Blockade of ionotropic glutamatergic transmission in the ventral tegmental area attenuates the physical signs of morphine withdrawal in rats

Progress in Neuro-Psychopharmacology & Biological Psychiatry 28 (2004) 1079 – 1087 www.elsevier.com/locate/pnpbp

Blockade of ionotropic glutamatergic transmission in the ventral tegmental area attenuates the physical signs of morphine withdrawal in rats Hui-Ling Wanga, Yan Zhaoa,*, Xiao-Hui Xianga, Hui-Sheng Wanga, Wei-Ran Wub a Department of Physiology, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China Department of Behavioral Neuroscience, Oregon Health and Science University, Portland, OR 97239, USA

b

Accepted 10 May 2004 Available online 31 July 2004

Abstract The present study sought to assess whether the blockade of ionotropic glutamate receptors in the ventral tegmental area (VTA) could modulate the morphine withdrawal in male Sprague–Dawley rats. The effects of dizocilpine (MK-801) or 6,7-dinitroquinnoxaline-2,3-dione (DNQX), ionotropic glutamate receptor antagonists, microinjected unilaterally into the VTA 30 min before naloxone [2 mg/kg, intraperitoneally (i.p.)] administration on the morphine withdrawal were assessed. Morphine dependence was developed with increasing morphine injection (i.p.), and morphine withdrawal was induced by injection of naloxone (2 mg/kg, i.p.). Jumping, wet-dog shakes, writhing posture, wall clamber, weight loss and Gellert–Holtzman scale were used as the indices to evaluate the intensity of morphine withdrawal. The results showed that unilateral microinjection of MK-801 or DNQX into the VTA significantly increased the incidence of wall clamber, had no effect on weight loss, and reduced all other symptoms of morphine withdrawal. These data suggest that the ionotropic glutamate receptors in the VTA are involved in mediating naloxone-precipitated opiate withdrawal. D 2004 Elsevier Inc. All rights reserved. Keywords: AMPA receptor; DNQX; MK-801; Morphine; Morphine withdrawal signs; NMDA receptor; VTA

1. Introduction In common with many other abused substances, the abrupt cessation of chronic opiate use results in a wellcharacterized withdrawal syndrome with symptoms that include pain sensitivity, dysphoria, irritability, restlessness, insomnia, diarrhea and hyperventilation. These syndromes are adverse enough to compel the use of opiates for relieving withdrawal symptoms. Studies have identified that the mesocorticolimbic (MCL) dopaminergic pathway Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; DA, dopamine; DNQX, 6,7-dinitroquinnoxaline-2,3-dione; i.p., intraperitoneally; MCL, mesocorticolimbic; MK-801, dizocilpine; mPFC, medial prefrontal cortex; NAcc, nucleus accumbens; NMDA, N-methyl-daspartic acid; VTA, ventral tegmental area. * Corresponding author. Tel.: +86 29 82655171; fax: +86 29 85267351. E-mail addresses: [email protected] (H.-L. Wang), [email protected] (Y. Zhao). 0278-5846/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2004.05.043

consisting of the ventral tegmental area (VTA)—the nucleus accumbens (NAcc)—medial prefrontal cortex (mPFC) as being involved in opiate withdrawal (Harris and Aston-Jones, 1994; Koob and Bloom, 1988). Dopaminergic neurons in the VTA mainly project into NAcc and mPFC and project into the lateral hypothalamus (LH) and the brainstem. The hypothalamus and the mPFC are components of the central autonomic network which controls both autonomic and endocrine functions (Kandel et al., 2001; Kitahama et al., 2000). Moreover, the studies of Swanson (1982) have demonstrated that dopaminergic efferent fibers from the VTA terminated at the locus coeruleus bilaterally. Therefore, the decreased dopaminergic activity in the VTA induced by morphine withdrawal increases the activity of noradrenalinergic and cholinergic system which are mainly involved in morphine withdrawal symptoms. These data support that dopaminergic activity in the VTA is directly or is indirectly involved in morphine withdrawal.

