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Ascorbate reduces morphine-induced extracellular DOPAC level in the nucleus accumbens: A microdialysis study in rats
Brain Research 1053 (2005) 62 – 66 www.elsevier.com/locate/brainres
Research Report
Ascorbate reduces morphine-induced extracellular DOPAC level in the nucleus accumbens: A microdialysis study in rats Z. Rajaei a,*, H. Alaei a, A. Nasimi a, H. Amini b, A. Ahmadiani b b
a Department of Physiology, Isfahan University of Medical Sciences, Isfahan, Iran Neuroscience Research Center, Shaheed Beheshti University of Medical Sciences, Tehran, Iran
Accepted 7 June 2005 Available online 26 July 2005
Abstract Most drugs of abuse increase dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) release in the shell of the nucleus accumbens. The effects of ascorbate, which is known to modulate dopamine neurotransmission, on the extracellular level of DOPAC in the nucleus accumbens of naive rats and of rats treated acutely with morphine were studied by using in vivo microdialysis and high performance liquid chromatography with electrochemical detection (HPLC-ECD). Acute morphine (20 mg/kg ip) treatment increased the level of DOPAC in the nucleus accumbens to approximately 170% of basal level. Acute treatment with ascorbate (500 mg/kg ip) alone did not alter nucleus accumbens’ DOPAC level, but pretreatment with ascorbate (500 mg/kg ip) 30 min before morphine administration attenuated the effects of acute morphine on the level of DOPAC. These results suggest that ascorbate modulates the mesolimbic dopaminergic pathway. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitters/modulators/transporters and receptors Topic: Opioids/anatomy, physiology and behavior—catecholamines Keywords: Ascorbate; DOPAC; Morphine; Microdialysis; Nucleus accumbens
1. Introduction Opioid agents such as morphine and heroin are readily self-administered by many species, including humans [16]. Injected or smocked, heroin produces a Frush_ within seconds or minutes. This pleasurable effect is caused by the interaction of heroin’s metabolites, 6-monoacetyl morphine and morphine, with opioid receptors, leading to immediate and specific changes in neurotransmission [16]. Dopaminergic as well as non-dopaminergic systems, including opioid, glutamate and GABA, have been postulated to underlie the rewarding effect of morphine [31]. The mesolimbic dopaminergic system, which originates in the ventral tegmental area (VTA) and projects to the nucleus accumbens, appears to be importantly involved [14].
* Corresponding author. Fax: +98 311 6688597. E-mail address: [email protected] (Z. Rajaei). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.06.040
Laboratory animals self-administer morphine into the VTA [1], and morphine infusions into the VTA lower brain stimulation reward thresholds [2]. Morphine, like other drugs that have abuse liability, increases extracellular dopamine level in the nucleus accumbens when administered systematically or infused directly into the VTA [5,15]. Ascorbate, the endogenous form of vitamin C, is found in high concentration with a heterogeneous distribution in the mammalian brain [18]. Ascorbate is released from glutamatergic neurons as part of the glutamate reuptake process, in which the high-affinity glutamate transporter exchanges ascorbate for glutamate [24]. Experimental evidence indicates that the ascorbate is capable of modulating the effects of dopamine in the mammalian brain. In many respects, it appears to act like a dopamine receptor antagonist. This conclusion first emerged from the dopamine receptor binding experiments that commonly included ascorbate in the assay as an antioxidant [24]. However, numerous laboratories reported that ascorbate actually inhibited the
Z. Rajaei et al. / Brain Research 1053 (2005) 62 – 66
binding of both dopamine agonists and antagonists in a dose-dependent manner [9,13,28]. Ample behavioral evidence support an antidopaminergic action of ascorbate, including the findings that ascorbate potentiates the catalepsy induced by the antipsychotic, haloperidol [7,25], and that the systemic and intraneostriatal administration of ascorbate blocks amphetamine-induced hyperactivity [25,29]. These results suggest that ascorbate inhibits dopamine neurotransmission. The aim of this study was to investigate whether ascorbate might prevent the increase of the dopamine system activity induced by morphine in the nucleus accumbens. Since DOPAC is indicating intraneuronal synthesis and metabolism of dopamine [10], therefore, the present study investigated the effect of ascorbate (administered ip) on the extracellular DOPAC level in the nucleus accumbens using in vivo microdialysis in rats. Additionally, the effect of ascorbate on the DOPAC level in the nucleus accumbens in response to a single morphine injection was also assessed.
2. Materials and methods 2.1. Drugs Morphine sulfate (Temad Company, Iran; 20 mg/kg ip) and ascorbate (BDH England; 500 mg/kg ip) were dissolved in normal saline.
