Blocking TRPV1 in nucleus accumbens inhibits persistent morphine conditioned place preference expression in rats

Abstract

The function of TRPV1 (transient receptor potential vanilloid subfamily, member 1) in the central nervous system is gradually elucidated. It has been recently proved to be expressed in nucleus accumbens (NAc), a region playing an essential role in mediating opioid craving and taking behaviors. Based on the general role of TRPV1 antagonist in blocking neural over-excitability by both pre- and post-synaptic mechanisms, TRPV1 antagonist capsazepine (CPZ) was tested for its ability to prohibit persistent opioid craving in rats. In the present study, we assessed the expression of TRPV1 in nucleus accumbens and investigated the effect of CPZ in bilateral nucleus accumbens on persistent morphine conditioned place preference (mCPP) in rats. We also evaluated the side-effect of CPZ on activity by comparing cross-beam times between groups. We found that morphine conditioned place preference increased the TRPV1 expression and CPZ attenuated morphine conditioned place preference in a dose-dependent and target-specific manner after both short- and long-term spontaneous withdrawal, reflected by the reduction of the increased time in morphine-paired side. CPZ (10 nM) could induce prolonged and stable inhibition of morphine conditioned place preference expression. More importantly, CPZ did not cause dysfunction of activity in the subjects tested, which indicates the inhibitory effect was not obtained at the sacrifice of regular movement. Collectively, these results indicated that injection of TRPV1 antagonist in nucleus accumbens is capable of attenuating persistent morphine conditioned place preference without affecting normal activity. Thus, TRPV1 antagonist is one of the promising therapeutic drugs for the treatment of opioid addiction.

Figure 1. Repeated morphine conditioning induced persistent conditioned place preference (CPP) during withdrawal.

Morphine conditioned place preference scores (time spent in drug-paired chamber) increased during morphine withdrawal. Sustained increase of mCPP score during morphine withdrawal occurred in high-dose morphine group (10 mg/kg) but not in low- (1 mg/kg) or moderate-dose (3 mg/kg) groups (n = 3). *p<0.01, vs SAL group. SAL, saline; MOR, morphine.

Figure 2. Analysis of western blot results of TRPV1 expression in the nucleus accumbens (NAc).

(A) Representative blots of TRPV1. β-actin showed similar protein loading in each group. (B) Densitometric analyses of TRPV1 expression in NAc. The changes were expressed as the ratio to β-actin. Data represent Mean±SEM (n = 3), *p<0.01, vs control group.

Figure 3. Quantitative changes in TRPV1 expression at the ultrastructural level in morphine withdrawal groups.

High-power electron micrographs from the NAc of different groups showing TRPV1 immunogold labeling in the axon terminals forming asymmetric, excitatory-type synapses (A) and symmetric, inhibitory-type synapses (B). In the morphine withdrawal groups the number of gold particles on terminals forming asymmetric synapses increased (A). Graph (C) shows changes in the average number of TRPV1-positive asymmetric and symmetric synapses in each group. The number of TRPV1-positive asymmetric synapses in the morphine withdrawal groups was significantly higher than the control group. However, no difference was found in the normalized number of TRPV1-positive symmetric synapses among all groups. The number of TRPV1-positive symmetric synapses of control were 4.72±0.77, 4.72±0.47 and 4.74±1.00 for 1 d, 1 w and 3 w group respectively. The number of TRPV1-positive asymmetric synapses of control were 7.74±1.38, 7.75±0.77 and 7.74±1.28 for 1 d, 1 w and 3 w group respectively. Graph (D) shows the normalized number of gold particles in TRPV1-positive asymmetric and symmetric synapses. No difference appeared in the normalized number of gold particles in TRPV1-positive symmetric synapses. In contrast, a significant increase occurred in the normalized number of gold particles in TRPV1-positive asymmetric synapses of morphine withdrawal groups versus control group. The normalized number of gold particles in TRPV1-positive symmetric synapses of control were 0.68±0.17, 0.68±0.03 and 0.69±0.04 for 1 d, 1 w and 3 w group respectively. The normalized number of gold particles in TRPV1-positive asymmetric synapses of control were 0.78±0.07, 0.79±0.13 and 0.79±0.03 for 1 d, 1 w and 3 w group respectively. Scale: 200 nm. Data represent Mean±SEM (n = 3), *p<0.05, vs control group.

