Mechanisms of activation of nucleus accumbens neurons by cocaine via sigma-1 receptor-inositol 1,4,5-trisphosphate-transient receptor potential canonical channel pathways

Abstract

Cocaine promotes addictive behavior primarily by blocking the dopamine transporter, thus increasing dopamine transmission in the nucleus accumbens (nAcc); however, additional mechanisms are continually emerging. Sigma-1 receptors (σ1Rs) are known targets for cocaine, yet the mechanisms underlying σ1R-mediated effects of cocaine are incompletely understood. The present study examined direct effects of cocaine on dissociated nAcc neurons expressing phosphatidylinositol-linked D1 receptors. Endoplasmic reticulum-located σ1Rs and inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) were targeted using intracellular microinjection. IP3 microinjection robustly elevated intracellular Ca(2+) concentration, [Ca(2+)]i. While cocaine alone was devoid of an effect, the IP3-induced response was σ1R-dependently enhanced by cocaine co-injection. Likewise, cocaine augmented the [Ca(2+)]i increase elicited by extracellularly applying an IP3-generating molecule (ATP), via σ1Rs. The cocaine-induced enhancement of the IP3/ATP-mediated Ca(2+) elevation occurred at pharmacologically relevant concentrations and was mediated by transient receptor potential canonical channels (TRPC). IP3 microinjection elicited a slight, transient depolarization, further converted to a greatly enhanced, prolonged response, by cocaine co-injection. The cocaine-triggered augmentation was σ1R-dependent, TRPC-mediated and contingent on [Ca(2+)]i elevation. ATP-induced depolarization was similarly enhanced by cocaine. Thus, we identify a novel mechanism by which cocaine promotes activation of D1-expressing nAcc neurons: enhancement of IP3R-mediated responses via σ1R activation at the endoplasmic reticulum, resulting in augmented Ca(2+) release and amplified depolarization due to subsequent stimulation of TRPC. In vivo, intra-accumbal blockade of σ1R or TRPC significantly diminished cocaine-induced hyperlocomotion and locomotor sensitization, endorsing a physio-pathological significance of the pathway identified in vitro.

Keywords: Calcium; Endoplasmic reticulum; Imaging; Nucleus accumbens; Sigma receptors; Transient receptor potential channels.

Figure 1. Neurons of interest

a, Nucleus accumbens neurons express both σ₁Rs and IP₃R3 proteins; NG108 cells were used as positive control; β-actin was used as an internal control. Results are representative for three independent experiments. b, Functional characterization of phosphatidylinositol-linked D₁ dopamine receptor expression in nAcc neurons: left panel -averaged Ca²⁺ responses induced by D₁ agonist SKF83959 (10 μM) upon extracellular administration to D₁-expressing nAcc neurons incubated in Ca²⁺-containing saline (left) or in Ca²⁺-free saline, in absence (middle) and presence of IP₃R blockers xestospongin C (XeC) and 2-APB (right); right panel – comparison of the Ca²⁺ responses produced by SKF83959 in the mentioned conditions; P < 0.00001 compared to basal Ca²⁺ levels (*) or to SKF83959 in Ca²⁺-free HBSS (**). Neurons not responding to SKF83959 with an increase in [Ca²⁺]_i were not used further for experiments. c, Lack of effect of cocaine microinjection alone on [Ca²⁺]_i of D₁-positive nAcc neurons: left - averaged tracings of the Ca²⁺ responses produced by intracellular microinjection of either control buffer or cocaine (C, 100 μM); right - Comparison of the amplitudes and areas under curve (A.U.C.) of the Ca²⁺ responses; lower concentrations of cocaine were similarly ineffective.

Figure 2. Cocaine shifts to the left the IP₃-induced concentration-Ca²⁺ response curve via σ₁R

a, Averaged Ca²⁺ responses elicited by IP₃ (20 nM) microinjection alone or in combination with cocaine (10 μM) or by IP₃ (20 nM) and cocaine (10 μM) coinjection upon σ₁R blockade with either BD-1063 (BD, 10 μM) or NE-100 (NE, 3 μM). b, Concentration-response curves indicating the effect of IP₃ (1, 10, 20, 30, 40, 50 and 60 nM) microinjection into D₁-expressing nAcc neurons (incubated in Ca²⁺-free saline) when injected alone (black) or in combination with 10 μM cocaine (red); P < 0.005(*), P <0.001(**) and P < 0.00001(***); comparison of the two data sets yielded statistical significance for the two fits: F = 5.1715; P = 0.0378 < 0.05. c, Comparison of the Ca²⁺ increases triggered by treatments mentioned in a; P < 0.00001 compared with IP₃ (*) or to combined cocaine and IP₃ microinjection (#).

