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. 2005 Nov;115(11):3104-16.
doi: 10.1172/JCI25509. Epub 2005 Oct 13.

Cannabinoids Promote Embryonic and Adult Hippocampus Neurogenesis and Produce Anxiolytic- And Antidepressant-Like Effects

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Cannabinoids Promote Embryonic and Adult Hippocampus Neurogenesis and Produce Anxiolytic- And Antidepressant-Like Effects

Wen Jiang et al. J Clin Invest. .
Free PMC article

Abstract

The hippocampal dentate gyrus in the adult mammalian brain contains neural stem/progenitor cells (NS/PCs) capable of generating new neurons, i.e., neurogenesis. Most drugs of abuse examined to date decrease adult hippocampal neurogenesis, but the effects of cannabis (marijuana or cannabinoids) on hippocampal neurogenesis remain unknown. This study aimed at investigating the potential regulatory capacity of the potent synthetic cannabinoid HU210 on hippocampal neurogenesis and its possible correlation with behavioral change. We show that both embryonic and adult rat hippocampal NS/PCs are immunoreactive for CB1 cannabinoid receptors, indicating that cannabinoids could act on CB1 receptors to regulate neurogenesis. This hypothesis is supported by further findings that HU210 promotes proliferation, but not differentiation, of cultured embryonic hippocampal NS/PCs likely via a sequential activation of CB1 receptors, G(i/o) proteins, and ERK signaling. Chronic, but not acute, HU210 treatment promoted neurogenesis in the hippocampal dentate gyrus of adult rats and exerted anxiolytic- and antidepressant-like effects. X-irradiation of the hippocampus blocked both the neurogenic and behavioral effects of chronic HU210 treatment, suggesting that chronic HU210 treatment produces anxiolytic- and antidepressant-like effects likely via promotion of hippocampal neurogenesis.

