Fig 2: Decreased expression of CAR1 in MDD patients and depression-like model rodents. (A) The protein level of CAR1 in healthy controls (n = 29) and MDD patients (n = 36) revealed by ELISA assay. (B) Western blot analysis for the expression level of CAR1 in the serum of mice after CSDS exposure. (C) Western blot analysis showed the changes of CAR1 in the hippocampus and prefrontal cortex of mice after CSDS or CRS (n = 6 for each group) treatment. (D) Western blot analysis of CA family isoforms in the hippocampi between CSDS mice model and control mice group. (E) Western blot analysis showed the changes of CAR1 in the hippocampus among CSDS susceptible group (n = 6), resilient group (n = 7) and control group (n = 6). (F) Q-PCR identification of car1 mRNA in the hippocampi among among CSDS susceptible group (n = 7), resilient group (n = 8) and control group (n = 7). (G) Western blot analysis showed the changes in the cerebellum and entorhinal cortex of CSDS mice. (H) The expression of CAR1 in the hippocampus and prefrontal cortex from the CUMS-treated rats. ß-actin or GAPDH was used as loading controls. Unpaired t-test analysis, *p < 0.05, **p < 0.01

Fig 3: Decreased the concentration of bicarbonate and proton in the extracellular space of the hilus of CAR1-/- mice. (A) Example mIPSC traces (top) and their average amplitude and frequency (bottom) of DG granule cells from WT mice underHCO3--free ACS for the normal ACSF condition. (B) Example mIPSC traces (top) and their average amplitude and frequency (bottom) of DG granule cells from WT mice and CAR1-/- mice under HCO3--free ACSF condition. (C) Schematic representation of a construct (top) and the timeline of the experimental procedure (middle). A representative image of the vHPC after bilateral infusions of AAV (bottom left). The infection of astrocytes was confirmed by co-immunostaining with EGFP (green) and GFAP (red) (bottom right). (D) The immobility time in the FST test after AAV-CAR1 infection. *p < 0.05, **p < 0.01 (Student’s t-test). (E) Representative mIPSC traces of DG granule cells from WT mice injected with AAV-control, CAR1-/- mice with AAV-control or AAV-CAR1 underHCO3--free ACSF condition (top) and their average amplitude and frequency analysis (bottom). (F) mIPSC traces of DG granule cells after CAR1 overexpression inCAR1-/-astrocytes with control, acetazolamide, and amiloride treatment as well as their average amplitude and frequency quantifications (bottom). (G) Representative images in the DG hilus after bilateral infusions of pHLIP at the vHPC (n = 4) (left); fluorescence density quantification of pHLIP labeled hilus interneurons from WT and CAR1-/- mice (right). (H) Representative images in the DG hilus after bilateral infusions of pHLIP at vHPC into WT mice infected with AAV-GFP, CAR1-/- mice with AAV-GFP or AAV-CAR1 and their fluorescence density quantification; boxed regions in the top panels were zoomed in the bottom panels. (I) The validation of pHLIP marked hilus interneuron identities by co-immunostaining with neuropeptide Y (NPY), Somatostatin (SST), and parvalbumin (PV). *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test and one-way ANOVA)

Fig 4: Specific expression of CAR1 in astrocytes. (A, B) A sample confocal image of a hippocampal slice stained with antibody against CAR1 (green) and DAPI (blue). Boxed area in A was zoomed in B. (C) Co-immunostaining with antibodies against CAR1 (green) and different cell type markers (red). Solid arrowheads: representative double-labeled cells. (D) The quantification of CAR1+ /GFAP+ cells in CAR1+ cells or GFAP+ cells. (E) RNAscope signals of CAR1 (white) in astrocytes labeled by anti-GFAP immunostaining (red) of the ventral hippocampus. (F, G) Zoomed in figures from E. F for the CA1 region and G for the DG region

Fig 5: A causal role of CAR1 in depression-like behaviors and the activity changes in granule cells. (A) Western blotting of hippocampal sample to validate the absence of CAR1 in CAR1-/- mice(top); depression-like behavioral tests including FST and TST for CAR1-/- mice and WT mice (bottom). (B, C) Example traces and their average amplitude and frequency of mEPSC (D) or mIPSC (E) in DG granule cells. (D) Representative responses of granule cells (black, WT; red, CAR1-/-) to perforant pathway stimulations (top); the peak response and the area under the curve quantification (bottom) of evoked responses (WT, n = 14; CAR1-/-, n = 17). (E) The representative mIPSC traces of DG granule cells from WT mice injected with AAV-control, CAR1-/- mice with AAV-control, or AAV-CAR1 (left), and their average amplitude and frequency of mIPSC of DG granule cells (right). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test and one-way ANOVA)