Localization and mechanisms of action of cannabinoid receptors at the glutamatergic synapses of the mouse nucleus accumbens

Abstract

Despite the role of excitatory transmission to the nucleus accumbens (NAc) in the actions of most drugs of abuse, the presence and functions of cannabinoid receptors (CB1) on the glutamatergic cortical afferents to the NAc have never been explored. Here, immunohistochemistry has been used to show the localization of CB1 receptors on axonal terminals making contacts with the NAc GABAergic neurons. Electrophysiological techniques in the NAc slice preparation revealed that cannabimimetics [WIN 55,212,2 (WIN-2) and CP55940] strongly inhibit stimulus-evoked glutamate-mediated transmission. The inhibitory actions of WIN-2 were dose-dependent (EC(50) of 293 +/- 13 nm) and reversed by the selective CB1 antagonist SR 141716A. In agreement with a presynaptic localization of CB1 receptors, WIN-2 increased paired-pulse facilitation, decreased miniature EPSC (mEPSC) frequency, and had no effect on the mEPSCs amplitude. Perfusion with the adenylate cyclase activator forskolin enhanced glutamatergic transmission but did not alter presynaptic CB1 actions, suggesting that cannabinoids inhibit glutamate release independently from the cAMP-PKA cascade. CB1 did not reduce evoked transmitter release by inhibiting presynaptic voltage-dependent Ca(2+) currents through N-, L-, or P/Q-type Ca(2+) channels, because CB1 inhibition persisted in the presence of omega-Conotoxin-GVIA, nimodipine, or omega-Agatoxin-IVA. The K(+) channel blockers 4-aminopyridine (100 micrometer) and BaCl(2) (300 micrometer) each reduced by 40-50% the inhibitory actions of WIN-2, and their effects were additive. These data suggest that CB1 receptors are located on the cortical afferents to the nucleus and can reduce glutamate synaptic transmission within the NAc by modulating K(+) channels activity.

Fig. 1.

Localization of CB1 receptors in the prelimbic cortex and the nucleus accumbens. Confocal images of single- or double-immunostained sections. A, Single immunostaining for CB1 shows that intense labeling is associated with perikarya located in the prelimbic cortex. B, CB1 labeling was also associated with large varicose axonal fibers that extend throughout the core of the NAc surrounding the anterior commissure.C, D, Double immunostaining for both CB1 (red) and GABA (green) shows that, within the NAc, CB1-IS fibers form terminal (arrows inC) or en passant (arrows inD) synaptic-like contacts with GABA-IS perikarya.AC, Anterior commissure. Scale bars, 25 μm.

Fig. 2.

CB1 receptor-mediated inhibition of evoked excitatory synaptic transmission in mice nucleus accumbens.A, The cannabimimetic WIN-2 (10 μm) reduced the fEPSP on average to 34 ± 5% (n = 12) of its basal value. Traces represent averages of 10 consecutive EPSPs. The effects of WIN-2 were reversed by the selective CB1 antagonist SR 141716A (10 μm). B, Preincubation of the slices with 10 μm of the CB1 antagonist SR 141716A was without effect on basal synaptic transmission (data not shown) but completely prevented the WIN-2-induced inhibition (n = 5).

Fig. 3.

Pharmacological characterization of the CB1-mediated inhibition. A, Dose–response curve for the CB1 agonist WIN-2. The EC₅₀ for WIN-2 was 291 ± 13 nm. Each point is expressed as the percentage of inhibition of basal evoked transmission, and the error bar represents the SEM. B, WIN 55,212,3 (10 μm), an enantiomer of WIN-2 inactive at CB1 binding sites, caused a small but significant fEPSP inhibition that, contrary to the WIN-2-induced inhibition, was not reversed by SR 141716A (10 μm; black bar). WIN-2-mediated fEPSP reduction was additive to the WIN 55,212,3 effect (hatched bar). These data are in agreement with the idea that WIN-2 at concentrations up to 10 μm is selective for CB1 receptors. C, The cannabinoid agonist CP 55940 (2 μm; n = 5), which is not structurally related to WIN-2, also inhibited fEPSP.

Fig. 4.

