Source Data file.