Gentiopicroside Attenuates Morphine Rewarding Effect through Downregulation of GluN2B Receptors in Nucleus Accumbens

Animals

Male adult C57BL/6 mice (8–10-weeks-old) weighing from 18 to 22 g were obtained from the Laboratory Animal Center of the Fourth Military Medical University. The animals were housed in plastic boxes in groups of four with food and water available ad libitum in a colony room with controlled temperature (24°C ± 2°C), humidity (50–60%), and a 12:12-h light–dark cycle. Mice were allowed to adapt to laboratory conditions for at least 1 week before the procedure. The experiments were performed during the light phase of the cycle. This study was performed in adherence with the National Institutes of Health guidelines for the use of experimental animals. The institutional ethical committee of the Fourth Military Medical University specifically approved this study.

CPP Procedures

CPP was performed in a CPP apparatus with a computer equipped with an automatic monitoring system (Jiliang, Shanghai, China). The apparatus comprised two compartments identical in size (30 × 30 × 40 cm each), separated by a platform with the sides painted white and black, respectively. Place conditioning was conducted as described previously with a minor modification [16]. The whole protocol consisted of the following five different phases: preconditioning test (1 trial), conditioning (2 trials/days for 7 days), Gent administration (i.p., twice daily for 8 days), 1 week no treatment, and morphine challenge on D21 (to test locomotor activity), over a total of 22 days (Figure 1A). The activity of each animal was recorded and measured on each test day to determine the time spent on each side of the experimental box when the guillotine doors were raised.

Slice Preparation and Whole-Cell Patch Clamp Recordings

Coronal brain slices (300 μm), containing the NAc, were prepared as previously described [18]. Slices were transferred to submerged recovery chamber with oxygenated (95% O₂ and 5% CO₂) artificial cerebrospinal fluid (ACSF) containing (in mM): 124 NaCl, 2.5 KCl, 2 CaCl₂, 2 MgSO₄, 25 NaHCO₃, 1 NaH₂PO₄, 10 glucose at room temperature for at least 1 h. Experiments were performed in a recording chamber on the stage of an Axioskop 2FS microscope with infrared DIC optics for visualization of whole-cell patch clamp recording. Excitatory postsynaptic currents (EPSCs) were recorded from the shell of NAc neurons with an Axon 200B amplifier (Axon Instruments, Foster City, CA, USA) and the stimulations were delivered by a bipolar tungsten stimulating electrode placed in the shell of NAc ∼100 μm apart from the recording electrode. AMPA receptor mediated EPSCs were induced by repetitive stimulations at 0.02 Hz and neurons were voltage clamped at −70 mV. The recording pipettes (3–5 MΩ) were filled with solution containing (mM) 145 K-gluconate, 5 NaCl, 1 MgCl₂, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, and 0.1 Na₃-GTP (adjusted to pH 7.2 with KOH). The NMDAR-mediated component of EPSCs was pharmacologically isolated in ACSF containing CNQX (20 μM), glycine (1 μM), and picrotoxin (100 μM). The patch electrodes contained 102 mM cesium gluconate, 5 mM TEA Chloride, 3.7 mM NaCl, 11 mM BAPTA, 0.2 mM EGTA, 20 mM HEPES, 2 mM MgATP, 0.3 mM NaGTP, and 5 mM QX-314 chloride (adjusted to pH 7.2 with CsOH) for the recording NMDAR-mediated EPSCs. Neurons were voltage clamped at −30 mV and NMDAR-mediated EPSCs were evoked at 0.05 Hz. Miniature excitatory synaptic currents (mEPSCs) were recorded in the neurons clamped at −70 mV with 0.5 μM TTX added in the ACSF. Access resistance was 15–30 MΩ and monitored throughout the experiment. Data were discarded if access resistance changed more than 15% during an experiment.