Figures

Figure 1
Figure 1
Expression of CB1 receptors in NS/PCs. (A) Coimmunofluorescent staining of CB1 and nestin in cultured hippocampal NS/PCs derived from E17 embryos. Hoechst staining was conducted to reveal the total cultured cells. The arrow indicates the glial-like cells, located in the center of a neurosphere, with CB1 staining and without nestin staining. Scale bar, 20 μm. (B) Western blot using cultured NS/PC reveals a 60-kDa protein band corresponding to CB1 receptor. (C) PCR indicates CB1 gene expression in NS/PCs (lane 2) using primers yielding a predicted product of 1,440 bp (i.e., the full encoding region of CB1 receptor) from embryonic NS/PCs. Lane 1: molecular weight standards; lane 2: CB1 receptor; lane 3: PCR reaction without sample added. (D) Confocal microscopic assessments of costaining of BrdU and CB1 receptors in the SGZ located between the hilus and the granule cell layer (granule) of the dentate gyrus in an adult rat. Scale bar, 10 μm.
Figure 2
Figure 2
Effects of the cannabinoid HU210 on proliferation of cultured hippocampal NS/PCs. (A) In the WST-8 assay, incubation of NS/PCs with 10 nM to 1 μM of HU210 for 48 hours significantly promoted NS/PC proliferation, which was blocked by the CB1 receptor antagonist AM281. AM281 alone significantly decreased NS/PC proliferation only with 10 μM, but this concentration of AM281 was not able to block the lethal effects of 10 μM of HU210 on NS/PCs. (B) BrdU incorporation assay confirmed the results obtained with the WST-8 assay shown in A. (C) Incubation of NS/PCs with 1 μM to 10 μM of AEA for 48 hours significantly promoted NS/PC proliferation in the WST-8 assay. (D) Application of 10 nM to 1 μM of HU210 significantly promoted NS/PC proliferation in both the presence and absence of the growth factors bFGF and EGF in the culture medium. (E) Pertussis (PTX; 100 ng/ml), a selective blocker for Gi/o protein activation, prevented the effects of 10 nM to 1 μM of HU210 on promoting NS/PC proliferation. (F) Incubation of NS/PCs with 1 mg/ml of cholera toxin, a selective Gs activator, stimulated a profound increase in cAMP accumulation in NS/PCs 0.5, 1, 2, and 24 hours after the addition of cholera toxin. (G) Incubation of NS/PCs with 1 mg/ml of cholera toxin for 0.5, 1, 2, 24, or 48 hours did not induce significant change in NS/PC proliferation. Error bars represent SEM. *P < 0.05 and **P < 0.01 by Tukey post-hoc tests after 1-way ANOVA.
Figure 3
Figure 3
Effects of the cannabinoid HU210 on PI3K/Akt and ERK signaling in cultured hippocampal NS/PCs. (A) There was no significant change in pAkt or actin in NS/PCs within the first hour after addition of 100 nM of HU210 to culture medium. (B) Application of 100 nM of HU210 rapidly induced phosphorylation of pERK1/2 in NS/PCs in the presence of bFGF and EGF in culture medium. (C) Application of 100 nM of HU210 3 hours after removal of bFGF and EGF from culture medium also induced phosphorylation of pERK1/2 in NS/PCs. (D) Application of the ERK signaling inhibitor U0126 blocked the promoting effects of 100 nM of HU210 on phosphorylation of pERK1/2 in NS/PCs 5 minutes after addition of HU210 to culture medium. (E) Addition of U0126 (10 μM) to the culture medium 1 hour before HU210 antagonized the promoting effects of 10 nM to 1 μM of HU210 on NS/PC proliferation. Error bars represent SEM. *P < 0.05 and **P < 0.01 by Tukey post-hoc tests after 1-way ANOVA. tERK1/2, total ERK1/2.
Figure 4
Figure 4
Effects of HU210 and AEA on neuronal differentiation of cultured hippocampal NS/PCs. (A) Incubation of NS/PCs with the culture medium containing either vehicle or 100 nM of HU210 without growth factors for 8 days produced similar density of neurons (pink cells) stained with TuJ1 antibody. The total cultured cells are labeled deep blue by Hoechst staining. (B) There was no significant difference in the ratio of TuJ1-labeled neurons to total cells following application of HU210 (10 nM to 1 μM) or AEA (1 or 5 μM) to culture medium.
Figure 5
Figure 5
Effects of HU210 treatment on cell proliferation in the dentate gyrus in adult rats (n = 5–7 rats in each group). Cell proliferation was assessed by BrdU labeling of dividing cells. (A) Representative microphotographs of the dentate gyrus show BrdU-positive cells clustered or aggregated in the SGZ in rats receiving an acute injection of vehicle or 100 μg/kg of HU210. Scale bar, 60 μm. (B) There was no significant difference in the average number of BrdU-stained cells in the dentate gyrus per section following 1 dose of acute vehicle, 100 and 25 μg/kg of HU210, and 3 mg/kg of AM281. (C) Representative microphotographs of the dentate gyrus show that twice-daily injections of 100 μg/kg of HU210 for 10 days apparently increased the density of BrdU-positive cells in the SGZ relative to chronic vehicle injection. Scale bar, 60 μm. (D) Relative to vehicle injection, there was a significant increase in the number of BrdU-immunoreactive cells in the dentate gyrus following chronic treatment with 100 μg/kg of HU210, but not 25 μg/kg of HU210 or 3 mg/kg of AM281. Error bars represent SEM. *P < 0.05 by Tukey post-hoc tests after 1-way ANOVA.
Figure 6
Figure 6
Fate and migration of BrdU-labeled cells in the dentate gyrus following chronic HU210 treatment. After receiving twice-daily injections of vehicle or 100 μg/kg of HU210 for 10 days, rats were given 4 BrdU injections, followed 1 month later by perfusion. (A) Representative confocal microscopic images show costaining (yellow) of BrdU (green) and NeuN (red) in the dentate granule cell layer. The majority of BrdU-stained cells are doubly labeled with the neuronal marker NeuN and located within the granule cell layer. 3D, 3 dimensional photograph of doubly stained neurons indicated with arrows. Scale bar, 20 μm. (B) Chronic HU210 significantly increased the number of BrdU-stained cells in the dentate gyrus (n = 5 in each group). (C) There was no significant difference in the proportion of cells doubly labeled with BrdU and NeuN to the total cells singly labeled with BrdU. Error bars represent SEM. **P < 0.01 by Student’s t test.
Figure 7
Figure 7
Effects of chronic HU210 on neuronal survival. (A) Both naive control rats and rats receiving twice-daily injections of HU210 (100 μg/kg) for 10 days showed similar density of NeuN-stained neurons in the dentate granule cell layer and CA3 pyramidal cell layer. (B) There was no significant difference in the total number of NeuN-stained cells in the dentate granule cell layer and CA3 pyramidal layer between naive and HU210-treated rats (n = 3 for each group). (C) While naive rats and chronic HU210-treated rats showed no TUNEL-stained cells in the hippocampus, kainic acid–treated (KA-treated) rats exhibited numerous TUNEL-positive neurons in the CA3 pyramidal cell layer and dentate granule cell layer. (D) While naive rats and chronic HU210-treated rats showed no Fluoro-Jade B–stained (FJB-stained) cells in the hippocampus, kainic acid–treated rats exhibited numerous Fluoro-Jade B–positive neurons in the CA3 pyramidal cell layer (n = 3 for each group). Scale bar, 60 μm.
Figure 8
Figure 8
Effects of chronic HU210 on the NSF test, the FST, and cell proliferation in the dentate gyrus. After receiving chronic vehicle, AM281, or HU210 injections for 10 days, rats were injected with BrdU to label dividing cells, followed 1 month later by behavioral testing and 1 day later by perfusion (n = 7–8 for each group in AC; n = 5 for each group in DF). (A) In the NSF test, rats receiving chronic HU210 (but not AM281) showed significantly shortened latency to feed in a novel environment but not in their home cages, suggesting anxiolytic effects produced by HU210. (B) In the FST, chronic HU210 (but not AM281) significantly shortened the duration of immobility (i.e., antidepressant-like effects). (C) Among the rats receiving vehicle, AM281, and HU210, there was no significant difference in the number climbing in the first 5 minutes in the pretest sessions of the FST. (D) Irradiation of the hippocampus prominently reduced cell proliferation in the SGZ. (E) Irradiation of the hippocampus blocked chronic HU210–induced shortened latency of rats to feed in novel environment but not in their home cages in the NSF test. (F) Irradiation of the hippocampus prevented chronic HU210–induced shortened duration of immobility in the FST. Error bars represent SEM. *P < 0.05 and **P < 0.01 by Tukey post-hoc tests after 1-way ANOVA.

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