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Dopaminergic neurons in the VTA receive a glutamatergic input from the mPFC, the pedunculopontine region and the subthalamic nucleus (Kalivas, 1993; White, 1996). Pharmacological activation of ionotropic or metabotropic glutamate receptors in the VTA elicits an increase in exploratory motor behavior and a dopamine (DA) transmitter release in the NAcc and the mPFC (Suaud-Chagny et al., 1992; Swanson and Kalivas, 2000). Moreover, chronically intermittent administration of abuse drugs, such as cocaine, morphine or alcohol, can increase extracellular levels of glutamate in the VTA, and the expression of GluR1 [an a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit1] and NMDAR1 [an N-methyld-aspartic acid (NMDA) receptor subunit 1] in the VTA (Fitzgerald et al., 1996; Kalivas and Duffy, 1998; Ortiz et al., 1995). Administration of both NMDA and AMPA receptor antagonists into the VTA reduces heroin reinforcement (Xi and Stein, 2002) and the former also reduces the expression of morphine-induced conditioned place preference (Popik and Kolasiewicz, 1999). These data suggest that an enhanced VTA glutamate receptor function is involved in opiate reinforcement. Accumulating evidence suggests that the glutamatergic system might be hyperactive during opioid withdrawal (Hong et al., 1993; Rasmussen, 1995; Sepulveda et al., 1998). Systemic or intracerebroventricular injection of NMDA and AMPA receptor antagonists prevents certain withdrawal symptoms in morphine-dependent rodent (Akaoka and Aston-Jones, 1991; Cappendijk et al., 1993; Popik and Skolnick, 1996; Rasmussen, 1995; Rasmussen et al., 1996; Trujillo and Akil, 1991). However, there is also evidence that withdrawal behaviors are not reduced by the AMPA-receptor antagonists, GYKI52466 and LY293588 (Fundytus and Coderre, 1994; Mclemore et al., 1997). Due to conflicting background literature, further studies need to be carried out to elucidate the function of glutamate in the VTA on opiate withdrawal. Therefore, the present study is undertaken to investigate whether the blockade of ionotropic glutamatergic receptors influences naloxone-precipitated symptoms of morphine withdrawal in dependent rats. Effects of dizocilpine (MK-801) and 6,7-dinitroquinnoxaline-2,3dione (DNQX) microinjected into the VTA on the morphine withdrawal signs were respectively examined. Data presented support the hypothesis that the glutamate receptor system in the VTA is involved in naloxoneprecipitated opiate withdrawal.

2. Materials and methods 2.1. Animals housing and surgery Seventy-six male Sprague–Dawley rats weighing 240– 280 g were acquired from the Medical Experimental Animal Center of Shaanxi Province, China. Animals were housed and maintained on a 12-h light/dark cycle (light on at