Western Blot

Equal amounts of protein (50 μg) from the NAc were separated and electrotransferred onto PDVF membranes (Invitrogen, San Diego, CA, USA), which were probed with antibodies for GluA1 (dilution ratio 1:300, Abcam), GluN2A (dilution ratio 1:200, Millipore, Billerica, MA, USA), GluN2B (dilution ratio 1:500, Millipore), D1 (dilution ratio 1:1000, millipore), D2 (dilution ratio 1:1000, Millipore), and with β-actin (dilution ratio 1:10000, Sigma) as a loading control. The membranes were incubated with horseradish peroxidase conjugated secondary antibodies (anti-rabbit/anti-mouse IgG for the primary antibodies), and bands were visualized using an ECL system (Perkin Elmer, Waltham, MA, USA).

Data Analysis

Results were expressed as mean ± SEM. Statistical comparisons were performed using a t-test or one-way ANOVA. In all cases, P < 0.05 was considered statistically significant.

Systemic Administration of Gent Attenuated Morphine-Induced Rewarding

The effects of Gent on morphine dependence were tested. Mice were first pretreated with morphine (s.c., 10 mg/kg) with conditioning place training for 7 days (D1–D7), followed by Gent (100 mg/kg, i.p., twice daily) or ifenprodil (5 mg/kg, i.p., twice daily) treatment for 8 days (D7–D14). Mice were then returned to their home cages for another 7 days (D15–D21) and were challenged with morphine (s.c., 10 mg/kg) on D21 (Figure 1A). No mice showed significant place preference for the drug-associated compartment before drug conditioning (D0), indicating that the CPP apparatus used was of a nonbiased design. After 7 days of conditioning with morphine (D1–D7, s.c., 10 mg/kg), mice showed significant place preference for the drug-paired compartment on D7. The mice were then administered with Gent (100 mg/kg) or ifenprodil (5 mg/kg) for 8 days (D7–D14) postmorphine withdrawal. The rewarding behaviors were tested on D7, D10, and D14 to evaluate the drug effects on morphine dependence. The mice still showed significant place preference on D10 and D14 even for 7 days with the administration of same volume of saline (0.5 mL/kg, i.p, twice daily) on D7–D14. However, the drug-seeking effect was attenuated on D10 and D14 (Figure 1B) when Gent (100 mg/kg) or ifenprodil (5 mg/kg) were administered on D7–D14 after morphine conditioning. Gent or ifenprodil itself did not show rewarding, aversive, or drug-seeking effect (Figure 1C).

Gent Reversal of Overexpression of GluN2B and D2 Receptors in the NAc of Morphine-Dependent Mice

Inotropic glutamate receptors, including NMDA and AMPA receptors, play pivotal roles in morphine rewarding [2, 5]. We examined the expression of NMDA and AMPA receptor subunits in total NAc homogenates by Western blot analysis to detect the effects of Gent on these receptors. Results showed that expression levels of AMPA receptor GluA1 subunit, as well as GluN2A- and GluN2B-NMDA receptors, increased after chronic morphine treatment compared with the saline control mice (Figure 2A). The increase in GluA1 and GluN2A subunits remained evident on D21 2 weeks postmorphine withdrawal or 1 week postGent withdrawal. Gent treatment (100 mg/kg) had no effect on the GluA1 and GluN2A subunit expression in the morphine-dependent mice (Figures 2B and C). However, GluN2B subunit expression was significantly reduced in the NAc of Gent-injected morphine-dependent mice (Figure 2D). Thus, the downregulated GluN2B subunit expression by Gent could explain the reduced NMDA receptor-mediated EPSCs in NAc synapses.

Next, we detected the expression of DA receptors, known as the major mediator for the morphine rewarding [7, 8]. The increase in the D1 and D2 receptors remained obvious on D21, 2 weeks postmorphine withdrawal or 1 week postGent withdrawal. Treatment of Gent (100 mg/kg, twice daily) had no effect on D1 receptor expression in the morphine-dependent mice (Figure 2E). However, D2 receptor expression was significantly reduced in the NAc of Gent-injected morphine-dependent mice (Figure 2F).