CB1-induced inhibition of action potential-independent glutamate release reveals a presynaptic site of action. A, B, Miniature EPSCs recorded in the presence of TTX are independent of Ca²⁺ channels activation. Bath application of cadmium chloride (100 μm) to block voltage-sensitive Ca²⁺ channels changed neither the amplitude distribution (A) nor the frequency distribution of mEPSCs (B).C, Representative consecutive 1 sec current sweeps from a cell (holding potential of −70 mV) in which mEPSCs were recorded in the absence or presence of 10 μm WIN-2. D, The distribution of mEPSC amplitude before and during the application of the CB1 agonist, in the seven cells recorded, was unchanged after 20 min bath perfusion of WIN-2. E, The distribution of the time intervals between successive mEPSCs in all of the neurons recorded (same as above) revealed that the mEPSCs frequency was reduced during WIN-2 application. For control conditions, a total of 1849 events were detected over a period of 9.14 ± 1.14 min (n= 7; range, 6–14 min). In the same neurons, a total of 1004 events were collected after 10–15 min WIN-2 perfusion over a period of 11.86 ± 2.20 min (range, 6–20 min).

Fig. 5.

CB1-mediated inhibition is independent of cAMP levels. A, The adenylate cyclase activator forskolin (FSK; 10 μm, 20 min) caused a robust augmentation of the fEPSP in NAc slices (n = 9).B, The WIN-2-mediated fEPSP inhibition was not affected when cAMP levels were augmented by bath application of forskolin (n = 7). Forskolin was applied 20 min before and during the WIN-2 application (10 μm).

Fig. 6.

The WIN-2-induced inhibition does not require N-, L-, or P/Q-type Ca²⁺ channels modulation.A, Typical experiment in which the slice was perfused with 1 μm ω-Conotoxin-GVIA, which blocked ∼60% of the fEPSP. It is clear that the fraction of synaptic transmission insensitive to ω-Conotoxin-GVIA was still sensitive to the CB1 agonist WIN-2 (10 μm). B, Summary of all of the experiments performed as above with specific blockers of N-type (ω-conotoxin-GVIA, 1 μm), L-type (nimodipine, 1 μm); and P/Q-type (ω-Agatoxin-IVA, 200 nN) voltage-sensitive Ca²⁺ channels. The histogram of the maximum inhibitions caused by perfusion of selective agents reveals how the different types of voltage-sensitive Ca²⁺channels contribute to the evoked release of glutamate in the NAc.C, Histogram of the maximum 10 μmWIN-2-mediated inhibition in the presence of Ca²⁺channel inhibitors. All experiments were performed as inA.

Fig. 7.

Effects of K⁺ channels blockade on the CB1-induced inhibition of glutamatergic synaptic transmission in the NAc. A, In standard ACSF (2.4 mm CaCl2;white bar), 4-AP (100 μm; hatched bar), and BaCl₂ (300 μm;dotted bar) reduced the WIN-2-induced fEPSP inhibition. WIN-2 reduced fEPSP by 39.1 ± 5.5% (n = 5;p = 0.015) and 32.3 ± 9.4% (n = 4; p = 0.029) in the presence of 4-AP and BaCl₂, respectively, compared with 65.6 ± 5.4% (n = 12) in control. When added together, 4-AP and BaCl₂ (black bar) completely prevented the WIN-2 (10 μm) inhibition (1.7 ± 11%; n = 4; p = 0.002). In contrast, the adenosine (200 μm; white bar)-induced inhibition was not affected by pretreatment with 4-AP and BaCl₂ (black bar). B, Representative experiment in which the large enhancement of the fEPSP caused by the combination of 4-AP and BaCl₂ was reduced by lowering extracellular Ca²⁺ concentration from 2.4 to 0.3 mm. In this condition, 4-AP and BaCl₂ still prevented the 1 μm WIN-2-induced inhibition. C, Summary of all the experiments performed as above (n = 8). It is clear that 4-AP (100 μm) and BaCl₂ (300 μm) blocked the CB1-mediated inhibition in low external Ca²⁺. Lowering the external Ca²⁺affected neither the time course nor the amplitude of the WIN-2 (1 μm)-mediated inhibition of the fEPSP [compare the inhibition in control ACSF (filled circles) with the inhibition in low external Ca²⁺ (open circles)].