Figure 3. σ₁R-mediated enhancement by microinjected cocaine of the IP₃-induced [Ca²⁺]_i increase

a, Averaged tracings of the Ca²⁺ responses elicited by intracellular microinjection of IP₃ (20 nM) alone or in presence of σ₁R antagonist BD-1063 (BD, 10 μM); or cocaine (C, 10μM) and IP₃ (20 nM) in absence, or presence of either BD (10 μM) or NE-100 (NE, 3 μM); the fast Ca²⁺ chelator BAPTA-AM (200 μM); or TRPC blocker SKF96365 (SKF, 2 μM). b, Comparison of the amplitudes and areas under curve (A.U.C.) of the Ca²⁺ responses; P < 0.00001 compared with IP₃ alone (*), with combined cocaine and IP₃ microinjection (#), or with all other treatment groups (+). c, Fura-2 AM fluorescence ratios (340 nm/380 nm) of cultured nAcc neurons before and after D₁ agonist SKF83959 (10 μM), after washing of SKF83959 and after microinjection of indicated compounds in absence and presence of the antagonist pretreatment indicated in the right side; cold colors indicate low levels of [Ca²⁺]_i; hot colors indicate high levels of [Ca²⁺]_i; fluorescence scale (0–2) is magnified in each panel showing the effect of injected compounds.

Figure 4. Cocaine does not enhance cADPR or OAG-mediated Ca²⁺ signaling in nAcc neurons

a, Averaged Ca²⁺ tracings indicating the response of Ca²⁺-free saline-incubated neurons to microinjection of 20 μM cADPR alone or co-injected with 10 μM cocaine or to microinjection of 50 μM cADPR. b, Comparison of the amplitudes and areas under curve of the Ca²⁺ responses to treatments indicated in a; P < 0.00001 compared to basal Ca²⁺ levels (*) or to the effect of 20 μM cADPR (**). c, Averaged Ca²⁺ tracings corresponding to the effects produced by OAG (75 μM) in Ca²⁺-free and Ca²⁺-containing saline, in absence and presence of cocaine (10 μM), or to the effect of 100 μM OAG in Ca²⁺-negative and Ca²⁺-positive conditions. d, Comparison of the effects in Ca²⁺-containing saline induced by treatments indicated in d; P < 0.00001 compared to basal Ca²⁺ levels (*) or to the effect of 75 μM OAG in presence of extracellular Ca²⁺ (**).

Figure 5. SKF96365 (2 μM) is devoid of inhibitory activity at voltage-gated Ca²⁺ channels in nAcc neurons

a, Averaged Ca²⁺ responses induced by 30 mM KCl in absence and presence of SKF96365 (2 μM). b, The amplitudes of the Ca²⁺ increases produced by KCl measured 357 ± 5.6 nM (n = 31 cells) in absence and 351 ± 5.9 nM (n = 37) in presence of SKF96365; the areas under curve were 828 ± 11.3nM x min and 817 ± 10.9 nM x min, respectively.

Figure 6. Cocaine potentiates the Ca²⁺ response of nAcc neurons to IP₃-generating molecules

a–b, ATP promotes Ca²⁺ mobilization from IP₃-sensitive pools in accumbens neurons: a, Averaged tracings of the Ca²⁺ responses elicited by ATP (20 μM) in Ca²⁺-free saline, in absence or presence of IP₃R blockers xestospongin C (XeC) and 2-aminoethoxydiphenyl borate (2-APB); ryanodine receptor blocker ryanodine (Ry); or lysosomal two-pore channel inhibitor Ned-19. b, Comparison of the amplitudes and the areas under curve (A.U.C.) of the Ca²⁺ increases produced by the indicated treatments; *P < 0.00001 compared with all other treatment groups. c–d, Cocaine produces σ₁R-dependent potentiation of the ATP-induced Ca²⁺ elevation in D₁-expressing accumbens neurons: c, Averaged tracings of the Ca²⁺ responses induced by bath application of ATP (20 μM) or cocaine (C, 10 μM) and ATP (20 μM), in naïve neurons or neurons incubated with either σ₁R antagonist BD-1063 (BD, 10 μM); or Ca²⁺ chelator BAPTA-AM (200 μM); or TRPC blocker SKF96365 (SKF, 2 μM). d, Comparison of the amplitudes and the areas under curve (A.U.C.) of the Ca²⁺ responses produced by the indicated treatments; P < 0.00001 compared with ATP alone (*), with combined cocaine and ATP administration (#), or with all other treatment groups (+).