Downregulation of Excitatory Synaptic Transmission by Gent

The input–output relationships measuring EPSCs amplitude (output) as a function of the afferent stimulus intensity (input) were compared in the shell of the NAc on D14 to detect the effects of Gent on synaptic transmission in morphine-dependent mice (Figure 3A). First, the slope of the AMPA receptor-mediated EPSC (AMPA-EPSC) curves was significantly greater in neurons from morphine-dependent mice compared with saline controls (Figures 2B and C). These data suggest an increase in synaptic efficacy in NAc neurons from morphine-dependent mice. Gent treatment (100 mg/kg) could significantly reverse the enhancement of AMPA receptor-mediated synaptic transmission induced by chronic morphine injection (Figures 3B and C). Then, we recorded the input–output curves of NMDA receptor-mediated EPSC (NMDA-EPSC) and found that Gent also notably reversed the enhancement of NMDA receptor-mediated synaptic transmission in the morphine-dependent mice (Figures 3D and E). These results suggest that Gent could partially downregulate the enhancement of excitatory synaptic transmission in the NAc by chronic morphine administration.

Effects of Gent on Basal Glutamatergic Synaptic Transmission

We recorded AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) in the shell of NAc on D14 to further determine the presynaptic or postsynaptic component associated with an increase in the synaptic transmission (Figure 4A). A significant increase in mEPSC frequency and amplitude was detected in morphine-dependent mice compared with saline controls (Figures 4B and C). Gent treatment (100 mg/kg, twice daily) could significantly reverse the enhancement of AMPA receptor-mediated mEPSC amplitude induced by chronic morphine injection. However, this compound had no effect on the mEPSC frequency (Figures 4B and C). These results indicate that downregulation of excitatory synaptic transmission by Gent is likely due to postsynaptic AMPA receptor modifications in the NAc synapses of morphine-dependent mice.

Noninhibition of Morphine-Induced Behavioral Sensitization by Gent

Repeated administration of morphine results in increased locomotor activity that persisted even after long-term drug abstinence. This mechanism is called “behavioral sensitization.” Locomotor activity was determined on D21 after morphine challenge (10 mg/kg). The 8-day administration (D7–D14) of saline (0.5 mL/kg, i.p., twice daily) or Gent (100 mg/kg, i.p., twice daily) did not induce locomotor sensitization (Figure 5A). The total travel distance significantly increased in the morphine-dependent mice after morphine challenge (10 mg/kg) on D21, indicating that the animals developed behavioral sensitization (Figure 5B). Gent (100 mg/kg) or ifenprodil (5 mg/kg) administered from D7 to D14 after morphine injection did not inhibit total locomotor activity during the morphine challenge on D21 (Figure 5B). These data suggest that Gent cannot inhibit morphine-induced behavioral sensitization, though it can inhibit morphine-rewarding behavior.

Expression of NMDA Receptors in Morphine Rewarding

The important roles of NAc in the processing of morphine dependence and behavioral sensitization in animals have been well-documented [14, 15]. In the past decade, accumulated evidence indicate that NMDA receptors play a pivotal role in the development of tolerance and physical dependence to opiates [2]. NAc NMDA receptor subunit expression and function are enhanced in morphine-dependent animals [3]. Our present study shows that the expression of GluN2A was significantly increased in the NAc in morphine-injected mice, consistent with previous reports [3, 19]. Furthermore, we also found that the expression of GluN2B and AMPA receptor GluA1 subunit were significantly increased in the NAc in morphine-dependent mice, despite the discrepancy of GluN2B expression in morphine dependence [3, 20, 21]. The present study reveals that treatment of mice with Gent systematically (i.p.) resulted in a significant inhibition on morphine rewarding and reversed the upregulation of GluN2B receptors in the NAc. Systemic administration of ifenprodil, one specific GluN2B receptors antagonist, significantly reduced rewarding behavior. This result suggests that Gent inhibits morphine rewarding, at least partially by reversing the overexpression of GluN2B receptors. Reports likewise show that Gent treatment significantly suppresses the increase in TNF-α and shows anti-inflammatory activities [22]. We could not exclude the possibility that Gent may take effect by inhibiting the release of other mediators, for example, TNF-α, substance P, bradykinin, or serotonin.

