The effect of cannabinoid type Ⅱ receptor on the excitability of substantia nigra dopaminergic neurons

Abstract

The biological effects of cannabinoids are mainly mediated by two members of the G-protein-coupled-receptor family: cannabinoid type 1 receptor (CB1R) and cannabinoid type 2 receptor (CB2R). Unlike CB1R, CB2R is considered a "peripheral" cannabinoid receptor. However, recent studies have found that CB2R is widely expressed in the central nervous system and is involved in dopamine related behavioral regulation, including dietary behavior, weight regulation, anxiety, and schizophrenia like behavior. Our previous laboratory research demonstrated that activating CB2R on dopaminergic neurons in the ventral tegmental area can regulate addictive behavior in animals by inhibiting neuronal excitability. However, it is currently unclear whether CB2R on dopaminergic neurons in the substantia nigra compacta (SNc) has similar therapeutic potential. Brain patch clamp results have shown that the CB2R agonist JWH133 significantly inhibits the discharge of SNc dopamine neurons in a concentration dependent manner. The pharmacological blocker AM630 of CB2R can reverse this inhibitory effect, indicating that the expression of CB2R in SNc dopaminergic neurons is functional. After treatment with JWH133, the number of induced action potentials decreased, and the peak potential interval time, action potential start time, and potential amplitude after hyperpolarization amplitude all increased. In addition, synaptic current results showed that JWH133 can significantly reduce the frequency of miniature excitatory postsynaptic currents, indicating that activating CB2R to some extent inhibits the release of presynaptic glutamate and indirectly excites postsynaptic neurons.

Keywords: AM630; JWH133; cannabinoid type II receptor; dopamine neurons; firing activity; substantial nigra.

FIGURE 1

Identification of dopaminergic neurons in the SNc of mice. (A) Green fluorescence represents the expression of CB2R-positive cells (top line). Red fluorescence represents the expression of TH-immunostaining neurons (middle line). Pictures in the bottom line indicated the co-localization of CB2R and TH positive cells. (B) The substantia nigra is located at the ventral midbrain. (C) IR-DIC video microscope of SNc neurons in the midbrain slice. (D) Electrophysiological characteristics of substantia nigra dopaminergic neurons. Current clamp recordings showed spontaneous low-frequency pacemaker activity and inward rectification property by injection of −150 pA hyperpolarized current. (E) In Gap free mode, SNc dopaminergic neurons exhibit slow and regular spontaneous firing activity. (F) The duration of the action potential (AP) is relatively long, with a half width of about 3 ms.

FIGURE 2

Effects of JWH133 on spontaneous firing of dopaminergic neurons in the SNc. The activation of CB2R reduces the discharge of dopamine neurons in vitro. (A) JWH133 significantly reduces the firing rate of dopaminergic neurons in the SNc. Representative pictures sowed the firing before (a), after JWH133 perfusion (b) and wash (c). (B) and (C) Summarized data showed that JWH133 dose-dependently inhibits firing frequency of dopamine neurons. The inhibitory effect of JWH133 on firing frequency was blocked by AM630 (1 μM) (n = 7, **P < 0.01, compared with control).

FIGURE 3

Effect of JWH133 on evoked firing of dopaminergic neurons in the SNc. Representative recordings (A)and summarized data (B) illustrated that JWH133 reduces evoked firing numbers of dopaminergic neuron when the neurons were injected 50 pA current. Representative recordings (C) and summarized data (C–G) illustrate that JWH133 increases ISI, AP initiation, AHP, and decreases AP numbers when neurons were injected with 50 pA current. (Figures 3C–F; paired t-test, ISI: t = 4.055, df = 6, P < 0.01; AP initiation: t = 3.392, df = 6, P < 0.05; AHP: t = 3.187, df = 6, P < 0.05; AP duration: t = 2.403, df = 6, P = 0.0531; AP number: t = 5.303, df = 6, P < 0.01). Data are presented as the mean ± SEM, compared with control.

FIGURE 4

The regulation of JWH133 on miniature excitatory/inhibitory postsynaptic current. (A) Typical traces showed the mEPSCs in the presence of TTX (1 μm) before (top trace) and after (bottom trace) JWH133 exposure. (B–D) Statistical analysis showed the influence of JWH133 on the frequency (n = 10, t = 3.146, df = 9, P < 0.05, Figure 4C), half-decay time (n = 10, t = 2.370, df = 9, P < 0.05, Figure 4D) and amplitude (n = 10, t = 0.09425, df = 9, P > 0.05, Figure 4B) of mEPSCs. (E) Typical traces of mIPSCs in the presence of NBQX (10 μM) and AP-5 (50 μM) before (top trace) and after (bottom trace) JWH133 (10 μmol/L) exposure using the whole cell patch-clamp recording in SNc slice. Cumulative probability analysis for mIPSC inter-event interval and amplitude under control conditions (baseline) and JWH133 exposure. (F–H). Comparison of mIPSC frequency, amplitude and half decay time before and after JWH133 exposure from SN slices tested (Amplitude: t = 0.1849, df = 9, P > 0.05; Frequency: t = 0.8929, df = 9, P > 0.05; Half decay time: t = 0.2911, df = 9, P > 0.05).