20:00), with constant temperature (22 8C) and free access to food and water at least 1 week before surgery. After surgery, rats were individually housed. Rats were anesthetized with sodium pentobarbital [50 mg/kg, intraperitoneally (i.p.)]. Guide cannulae were placed 2 mm dorsal to rat VTA (from bregma: AP 5.3 mm; ML, +0.7 mm; DV, 7.7 mm; Paxinos and Watson, 1986) and anchored to the skulls. During surgery, the rectal temperature was monitored and kept between 37 and 38 8C by a thermostatically controlled heating pad. After the stereotaxic surgery, rats were housed individually and allowed to recover for 7 days before any treatment. Rats were given sodium penicillin (0.2 million units per day, i.p.) during the first 3 days of recovery to prevent wound and intracerebral infections. 2.2. Drugs Morphine hydrochloride was purchased from the Shengyang Pharmaceutical Factory (Shengyang, China). (+) MK-801, DNQX and naloxone hydrochloride were from Sigma (St. Louis, MO, USA). The drugs were dissolved in sterile saline. Morphine hydrochloride, naloxone hydrochloride and saline were injected i.p. at a dosage of 2 ml/kg, respectively. (+) MK-801, DNQX and saline were given 0.5 Al/side, respectively, into the VTA at a speed of 0.25 Al/min. The injectors were maintained in the position for an additional 2 min to allow drug diffusion. 2.3. Experimental procedure 2.3.1. Morphine dependence and withdrawal An adapted method described by Chou et al. (2002) and Valverde and Roques (1998) was used. Rats received morphine i.p. injections twice a day at 08:30 and 16:30 for 7 days. The daily dosage was 10, 20, 30, 40, 50, 50 or 50 mg/kg/injection, respectively. One hour after the last injection, rats were microinjected with saline or (+) MK801 (1, 2.5 or 5 Ag/0.5 Al) or DNQX (1, 2 or 4 Ag/0.5 Al) into the VTA. Thirty minutes later, rats were given naloxone hydrochloride (2 mg/kg, i.p.; Xavier et al., 1997; AriciogluKartal et al., 2003) and immediately placed into an observation tank for behavior analysis. The saline control rats received saline injection instead of morphine on the same schedule. Animals were divided into nine treatment groups shown as Table 1. 2.3.2. Behavior analysis The withdrawal intensity was evaluated with 10 withdrawal signs including wall clamber, jumping, wet-dog shake, writhing posture, weight loss, genital grooming, teeth-chattering, ptosis, diarrhea and irritability. The characteristics of these behaviors were described by FernandezEspejo et al. (1995) as shown in Table 2. The rats were placed into a clear, Plexiglas observation tank (505060 cm) and were rated for withdrawal severity for 60 min by a

H.-L. Wang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 28 (2004) 1079–1087 Table 1 Test group Groups

Control groups Saline control (sal+sal+sal, n=8) Dependence control (mor+sal+sal, n=8) Withdrawal control (mor+sal+nal, n=12)

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violet to determine locations of injector tips (Fig. 1, Xi and Stein, 2002; Zhou et al., 2000). Day (1st–7th) administration (i.p.)

VTA administration

Test administration (i.p.)

saline

0.5 Al saline

3 ml/kg saline

morphine

0.5 Al saline

3 ml/kg saline

morphine

0.5 Al saline

2 mg/kg naloxone

MK-801 treatment groups (n=8, each group) (mor+MK morphine 1.0 Ag/0.5 Al 1 Ag+nal) MK-801 (mor+MK morphine 2.5 Ag/0.5 Al 2.5 Ag+nal) MK-801 (mor+MK morphine 5.0 Ag/0.5 Al 5 Ag+nal) MK-801

2 mg/kg naloxone 2 mg/kg naloxone 2 mg/kg naloxone

DNQX treatment groups (n=8, each group) (mor+DNQX morphine 1 Ag/0.5 Al 1 Ag+nal) DNQX (mor+DNQX morphine 2 Ag/0.5 Al 2 Ag+nal) DNQX (mor+DNQX morphine 4 Ag/0.5 Al 4 Ag+nal) DNQX

2 mg/kg naloxone 2 mg/kg naloxone 2 mg/kg naloxone

rater blind to the treatment. The frequency of wall clamber, jumping, body shake and writhing posture was evaluated; the presence of ptosis, genital grooming, teeth-chattering, irritability and diarrhea was recorded; and the percentage of weight loss was also calculated. Furthermore, a score for each group was calculated using a modification of the scale described by Gellert and Holtzman (1978) in which two classes of signs were distinguished. Graded signs were considered as: weight loss (each 1% of weight loss quantified as 1), number of jumps, writhing posture and wall clamber (1–4 times quantified as 1, 5–9 quantified as 2 and 10 or more quantified as 3), number of body shakes (1–2 quantified as 2, 3 or more quantified as 4). (II) Checked signs, in which only the presence or absence was evaluated, were considered as: diarrhea (2), ptosis (2), teeth-chattering (2), irritability (3) and erection or genital grooming (3).