Figure 7. Intracellular microinjection of cocaine enhances IP₃-induced depolarization in the nAcc

a–b, Cocaine microinjection alone does not modify neuronal membrane potential: a, Representative recordings of the resting membrane potential of D₁-expressing nAcc neurons treated intracellularly with either control vehicle or cocaine (C, 10 μM, final intracellular concentration). b, Neither control vehicle microinjection or cocaine microinjection did produce a significant change in neuronal membrane potential. c-d, Cocaine potentiates the IP₃-dependent depolarization of D1-expressing accumbens neurons: c, Characteristic changes in resting membrane potential of neurons microinjected with either IP₃ (20 nM) or IP₃ (20 nM) and cocaine (C, 10 μM) in absence and presence of bath-applied σ₁R inhibitor BD-1063 (BD, 10 μM), of the Ca²⁺ chelator BAPTA-AM (200 μM) or of TRPC blocker SKF96365 (SKF, 2 μM). d, Comparison of the amplitudes of the depolarizations induced by treatment conditions described in c; P < 0.00001 compared with IP₃ injection alone (*), with combined cocaine and IP₃ microinjection (#), or with all other treatment groups (+).

Figure 8. Cocaine enhances the ATP-induced depolarization

a, Typical recordings of membrane potential modifications of D1-expressing accumbens neurons treated extracellularly with ATP (20 μM) or ATP (20 μM) and cocaine (C, 10 μM) in absence and presence of the σ₁R inhibitor BD-1063 (BD, 10 μM), of the Ca²⁺ chelator BAPTA-AM (200 μM) or of the TRPC blocker SKF96365 (SKF, 2 μM). b, Comparison of the amplitudes of the depolarizations induced by treatments described in a; P < 0.00001 compared with ATP alone (*) or with combined cocaine and ATP administration (#).

Figure 9. Cocaine-induced behavioral hyperactivity and sensitization involves σ₁R and TRPC

a, Coronal section indicating the distribution of infusion sites in the nAcc for the 35 experimental animals (aCSF-saline, n = 5; aCSF-cocaine, n = 7; BD-1063-saline, n = 5; BD-1063-cocaine, n = 6; SKF96365-saline, n = 6; SKF96365-cocaine, n = 6). Injection sites may appear fewer than the reported number of rats because of overlap of placements. b, Pretreatment with BD-1063 (80 μg/side) or SKF96365 (20 μg/side) administered 20 min prior to each cocaine injection for 5 days significantly inhibited cocaine-induced ambulatory activity on Days 2–5, and the development of repeated cocaine-induced locomotor sensitization on Day 12. Repeated administration of either antagonist alone did not alter levels of locomotion; *P < 0.05, aCSF-cocaine rats were significantly different from all other experimental groups; # SKF96365-cocaine rats were significantly different from aCSF-saline, BD-1063-saline and SKF96365-saline on day 5. Data are presented as mean +/− sem ambulatory counts/60 minutes; abbreviations: aV, intra-nAcc aCSF + ip saline vehicle; BV, intra-nAcc BD-1063 + ip saline vehicle; SVm intra-nAcc SFK96365 + ip saline vehicle; aC, intra-nAcc aCSF + ip cocaine; BC, intra-nAcc BD1063 + ip cocaine; SC, intra-nAcc SKF-96365 + ip cocaine.

Figure 10. Mechanism of cocaine-induced activation of D₁-expressing nAcc neurons

Cocaine activates endoplasmic reticulum (ER)-located σ₁Rs and potentiates Ca²⁺ release from the ER via IP₃ receptors type 3 (IP₃ R3) promoted by GPCR agonists (Gq-coupled, such as ATP). The increase in cytosolic Ca²⁺ triggers activation of TRPC and additional Ca²⁺ entry, as well as Na²⁺ entry, followed by depolarization and activation of these neurons, triggering hyperlocomotion and behavioral sensitization in vivo.