doi: 10.1111/adb.12367. Epub 2016 Feb 1.
Expression of functional cannabinoid CB2 receptor in VTA dopamine neurons in rats
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- PMID:26833913
- PMCID: PMC4969232
- DOI: 10.1111/adb.12367
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Expression of functional cannabinoid CB2 receptor in VTA dopamine neurons in rats
Hai-Ying Zhang et al. Addict Biol.2017 May.
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Abstract
We have recently reported the expression of functional cannabinoid CB2 receptors (CB2 Rs) in midbrain dopamine (DA) neurons in mice. However, little is known whether CB2 Rs are similarly expressed in rat brain because significant species differences in CB2 R structures and expression are found. In situ hybridization and immunohistochemical assays detected CB2 gene and receptors in DA neurons of the ventral tegmental area (VTA), which was up-regulated in cocaine self-administration rats. Electrophysiological studies demonstrated that activation of CB2 Rs by JWH133 inhibited VTA DA neuronal firing in single dissociated neurons. Systemic administration of JWH133 failed to alter, while local administration of JWH133 into the nucleus accumbens inhibited cocaine-enhanced extracellular DA and i.v. cocaine self-administration. This effect was blocked by AM630, a selective CB2 R antagonist. These data suggest that CB2 Rs are expressed in VTA DA neurons and functionally modulate DA neuronal activities and cocaine self-administration behavior in rats.
Keywords: CB2 receptor; JWH133; cannabinoid; cocaine; dopamine; self-administration.
© 2016 Society for the Study of Addiction.
Figures
RNAin situ hybridization results, illustrating cannabinoid CB2 mRNA expression (green) in VTA DA neurons of naïve rats (A) and cocaine self-administration rats (B) measured by RNAscope ISH. A DAT probe was used to label VTA DA neurons (red) and DAPI was used to label cell nuclei (blue).C: Quantitative assay results in RNAscope ISH assays, illustrating that cocaine self-administration significantly increased CB2 mRNA expression in VTA DA neurons.D: Quantitative RT-PCR results, illustrating that chronic cocaine self-administration (4 weeks) significantly up-regulated CB2 mRNA expression in prefrontal cortex (PFC), striatum (STR) and midbrain. *p<0.05, ***p<0.001, compared with naïve rats or sucrose SA rats.
CB2R-immunostaining in VTA of rats, illustrating the binding sites (epitopes) of Alomone rCB2 antibody on CB2Rs (A). Preabsorption of Alomone rCB2-antibody by immune peptide blocked rCB2-staining in VTA DA neurons (B). CB2R- and tyrosine hydroxylase (TH)-staining in midbrain VTA and SNc under low magnification (10×) (C).D: The Alomone rCB2R-antibody detected rCB2R-immunostaining under high magnification (40×) in VTA DA neurons (TH-positive, marked by white filled arrows), non-DA neurons (TH-negative, green cells indicated by white triangles) and glial cells (indicated by white open arrows). Ab=antibody.
Electrophysiological assays in rats, illustrating that activation of CB2Rs by JWH133 inhibits neuronal firing in single dissociated VTA DA neurons.A: Identification of VTA DA neurons, illustrating a representative dissociated DA neuron (phase contrast, Lucifer yellow labeled and TH stained images,Aa), the effect of DA on neuronal firing (Ab), and on action potential (AP) profiles. VTA DA neurons exhibited low firing rates (1–3 Hz) with relatively long AP duration (Ac) and a characteristic H-current (Ad).B: Identification of VTA GABA neurons, illustrating a representative dissociated GABA neuron (Ba), the effect of DA on neuronal firing (Bb), and AP profiles. VTA GABA neurons exhibited high firing rates (>7 Hz,Bb) with short AP durations (Bc) and did not show H-currents (Bd).C: Representative trace of spontaneous neuronal firing before (Ca) and after (Cb) JWH133 (1 µM).D: group mean data, illustrating that JWH133 dose-dependently inhibited VTA DA neuronal firing, an effect that was blocked by AM630.E: Bath-applied JWH133 (1 µM) hyperpolarized resting membrane potential (RP), potentiated the after-hyperpolarization potential (AHP), but had no effect on AP amplitude and duration. The number in the each column indicates neuronal numbers tested for each treatment. *p<0.05, compared to baseline.
Effects of JWH133 on basal and cocaine-enhanced extracellular DA in the NAc of rats.A: Systemic administration of JWH133 did not significantly alter basal extracellular DA as compared to pre-JWH133 baseline.B: Intra-NAc local perfusion of JWH133 significantly reduced extracellular DA.C: Systemic administration of JWH133 failed to alter cocaine-enhanced extracellular DA.D: Local perfusion of JWH133 (100 µM) into the NAc attenuated cocaine-induced increases in extracellular DA. *p<0.05, **p<0.01, ***p<0.001, compared to pre-cocaine or pre-JWH133 baseline;##p<0.01,###p<0.001, compared to vehicle control group.
Effects of local intra-NAc administration of JWH133 on cocaine self-administration in rats.A: Microinjections of JWH133 into the NAc inhibited cocaine self-administration under FR2 reinforcement;B: intra-NAc microinjections of JWH133 lowered break-point levels for cocaine self-administration under PR reinforcement conditions. *p<0.05, compared to vehicle control group.
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