2.4. Statistical analysis Nonparametric statistics were performed for all behavioral patterns. Kruskal–Wallis tests were used to assess the variance of the behavioral measures over groups using the medians for times allocated to each behavioral category. The two-tailed Mann–Whitney U test was used to examined the differences between groups. Data concerning the Gellert– Holtzman scale and weight loss were analyzed by an analysis of variance (ANOVA) with one factor. Post hoc comparisons (Student–Newman–Keuls) were subsequently carried out for the differences between means.

3. Results 3.1. Gellert–Holtzman scale Naloxone administration (2 mg/kg, i.p.) to morphinedependent rats induced significant withdrawal signs, with a Gellert–Holtzman score of 29.64F4.09 (n=12). In comparison, (+) MK-801 (1, 2.5 or 5 Ag/0.5 Al) or DNQX (1, 2 or 4 Ag/0.5 Al) microinjected into the VTA 30 min before a naloxone injection substantially attenuated morphine withdrawal signs, with Gellert–Holtzman scores being reduced to 20.44F0.97, 18.17F1.74, 19.14F0.74 (n=8) and 18.40F0.90, 16.00F0.86, 18.67F1.28 (n=8), respectively. Saline microinjection into the VTA did not influence the Table 2 Ethogram of the rats’ behavior during morphine withdrawal Pattern

Category

Description

Wall clamber

Exploration

Body shake

Wet-dog shakes

Writhing posture

Writhing behavior

Jumping

Escape

Genital grooming

Self care

Erect posture with forelegs on the wall of the cage The rat shakes its body or moves the hand with sideways movement The rat lies on the floor, while the belly is firmly pressing the surface Abdominal contractions are usually present Leaping off the surface of the cage The rat licks its genitalia. It usually follows ejaculation The rat rapidly clicks teeth together Closing of the eyelids Presence of soft or formless stool Attack or vocalization when touched at the end of the period The percentage of weight loss before and 1 h after each test

(I)

2.3.3. Histology Anaesthetised with sodium pentobarbital (60 mg/kg, i.p.) at the end of the experiment, rats were perfused with phosphate-buffered formalin. After post-fixed in the formalin, rat brains were placed into a buffered 30% sucrose solution for at least 2 days, and cut on a cryostat into 50-Am coronal sections. Sections were stained with 0.5% Cresyl

Teeth-chattering Ptosis Diarrhea Irritability

Weight loss

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(1, 2 or 4 Ag/0.5 Al) microinjected into the VTA significantly reduced the incidence of wet-dog shakes in morphine withdrawal rats (Fig. 2B). The incidence was reduced to 7.0 (0.5–25.5), 8.0 (0.0–15.5), 7.0 (4.0-13.0) and 4.0 (0.0–7.0), 4.5 (1.75–7.25), 9.0 (2.5–19.75), respectively. However, the effects of (+) MK-801 and DNQX on attenuating wet-dog shakes in morphine withdrawal rats were not dose-dependent, as shown in Fig. 2B. 3.3. Writhing posture Saline injection into the VTA did not induce writhingposture in either morphine dependent rats or non-dependent rats. Naloxone (2 mg/kg, i.p.) administration induced writhing posture in morphine-dependent rats with an incidence of 38.0 (30.0–44.0, n=12). Different doses of (+) MK-801 (1, 2.5 or 5 Ag/0.5 Al) or DNQX (1, 2 or 4 Ag/ 0.5 Al) microinjected into the VTA significantly reduced the incidence of writhing posture (Fig. 2C) in the morphine withdrawal rats to 13.0 (8.5–58.5), 7.0 (0.0–33.25), 3.0 (0.0–27.0) and 16.0 (3.0–60.0), 8.0 (0.0–21.0), 3.5 (0.0– 22.25), respectively. The inhibiting effects of different doses of MK-801 or DNQX on writhing posture were dosedependent in withdrawal rats. The two groups receiving a high dose of either (+) MK-801 (2.5, 5 Ag/0.5 Al) or DNQX (2, 4 Ag/0.5 Al) showed a significantly lower incidence of writhing posture than those receiving a low dose of (+) MK801 (1 Ag/0.5 Al) or DNQX (1 Ag/0.5 Al; Pb0.05), as shown in Fig. 2C. 3.4. Jumping

