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Cannabinoid CB2 receptors modulate midbrain dopamine neuronal activity and dopamine-related behavior in mice - PubMed

  • ️Wed Jan 01 2014

Cannabinoid CB2 receptors modulate midbrain dopamine neuronal activity and dopamine-related behavior in mice

Hai-Ying Zhang et al. Proc Natl Acad Sci U S A. 2014.

Abstract

Cannabinoid CB2 receptors (CB2Rs) have been recently reported to modulate brain dopamine (DA)-related behaviors; however, the cellular mechanisms underlying these actions are unclear. Here we report that CB2Rs are expressed in ventral tegmental area (VTA) DA neurons and functionally modulate DA neuronal excitability and DA-related behavior. In situ hybridization and immunohistochemical assays detected CB2 mRNA and CB2R immunostaining in VTA DA neurons. Electrophysiological studies demonstrated that activation of CB2Rs by JWH133 or other CB2R agonists inhibited VTA DA neuronal firing in vivo and ex vivo, whereas microinjections of JWH133 into the VTA inhibited cocaine self-administration. Importantly, all of the above findings observed in WT or CB1(-/-) mice are blocked by CB2R antagonist and absent in CB2(-/-) mice. These data suggest that CB2R-mediated reduction of VTA DA neuronal activity may underlie JWH133's modulation of DA-regulated behaviors.

Keywords: CB2 receptor; JWH133; cannabinoid; cocaine; dopamine.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

CB2 mRNA expression in WT, CB1−/−, and CB2−/− mice. (A) Mouse CB2 genomic structure and transcripts (mRNAs), illustrating that the CB2 gene contains three exons with two separate promoters (P1 and P2). (B) CB2A and CB2B transcripts and the binding sites of three TaqMan probes used to detect CB2 mRNA by RT-PCR. The CB2A and CB2B probes target the 5′ UTR, whereas the CB2-KO probe targets the CB2-deleted gene sequence in the Zimmer strain of CB2−/− mice. (C) CB2 mRNA was detectable in WT, CB1−/−, and CB2−/− mice when using the CB2A or CB2B probe, but was not detectable in CB2−/− mice when using the CB2-KO probe. The CB2 mRNA levels in each brain or spleen tissue are the relative levels (folds) compared with those in cortex of WT mice (defined as 1). All quantificated data are normalized to control (cortex). Error bars indicate ±SEM. *P < 0.05, compared with WT mice. NM_009924.2 and AK036658.1 are the GenBank cDNA codes.

Fig. 2.
Fig. 2.

CB2 mRNA expression in VTA DA neurons by ISH assays. (A, a) CB2A mRNA and the location detected by a CB2-riboprobe. (A, b) The antisense, but not the sense (control), riboprobe detected CB2 mRNA in midbrain neurons in WT and CB1−/− mice, but not in CB2−/− mice. (A, c) Double-label fluorescent images of CB2 mRNA (by ISH) and TH (by IHC) staining, illustrating CB2 mRNA staining in individual TH-positive VTA DA neurons in WT mice, but not in CB2−/− mice (Zimmer strain). (B, a) CB2A mRNA and the location detected by a CB2-RNAscope probe. (B, b and c) CB2-RNAscope probe-detected CB2 mRNA in VTA DA neurons in WT and CB2−/− mice. This probe detected CB2 mRNA in CB2−/− mice because it targets the downstream UTR region rather than the upstream gene-deleted region. Scales are shown in the figures. Also see Fig. S1.

Fig. 3.
Fig. 3.

