Fig 1: CAPS2 gene expression in the animal models of depression, MDD patients, and identification of CAPS2 expressing cells. (a–d) qRT-PCR analysis showed that the CAPS2 mRNA expression level decreased in the habenula of rats exposed to CRS (a), mice exposed to electric shock (b). CAPS2 mRNA expression showed a tendency to decrease in the habenula of MDD patients who died by suicide (c and d). CAMK2B levels did not show any difference between the two groups. The relative abundances of mRNA were normalized to the amount of GAPDH using the comparative threshold cycle method. (e–h) Photomicrographs of the double-labeled fluorescent in situ hybridization for CAPS2, TAC1, TAC2, CHAT, and VGLUT1. Antisense riboprobe-labeled were for CAPS2/TAC1 (e), CAPS2/CHAT (f), CAPS2/TAC2 (g), and CAPS2/VGLUT1 (h). These genes were co-expressed in the MHb. Data represent mean ± SEM, *P < 0.05 (a, NS, n = 4; CRS, n = 4 rats; P = 0.029, Mann–Whitney U-test; b, CON, n = 8; LH, n = 8 mice; P = 0.038, c, d, CON, n = 11; MDD, n = 12 subjects; CAPS2: P = 0.279; CAMK2B: P = 0.483; Student’s t-test; Scale bars, 200 μm; 10 μm in the magnified image).
Fig 2: CAPS2 knockdown in the MHb induces altered neuronal activity in the VTA and the DRN. (a) Photomicrographs of VTA immunohistochemistry slices from mice injected with AAV-sh-vehicle (CON) or AAV-sh-CAPS2 (KD). MHb CAPS2 knockdown reduced c-Fos positive cells (red) in the VTA neurons. (b and c) C-Fos-positive cell counts and c-Fos/TH double-positive cell counts in VTA. (d) DRN slices from mice injected with AAV-sh-vehicle (CON) or AAV-sh-CAPS2 (KD) were used for immunohistochemistry. (e and f) c-Fos-positive cell count in DRN was reduced in the knockdown group, but c-Fos/TPH double-positive cell count showed no significant difference. (g and h) C-Fos/CAMK2B double-positive cells were reduced in DRN of the knockdown mouse. Data represent mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 (CON, n = 6; KD, n = 6 mice; b, Student’s t-test, P < 0.001, c, Mann–Whitney U-test, P = 0.002; Scale bars, 200 μm; 50 μm in the magnified image, 3 sections per mouse is averaged. e and f, Student’s t-test, c-Fos: P = 0.049, c-Fos/TPH: P = 0.121 each; Scale bars, 200 μm; 50 μm in the magnified image, h, Student’s t-test, P = 0.007; Scale bars, 50 μm).
Fig 3: Effects of CAPS2 knockdown in the MHb on anxiety- and depression-like behaviors. (a) Experimental paradigm for behavioral testing of mice infected with the virus. (b) Schematic representation of the AAV vector engineered to induce CAPS2 knockdown. (c) GFP expression of AAV-sh-vehicle (CON) or AAV-sh-CAPS2 (KD) after 4 weeks of injection. Scale bars, 100 μm. (d) Diagram showing viral spread in the mouse brain. (e–l) Despair-like symptom and anhedonia-like symptom were examined by the tail-suspension test (TST, e), forced swim test (FST, f), and sucrose preference test (SPT, g and h). Behavioral effects of expressing AAV-sh-CAPS2 in the MHb on anxiety levels were performed in the open field test (OFT, i and j) and the elevated zero maze test (EZM, k and l) and analyzed with Ethovision XT 12. AAV-sh-CAPS2 (KD) injected group showed increased immobility time in TST and FST, indicating despair-like symptoms, but no difference in anxiety-like and anhedonia-like symptom. Data represent mean ± SEM, **P < 0.01, ***P < 0.001 (e–f, i–l; CON, n = 26; KD, n = 29 mice; TST: P < 0.001; FST: P = 0.001; OFT, frequency to center: P = 0.924, OFT, center time: P = 0.379; EZM, frequency to open arm: P = 0.807, EZM, time in open arm: P = 0.942; g and h; CON, n = 18; KD, n = 20 mice; SPT, fluid consumption: P = 0.637, SPT, sucrose preference: P = 0.892; Student’s t-test).
Fig 4: DCVs are accumulated in IPN of MHb CAPS2 knockdown mice. (a) Representative electron micrographs of IPN slices from the mice injected with AAV-sh-vehicle (CON) or AAV-sh-CAPS2 (KD). Closed arrowheads indicate DCVs. (b–g) MHb CAPS2 knockdown effect in IPN synapses was examined by measuring the area of presynaptic bouton, PSD length, DCV, SV number, and density. Decreased CAPS2 expression in the MHb led to increased DCV number and density in the IPN. Data represent mean ± SEM, *P < 0.05 (CON, n = 4; KD, n = 4; 50 synapses counted and averaged per mice; presynaptic area: P = 0.486; PSD length: P = 1.000; DCV number: P = 0.029; DCV density : P = 0.029; SV number: P = 0.200; SV density: P = 0.686; Mann–Whitney U-test; Scale bars, 1 μm).
Fig 5: CAPS2 knockdown in the MHb leads to decreased neuronal activity in the IPN. (a) Western blot analysis from the IPN tissue of MHb AAV-sh-vehicle (CON) or AAV-sh-CAPS2 (KD) injected mice. (b–e) Western blot band intensity for CAPS2, c-Fos, p-ERK, ERK. AAV-sh-CAPS2 (KD) virus injection reduced the CAPS2 protein levels in IPN and diminished c-Fos and p-ERK protein levels. Uncropped images are presented in Supplementary Figure S5. (f and g) Representative immunohistochemistry image (f) and c-Fos-positive cell number (g) in IPN of control and MHb CAPS2 knockdown mouse. The number of c-Fos-positive cells was decreased in the MHb CAPS2 knockdown group. Data represent mean ± SEM, *P < 0.05, **P < 0.01 (WB, CON, n = 5; KD, n = 5 mice; CAPS2: P = 0.016; c-Fos: P = 0.016; p-ERK: P = 0.008; ERK: P = 0.222; Mann–Whitney U-test; IHC, CON, n = 6; KD, n = 6; P = 0.041, Student’s t-test; Scale bars, 100 μm).
Supplier Page from Abcam for Anti-CAPS2 antibody