Fig. 1. Drawings show the injector tip locations for physiological saline, (+) MK-801, or DNQX into the VTA. The pentacles or circularities represent the tip locations in the VTA for physiological saline/0.5 Al: (B) saline control group; (1) dependence control group; ( ) withdrawal control group; (D) location of injector tips in VTA for 1, 2.5, 5 Ag/0.5 Al MK-801; (E) location of injector tips in VTA for 1, 2, 4 Ag/0.5 Al DNQX. (n) Injection tips outside of the VTA region, which were not included in statistical analyses. Abbreviations: VTA, ventral tegmental area; MK-801, dizocilpine; DNQX, 6,7-dinitroquinoxaline-2,3-dione.

!

Gellert–Holtzman score in either the morphine dependent or nondependent rats (Fig. 2A). However, the blockade of morphine withdrawal signs by (+) MK-801 or DNQX showed no dose-dependent tendency, as shown by Fig. 2A. 3.2. Wet-dog shakes No wet-dog shakes were observed with saline injection into the VTA either in the morphine dependent rats or in the nondependent rats. Naloxone (2 mg/kg, i.p.) administration induced wet-dog shakes in morphine dependent rats with an incidence of 36.0 (31.0–47.0, n=12). In comparison, different doses of (+) MK-801 (1, 2.5 or 5 Ag/0.5 Al) or DNQX

No jumping was observed after a saline injection into the VTA either in the morphine-dependent rats or in the nondependent rats. However, naloxone (2 mg/kg, i.p.) administration induced jump behavior in morphine-dependent rats with a jumping incidence of 12.0 (11.0–14.0, n=12). Different doses of (+) MK-801 (1, 2.5 or 5 Ag/0.5 Al) or DNQX (1, 2 or 4 Ag/0.5 Al) microinjected into the VTA significantly attenuated the jump behavior in morphine withdrawal rats (Fig. 2D). The incidence of jump behavior reduced to 4.0 (0.0–5.0), 1.0 (0.0–6.25), 0.0 (0.0–4.0) and 0.0 (0.0–3.0), 0.0 (0.0–2.5), 0.0 (0.0–0.0), respectively. In addition, the inhibiting effects of (+) MK-801 on jump behavior were dose-dependent. 3.5. Wall clamber Wall clamber was observed in both morphine-dependent and nondependent rats with incidences of 7.0 (2.0–18.5; n=8) and 6.0 (2.5–12.5; n=8), respectively. Naloxone (2 mg/ kg, i.p.) administration had no significant effect on this behavior in morphine-dependent group with an incidence of 11.0 (7.0–17.0; n=12). Different doses of (+) MK-801 (1, 2.5 or 5 Ag/0.5 Al) or DNQX (1, 2 or 4 Ag/0.5 Al) microinjected into the VTA 30 min before naloxone

H.-L. Wang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 28 (2004) 1079–1087