Figure 4. TRPV1 antagonist CPZ administered in bilateral NAc core decreased persistent morphine CPP after both short-(1 w) and long-term(3 w) withdrawal.

(A) Dose-dependent effect of CPZ on persistent mCPP expression. Repeated high-dose CPZ (10 nM) injection during short-term morphine withdrawal could induce a complete and prolonged inhibition on mCPP expression. The moderate-dose CPZ (1 nM) has similar but temporary prohibiting effect on mCPP expression, as it could only be observed at 1 w but not 3 w of morphine withdrawal. The low-dose CPZ (0.1 nM) has no influence on mCPP. The upper schedule panel shows the rats were trained conditioned to morphine and received repeated CPZ injections in NAc core after short-term(1 w) withdrawal (n = 11 of Vehicle and CPZ 0.1 nM group, n = 10 of CPZ 1 nM group, and n = 9 of CPZ 10 nM group). (B) CPZ (1 nM and 10 nM) administration eliminated mCPP (n = 7). The upper schedule panel shows the rats were conditioned to morphine and received CPZ injections in NAc core after long-term(3 w) withdrawal. *p<0.01, vs control group; ^# p<0.05, vs control group. MOR, morphine; Veh, vehicle; CPZ, capsazepine; NAc, nucleus accumbens.

Figure 5. Distribution of infusion sites in nucleus accumbens core.

(A) Photograph of representative cannula placements. The cannulas are implanted 1.5 mm above the target. (B) Schematic representations of vehicle or capsazepine injection sites in nucleus accumbens core at the labeled coronal planes. The numbers are the coordinates anterior (+) to bregma in millimeters according to Paxinos and Watson (2005).

Figure 6. Injection of TRPV1 antagonist CPZ in unilateral of NAc or DS did not change persistent mCPP expression after short-term(1 w) withdrawal.

(A) Unilateral NAc CPZ injection (10 nM) or one side off-target during bilateral NAc CPZ infusion (1 nM or 10 nM) failed to affect the sustained mCPP. The upper panel shows the scheme for morphine conditioning, CPZ treatment and mCPP test. Values are Mean ±SEM (n = 7 of Vehicle and Uni-NAc+CPZ group and n = 3 of Uni-off NAc+CPZ group). (B) CPZ (10 nM) injection in DS failed to influence persistent morphine CPP. The upper panel shows rats were conditioned to morphine and received CPZ injections in bilateral DS after short-term withdrawal. Values are mean ±SEM (n = 6 of DS-Vehicle group and n = 7 of DS-CPZ 10 nM group). MOR, morphine; Veh, vehicle; CPZ, capsazepine; NAc, nucleus accumbens; Uni, unilateral; DS, dorsal striatum.

Figure 7. TRPV1 antagonist CPZ administered in bilateral NAc fail to affect normal activity after both short-(1 w) and long-term(3 w) withdrawal.

(A) All doses of CPZ tested failed to influence the activity of animal measured by photobeam breaks during the 15-min mCPP test (n = 11 of Vehicle and CPZ 0.1 nM group, n = 10 of CPZ 1 nM group, and n = 9 of CPZ 10 nM group). The upper schedule panel shows the rats were trained conditioned to morphine and received repeated CPZ injections in NAc core after short-term (1 w) withdrawal. (B) Both effective doses of CPZ (1 nM and 10 nM) in eliminating sustained mCPP expression fail to affect activity (n = 7). The upper schedule panel shows the rats were conditioned to morphine and received CPZ injections in NAc core after long-term (3 w) withdrawal.Values are mean ±SEM. MOR, morphine; Veh, vehicle; CPZ, capsazepine; NAc, nucleus accumbens.