Selective overexpression of hippocampal CB1R alleviates chronic stress-induced neurogenesis impairment and behavior disorder in rats

To investigate the effects of selective overexpression of hippocampal cannabinoid receptor 1 (CB1R) on newly generated cells and newly matured neurons in the hippocampus of stress-induced rats.

Thirty-two 6 -8 week -old male SD rats (body weight 180 to 220 g) were randomly assigned to a Control group (n=10) and a Stress group (n=22). After the Stress group underwent 3 weeks of chronic restraint stress (CRS), 11 rats were randomly selected to form the Stress+CB1R-OE group, receiving hippocampal injections of an adeno-associated virus (AAV) for CB1R overexpression via stereotaxic surgery. The Control group and the remaining Stress group received a null AAV, designated as Control+AAV-NC and Stress+AAV-NC groups, respectively; viral transfection lasted 3 weeks. Body weight was measured every Sunday morning for weeks 1 to 7. Behavioral levels were assessed using the novel environment feeding suppression test. Immunofluorescence staining was used to detect the expression of 5-bromo-2’-deoxyuridine (BrdU), Neuronal Nuclei (NeuN), and CB1R in the hippocampal dentate gyrus (DG) (n=5 per group, 3 slices per rat). A NOVEL confocal microscope was used for scanning. Imaris software calculated the density of BrdU⁺ and BrdU⁺/NeuN⁺ cells per unit volume in the DG, while Image J software measured the mean fluorescence intensity of CB1R and NeuN in the DG. Western blot assay was used to detect hippocampal CB1R protein expression levels.

After 3 weeks of CRS, the body weight of the Stress+AAV-NC group was significantly lower than that of the Control+AAV-NC group (P=0.001). Selective hippocampal CB1R overexpression for 3 weeks partially ameliorated body weight gain in the Stress+CB1R-OE group, but their weight remained significantly lower than the Control+AAV-NC group (P=0.004). The novel environment feeding suppression test showed that compared to the Control+AAV-NC group, the Stress+AAV-NC group had a significantly prolonged feeding latency (P<0.001), with no significant difference in feeding amount within 5 min. After selective CB1R overexpression, the Stress+CB1R-OE group showed a significantly shortened feeding latency (P=0.039), with no significant differences in feeding amount among the three groups. Immunofluorescence result indicated that compared to the Control+AAV-NC group, the Stress+AAV-NC group had a significantly reduced mean fluorescence intensity of CB1R⁺ cells in the DG (P=0.008). After selective CB1R overexpression, the Stress+CB1R-OE group showed a significantly increased mean CB1R fluorescence intensity in the DG compared to the Stress+AAV-NC group (P<0.001). Western blotting result showed that hippocampal CB1R protein expression was significantly lower in the Stress+AAV-NC group than in the Control+AAV-NC group (P=0.001). Compared to the Stress+AAV-NC group, the Stress+CB1R-OE group had a significantly increased hippocampal CB1R expression level (P<0.001). Compared to the Control+AAV-NC group, the Stress+AAV-NC group had reduced densities of BrdU⁺ newborn cells (P=0.003) and BrdU⁺/NeuN⁺ newly matured neurons (P=0.001) in the DG. After selective CB1R overexpression, the Stress+CB1R-OE group showed significantly increased densities of BrdU⁺ cells (P=0.035) and BrdU⁺/NeuN⁺ neurons (P=0.028) in the DG. No significant difference was observed in the mean NeuN fluorescence intensity in the DG among the three groups.

Selective overexpression of hippocampal CB1R can alleviate stress-mediated reduction in hippocampal neurogenesis and subsequently improve behavioral deficits in stress-induced rats.