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Fig. 2. Bar graph showing the effects of MK-801 or DNQX unilaterally microinjected into the VTA on the naloxone-precipitated withdrawal signs in morphinedependent rats. Effects of MK-801 or DNQX microinjection into the VTA on the naloxone-precipitated increase of the mean Gellert–Holtzman score (A); on the naloxone-precipitated increase of the median of wet dog shake (B), writhing posture (C), jumping (D) and wall clamber (E); and on the naloxoneprecipitated increase of the mean percentage of weight loss (F). (A) by using one-way ANOVA analysis; (B–E) by using Mann–Whitney U tests; (F) by using one-way ANOVA in all groups and then using Student–Newman–Keuls analysis for differences between groups. In graphs A–D, ***Pb0.001 indicates the significant difference compared with that of saline (sal+sal+sal) or morphine (mor+sal+sal) injection group; *Pb0.05 or **Pb0.01 indicates the significant difference compared with that of naloxone (mor+sal+nal) or saline (sal+sal+sal) injection group. In graph E, *Pb0.05 or **Pb0.01 indicates the significant difference compared with that of saline (sal+sal+sal) or morphine (mor+sal+sal) or naloxone (mor+sal+nal) injection group. In graph F, *Pb0.05 indicates the significant difference compared with that of saline (sal+sal+sal) or morphine (mor+sal+sal) injection group.

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injection increased the wall clamber behavior to 20.0 (13.5– 28.0), 26.5(11.75–46.5), 39.0 (18.0–43.0; n=8) and 19.0 (13.0–58.0), 52.5 (17.0–76.75), 65.5 (8.25–102.25; n=8), respectively, which were significantly higher than that of the group receiving only a naloxone injection without any (+) MK-801 or DNQX injection ( Pb0.01 or Pb0.05). This effect was dose-dependent both in (+) MK-801 and DNQX groups, as shown in Fig. 2E. 3.6. Weight loss Naloxone (2 mg/kg, i.p.) administration induced significant weight loss by 4.09F0.55% in morphine-dependent rats (n=12). Microinjection of (+) MK-801 (1, 2.5 or 5 Ag/ 0.5 Al) or DNQX (1, 2 or 4 Ag/0.5 Al) into the VTA 30 min before naloxone administration also induced the weight loss in withdrawal rats, but the weight loss (3.44F0.29%, 3.17F0.40%, 3.29F0.29% and 2.90F0.23%, 2.36F0.34%, 2.67F0.33%, respectively) was not significantly different from that of the group only receiving naloxone injection. Saline microinjection into the VTA did not influence weight loss in either morphine-dependent or nondependent rats, as shown in Fig. 2F. Other withdrawal signs, such as diarrhea, ptosis, teethchattering, irritability and genital grooming, were also noted early. Irritability, diarrhea and ptosis were presented in morphine-dependent rats with only naloxone injection. Nevertheless, teeth-chattering and genital grooming was presented both in morphine-dependent rats and in nondependent (saline, i.p.) rats. Different doses of (+)MK-801 or DNQX microinjected into the VTA diminished the present percentage of ptosis, irritability and genital-grooming, but did not influence the present percentages of diarrhea and teeth-chattering as showed in Table 3. Because these withdrawal signs were applied to calculate Gellert–Holtzman scale score, the individual statistical analyses were not performed. Table 3 Percentage of animals with diarrhea, ptosis, chattering-teeth, irritability, or genital-grooming for nine groups in the test Groups

Diarrhea

Ptosis

Chatteringteeth

Irritability

Genitalgrooming

Sal+sal+sal Mor+sal+sal Mor+sal+nal Mor+MK 1 Ag+nal Mor+MK 2.5 Ag+nal Mor+MK 5 Ag+nal Mor+DNQX 1 Ag+nal Mor+DNQX 2 Ag+nal Mor+DNQX 4 Ag+nal