Mouse CB2 immunostaining in VTA DA neurons. (A and B) Representative confocal images of CB2R immunostaining in VTA DA neurons, illustrating that the NIH-5633 (A) and Alomone (B) mCB2 antibodies (with epitopes in the deleted portion of the receptor in Zimmer CB2−/− mice) were detected by CB2 immunostaining in VTA DA neurons in WT and CB1−/− mice, but were barely detectable in CB2−/− mice. Preabsorption of the antibody by specific immune peptide blocked CB2R immunostaining. (C and D) Mean densities of CB2R immunostaining in VTA DA neurons of WT, CB1−/−, and CB2−/− mice. The numbers in the graph bars are numbers of TH-positive VTA DA neurons. All quantificated data are means ± SEM. ***P < 0.001, compared with WT mice. Also see Figs. S2–S5.

Fig. 4.
Fig. 4.

Activation of CB2Rs reduces VTA DA neuronal firing ex vivo. (A) Phase-photo image showing a dissociated VTA DA neuron. (B and C) Representative recording and summarized data illustrating that JWH133 and additional four CB2R agonists (GW405833, SER601, CB65, and HU308) inhibited VTA DA neuronal firing similarly in WT mice, but not in Zimmer CB2−/− mice. This inhibitory effect was blocked by coadministration of AM630 (1 μM). (D and E) Representative AP traces and summarized group data illustrating that JWH133 altered membrane potential (MP), AP firing rate, AP initiation, AP duration, and AHP in WT mice. (F and G) Representative depolarizing current-induced AP firing and summarized data illustrating that JWH133 decreased VTA DA neuronal excitability. (H and I) Representative records and summarized data illustrating that JWH133 or GW405833 (GW) inhibited VTA DA neuronal firing in brain slices in a concentration-dependent manner. This effect was blocked by coadministration of AM630 and was absent in CB2−/− mice. All quantificated data are normalized to control (predrug baseline). Error bars indicate ± SEM. *P < 0.05; **P < 0.01, compared with predrug controls. Also see Fig. S6.

Fig. 5.
Fig. 5.

CB2R activation inhibits VTA DA neuronal firing in vivo. (A) Brain section image illustrating the track of a recording electrode and tips (recording sites) of the electrodes in the brain, and a characteristic action potential in a VTA DA neuron. (B) Representative extracellular single unit recording illustrating that JWH133 (10 or 20 mg/kg i.p.) dose-dependently inhibited basal FR and BS of VTA DA neurons in an anesthetized WT mouse. This effect was reversed by AM630 (10 mg/kg) administered 10 min after JWH133 injection. (C) Normalized FRs over the pre-JWH133 baseline, illustrating that JWH133 dose-dependently inhibited VTA DA neuronal firing in WT and CB1−/− mice, but not in CB2−/− mice. (D and E) Representative single-unit recording and summarized data illustrating that AM630 (10 mg/kg) alone failed to alter basal FR or ISI CV, but slightly potentiated BS. AM630 pretreatment prevented 20 mg/kg JWH133-induced inhibition of neuronal firing. Subsequent administration of quinpirole (a DA D2R agonist, 0.1 mg/kg) inhibited VTA DA neuronal firing, which was reversed by haloperidol (a D2R antagonist, 0.1 mg/kg). All quantificated data are normalized to control. Error bars indicate ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 compared with predrug controls.

Fig. 6.
Fig. 6.

Microinjections of JWH133 into the VTA inhibit i.v. cocaine self-administration. (A, a) Brain section image illustrating the track of representative guide cannulae and representative microinjection sites in the VTA. (A, b and c) Representative cocaine self-administration records and summarized data, illustrating that intra-VTA JWH133 microinjections significantly inhibit cocaine self-administration in WT mice, but not in CB2−/− mice. (A, d) Bilateral microinjections of JWH133 into the VTA failed to alter oral sucrose self-administration behavior. (B, a and b) Microinjections of the same doses of JWH133 into a brain region adjacent and dorsal to the VTA (B, a) had no effect on cocaine self-administration (B, b). Up arrows indicate the last cocaine infusion allowed. All quantificated data are means ± SEM. *P < 0.05, compared with vehicle group. RN, red nucleus; SNr, substantia nigra pars reticulata.

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