DA Receptors in Morphine Rewarding

DA receptor is known for its crucial role in behavioral responses to morphine [6–8], and the subunits of DA receptor play different roles in morphine tolerance or dependence [9]. D2 receptor deletion significantly alters the reward-enhancing effects of morphine, suggesting that it plays an important role in rewarding effects [23, 24]. In the present study, we found that the expression of D1 and D2 receptors increased the NAc of mice with morphine dependence. Treatment of mice with Gent systematically (i.p.) reversed the upregulation of D2 receptor, but not the D1 receptor in the NAc. However, whether there are functional consequences of such differences of Gent on DA receptor expression remains unclear.

Effects of Gent on the Synaptic Transmission in the NAc of Morphine-Dependent Mice

Previous reports have revealed that the shell and core subdivisions of the NAc play different functional roles in motivated behavior [25–27]. The in vivo neurochemical evidence shows the functional compartmentation within the NAc and the preferential effect of psychostimulants and morphine in the shell of the NAc at doses known to sustain intravenous drug self-administration [25]. In present study, we selected to record in the shell of the NAc because of the more pronounced functional alteration in the shell compared with the core. Numerous studies on morphine dependence on synaptic transmission are focused on the NMDA-EPSCs. However, documents on morphine dependence on NMDA synaptic transmission are not consistent. For example, Martin et al. reported that chronic morphine treatment significantly decrease NMDA receptor-mediated synaptic transmission in the accumbens, and these effects persist for 1 week postwithdrawal [28]. By contrast, recent studies have reported that NMDA receptor subunit expression and function in NAc are enhanced in morphine-dependent rats [3, 20]. In the different brain regions, chronic morphine administration significantly increased protein levels of NR1 and GluN2B subunits in NAc but not in the central medial amygdala. This finding indicates a region-specific response of NMDA receptor subunits to chronic morphine administration at protein levels [29]. In the present study, we found that both AMPA- and NMDA-EPSC were increased in the morphine-dependent mice when recorded in the first week of morphine withdrawal. Gent treatment (100 mg/kg, twice daily) significantly reversed the enhancement of synaptic transmission. Quantal release analysis by AMPA mEPSC recordings revealed that downregulation of excitatory synaptic transmission by Gent was likely due to postsynaptic AMPA receptor modifications in the NAc synapses of morphine-dependent mice. This mechanism was demonstrated by unchanged mEPSCs frequency and decreased mEPSCs amplitude in the recordings and raises the possibility that Gent targets the postsynaptic modulation of GluN2B receptors in the NAc. AMPA receptors are heteromeric complexes composed of four distinct subunits of GluA1–4. AMPA receptor trafficking is perceived to be critical for long-term potentiation and depression [4, 5]. The phosphorylation of the AMPA receptor GluA1 subunit plays a key role in its synaptic expression and synaptic plasticity [6, 7]. In present study, we found that Gent had nonsignificant effect on the increased GluA1 expression caused by morphine as shown by Western Blot analysis. However, whole-cell recording revealed that the enhanced amplitude of AMPA-mEPSCs was significantly reduced by Gent treatment. These results imply that Gent might depress the AMPA receptor-mediated synaptic transmission by attenuating the phosphorylation level of GluA1 or inhibiting the AMPA receptors trafficking to the membrane by morphine.

Effects of Gent on the Morphine Dependence and Locomotor Sensitization

CPP test and behavioral sensitization of locomotor activity were used to investigate drug-seeking related behaviors, which correlate with psychological dependence. Results showed that Gent could completely abolish the CPP effect induced by morphine, but had no effect on morphine-induced behavioral sensitization. These results are consistent with the reports that dextromethorphan (a noncompetitive NMDA antagonist) or siRNA against the GluN2B subunit can completely abolish the CPP effect induced by morphine, but had no effect on morphine-induced behavioral sensitization [30, 31].

Hence, our findings provide evidence that Gent inhibits morphine dependence through the downregulation of GluN2B-containing NMDA receptors in the NAc. Hopefully, this study can help further elucidate the underlying mechanisms of Gent during morphine dependence and aid on the clinical treatment of drug rewarding with traditional herbs.