0.00 0.00 91.67 100.00

0.00 0.00 72.70 75.00

0.00 40.00 83.33 72.50

0.00 0.00 83.33 50.00

0.00 25.00 58.33 37.50

87.50

66.50

83.33

50.00

37.50

87.50

62.50

83.33

37.50

37.50

100.00

40.00

83.33

37.50

37.50

100.00

40.00

72.50

25.00

25.00

87.50

54.50

72.50

37.50

25.00

4. Discussion 4.1. An interaction between the VTA glutamate and DA systems in morphine withdrawal Previous studies have shown that morphine withdrawal induces glutamate release in the VTA and NAcc (Aghajanian et al., 1994; Jhamandas et al., 1996; Tokuyama and Ho, 1996) and increases the expression of NMDA and nonNMDA receptor subunits in the VTA (Fitzgerald et al., 1996; Ortiz et al., 1995). Overexpression of GluR1 in the VTA induced viral-mediated gene transfer and increased the morphine’s stimulant and rewarding properties (Carlezon et al., 1997, 2000), while lack of gene encoding epsilonl subunit of the NMDA receptor in mice markedly eliminates typical withdrawal behaviors induced by chronic morphine treatment (Inoue et al., 2003). Furthermore, systemic and intracerebroventricular injections of NMDA receptor antagonists (MK-801, memantine and dextromethorphan, etc.) and non-NMDA receptor antagonists (DNQX, CNQX, LY293558 and LY215490) prevent some features of morphine withdrawal symptoms in rats and mice (Bristow et al., 1997; Fundytus and Coderre, 1994; Leal et al., 2003; Manning et al., 1996; Popik et al., 1998; Rasmussen and Vandergiff, 2003; Thorat et al., 1994). Morphological experiment revealed a decrease of DA neuron numbers and perimeter in the VTA in morphine-withdrawn rats (Spiga et al., 2003). Taken together, these data support a role of brain glutamate receptors and DA neurons in opiate withdrawal and suggest an interaction between VTA glutamate and DA systems in the withdrawal. However, the role of VTA glutamatergic transmission in opiate withdrawal is less well understood. 4.2. The method evaluation In the present study, animals receiving morphine injection twice a day developed a intense withdrawal syndrome precipitated by naloxone (2 mg/kg, i.p.; Broseta et al., 2002; Chou et al., 2002; Valverde and Roques, 1998). Jumping, wet-dog shakes, writhing posture, wall clamber, weight loss and Gellert–Holtzman scale, which are all sensitive and reliable indicators of the degree of morphine dependence for rodent (Bristow et al., 1997; Caille et al., 1999; Fernandez-Espejo et al., 1995; Jones et al., 2002; Tokuyama et al., 1996, 2001), were employed to evaluate the intensity of morphine withdrawal. Characteristic signs for morphine withdrawal were not observed in either nondependent or morphine-dependent rats without administration naloxone. Results in this study demonstrated that unilateral microinjections of both (+) MK-801 and DNQX into the VTA significantly reduced the total Gellert–Holtzman scale score and the incidence of jumping, wet-dog shakes and writhing posture in morphine withdrawal rats, but did not significantly influence the weight loss of the rats. On the contrary, DNQX (1, 2, 4 Ag) or (+) MK-801 (1, 2.5

H.-L. Wang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 28 (2004) 1079–1087

and 5 Ag) significantly increased the incidence of wall clamber. Our findings are in line with the hypothesis that endogenous glutamatergic transmission within the VTA plays an important role in mediating opiate withdrawal. 4.3. The effects of MK-801 on morphine withdrawal signs Unilateral microinjections of MK-801 (1, 2.5 or 5 Ag/0.5 Al) into the VTA 30 min before naloxone administration in morphine-dependent rats significantly attenuated the Gellert–Holtzman scale score ( Pb0.001), the incidence of jump, wet-dog shakes and writhing posture ( Pb0.001). Moreover, the inhibition on jumping by (+) MK-801 showed a dose-dependent tendency that was not significant statistically. These results are consistent with other findings using (+) MK-801 pretreatment before naloxone precipitation in morphine-dependent rodents (Gonzalez et al., 1997; Higgins et al., 1992; Maldonado et al., 2003; Manning et al., 1996; Trujillo and Akil, 1991). Experiments of Narayanan et al. (1995) also demonstrated that stimulatory effects of (+) MK-801 injected into VTA closely resemble those produced by systemic administration of a low dose of MK-801. Kalivas and Alesdatter (1993) reported that microinjections of MK-801 or D1 antagonist into the VTA disrupted the development of cocaine and amphetamine sensitization. Lesion of the prefrontal cortex region or local application of glutamate receptor antagonists into the prefrontal cortex region can attenuate the development of morphine sensitization (Carr and Sesack, 2000; Kalivas and Alesdatter, 1993; Vezina, 1996). These data suggest that complicated interplays between mesocorticolimbic glutamate and DA systems mediate morphine dependence. The effects of (+) MK-801 on weight loss of morphine withdrawal rats are contradictory, with some showing an effect of blockade (Gonzalez et al., 1997; Tanganelli et al., 1991; Tokuyama et al., 1996; Trujillo and Akil, 1991) and others showing no blockade (Bell and Beglan, 1995; Zhu and Barr, 2000). In the present study, MK-801 microinjected into the VTA inhibited weight loss, although there was no significant difference between MK-801 treatment groups and naloxone control group. More interestingly, the highest dose of MK801 increased wall clamber in morphine withdrawal rats. Our finding is in line with other reports demonstrating that MK-801 given by either systemic injection or intrategmental infusion could enhance motor behavior and DA release in the VTA (Carlsson and Carlsson, 1989; Kretschmer, 1999). In summary, these results support that NMDA receptors in VTA are involved in opiate withdrawal.

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dependent mice. Recently LY293558 and LY300168 were shown to attenuate the severity and occurrence of naloxone precipitated morphine withdrawal signs in rats and in mice (Mclemore et al., 1997; Rasmussen et al., 1996; Rasmussen and Vandergiff, 2003). A unilateral injection of DNQX (1, 2 or 4 Ag/0.5 Al) into the VTA significantly diminished the total Gellert–Holtzman scale score and the incidence of jumping, wet-dog shakes or writhing posture. The decreases in physical signs of morphine withdrawal did not show dose dependence. DNQX did not significantly inhibit weight loss in morphine withdrawal rats. In contrast, DNQX into the VTA 30 min before naloxone administration significantly increased the incidence of wall clamber in a dose-dependent manner. It also induced contralateral circling within the first half an hour, which was consistent with other findings (Dalia et al., 1996). Furthermore, the dose–response data show that minimal effective dose (1 Ag) in the present studies is the same as that required for DNQX in NAcc to attenuate reward responses elicited by systemic amphetamine and cocaine (Kaddis et al., 1995; Layer et al., 1993; Willins et al., 1992). Together, these data suggest that AMPA/kainate receptors in the VTA paly an important role in opiate withdrawal. Nonetheless, it should be noted that not all data are consistent with the above conclusion because interactions between glutamate receptor antagonists and drugs of abuse appear to be rather complex (Nestler and Aghajanian, 1997). The bases for conflicting data are not clear; hence, more studies are required for clarifying the roles and mechanisms of VTA glutamatergic transmission in opiate tolerance, dependence and withdrawal.

5. Conclusion In conclusion, MK-801 (1, 2.5 or 5 Ag/0.5 Al) or DNQX (1, 2 or 4 Ag/0.5 Al) unilaterally administrated into the VTA partially reduced the intensity of physical signs in morphine withdrawal rats. These data support a role for VTA glutamatergic transmission in opiate withdrawal syndrome and suggest the usefulness of NMDA and/or AMPA/Kaite antagonists in the therapeutic treatment of opiate abuse.

Acknowledgements We would like to thank Professors Jingshi Tang and Jianqun Yan for their comments on the drafts of this paper. This work was supported by the Shaanxi Province Natural Science Grants of China (No. 2002C122).

4.4. The effects of DNQX on morphine withdrawal signs In contrast to the extensive studies with the NMDA receptor, less is known about the specific contribution of AMPA receptor to opiate dependence, especially to morphine withdrawal. Cappendijk et al. (1993) first reported that DNQX inhibited withdrawal jumping in morphine-

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