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NaH2PO4, 7.2 mM glucose, pH 7.4) [26], at a flow rate of 2 Al/min, using a syringe pump (model 100, Stoelting). After a 60 min washout period, dialysis samples were collected for 20 min periods successively in vials containing 10 Al of 0.5 M acetic acid to minimize decomposition. Up to six baseline samples were collected prior to drug injections, and samples were collected for 2 h following the drug injection. The microdialysis samples were stored at 70 -C until analyzed. 2.4. Experimental groups Experimental groups consisted of (1) saline group that was injected with saline following the collection of the baseline samples. (2) ascorbate group that was injected with ascorbate (500 mg/kg ip) following the collection of the baseline samples. (3) saline + morphine group that was injected with saline 10 min after the collection of the fourth baseline sample and, 30 min later, received an injection of morphine (20 mg/kg ip). (4) ascorbate + morphine group that was injected with ascorbate (500 mg/kg) 10 min after the collection of the fourth baseline sample and, 30 min later, received an injection of morphine (20 mg/kg ip). In all groups, the effects of the drug were monitored for 2 h. 2.5. Histology
2.2. Subjects and surgery Experiments were performed on 21 male Wistar rats (240 –280 g). They were housed in groups of four per cage in a colony room controlled for temperature with a normal 12 h light/12 h dark cycle. Food and water were available ad libitum. Animals were anesthetized with chloral hydrate (450 mg/ kg, ip), and supplementary doses were administered if needed. Body temperature was maintained at 37 T 0.1 -C with a temperature control unit (Letica, Spain). The rats were implanted stereotaxically with a guide cannula unilaterally, with tips aimed over the nucleus accumbens. The coordinates were chosen such that the tip of the dialysis probes (220 Am outer diameters and a 1 mm active membrane, AgnTho’s, Lidingo, Sweden) was located in the medial portion of the shell of the accumbens (Ap = 1.7 mm, L = 0.8 mm from bregma, DV = 7.5 mm from the surface of skull) [19]. The cannula was fixed firmly to the skull with dental cement. 2.3. In vivo microdialysis The microdialysis probe was inserted through the guide cannula and was continuously perfused with artificial cerebrospinal fluid (140 mM Nacl, 3 mM KCl, 2.4 mM CaCl2, 1 mM MgCl2, 1.2 mM Na2HPO4, 0.27 mM
Upon completion of a microdialysis experiment, the animal was sacrificed by a high dose of the anesthetic then transcardially perfused with 100 ml of saline followed by 100 ml of 10% formalin. The brain was removed and stored in 10% formalin for at least 24 h. Brains were sectioned coronally at 30 Am by a freezing microtome (Leica, Germany). Sections were mounted on gelatin-coated slides and stained with cresyl violet. The tracks left by the probes were identified using a light microscope, and their exact positions determined by reference to a rat brain atlas [19]. Only the dialysates of animals whose probe were within the shell of the nucleus accumbens were analyzed. 2.6. Catecholamine assay Dialysate samples were analyzed for DOPAC by high performance liquid chromatography (HPLC) with electrochemical detection. The HPLC system consisted of a Shimadzu C18 column (250 4.6 mm 5Am), a Shimadzu solvent delivery system (LC 10 ADvp) and a Shimadzu electrochemical detector (LC-ECD 6 A) with a working electrode set at a potential of 0.75 V. The mobile phase consisted of 0.07 M sodium acetate, 0.1 mM sodium octyl sulfate, 0.1 mM EDTA in 10% HPLC grade acetonitrile, adjusted to pH 3.7 with acetic acid. The flow rate was set at 1 ml/min.
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2.7. Statistical analysis Baseline values for DOPAC (mean of the value of the six 20-min samples T SEM), expressed as pmol/10 Al. The data were expressed as percentage of baseline values. The values at different intervals were compared both within each experimental group and between 4 experimental groups using repeated measures ANOVA. A Tukey test for post-hoc comparisons was performed when appropriate. A P < 0.05 was considered as significant.
3. Results 3.1. Acute effect of ascorbate Baseline values for DOPAC did not significantly differ between the two groups of saline (2.061 T 0.31 pmol/10 Al) and ascorbate (2.3 T 0.31 pmol/10 Al). As shown in Figs. 1 and 2, two-way ANOVA with repeated measures revealed that extracellular levels of DOPAC in the nucleus accumbens did not significantly change due to treatment with saline or ascorbate (500 mg/kg). 3.2. Effect of ascorbate in rats treated acutely with morphine The baseline values of DOPAC did not significantly differ between the two groups of saline + morphine (2.85 T 0.38 pmol/10 Al) and ascorbate + morphine (2.36 T 0.39 pmol/10 Al). As shown in Figs. 3 and 4, one-way ANOVA with repeated measures revealed that acute treatment with morphine (20 mg/kg ip) significantly increased the extracellular levels of DOPAC ( P < 0.05) in the nucleus accumbens. Two-way ANOVA with repeated measures revealed that pretreatment with 500 mg/kg ascorbate significantly attenuated the effect of morphine on the
Fig. 1. The extracellular levels (mean T SEM) of DOPAC in the nucleus accumbens, expressed as the percentage of baseline, prior to and following saline (n = 5) or ascorbate (500 mg/kg ip; n = 6) administration at time 0 (arrow). There was no significant change due to saline or ascorbate (ANOVA, P > 0.05).
Fig. 2. Average (TSEM) of the DOPAC changes over 2 h induced by saline and ascorbate (500 mg/kg ip) in the nucleus accumbens, expressed as percentage of baseline. There was no significant changes due to saline or ascorbate (ANOVA, P > 0.05).
extracellular levels of DOPAC ( P < 0.05) in the nucleus accumbens. 3.3. Locations of the accumbal dialysis probes All animals that were included in the biochemical analysis had appropriate probe placements within the shell region of the nucleus accumbens. Representative of the accumbal probe placements are shown in Fig. 5.
4. Discussion The mesolimbic pathway is often thought of as the ‘‘reward pathway’’ and connects the VTA with the nucleus
Fig. 3. The extracellular levels (mean T SEM) of DOPAC in the nucleus accumbens, expressed as the percentage of baseline, prior to and following saline (n = 5) or ascorbate (500 mg/kg ip; n = 5) pretreatment before an acute dose of morphine (20 mg/kg ip). Saline or ascorbate administered at time 30 (first arrow), 20 mg/kg morphine administered at time 0 (second arrow). Ascorbate significantly attenuated the effect of morphine on the levels of DOPAC (ANOVA, *P < 0.05).
Z. Rajaei et al. / Brain Research 1053 (2005) 62 – 66
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Fig. 4. Average (TSEM) of the DOPAC changes over 2 h induced by saline or ascorbate pretreatment (500 mg/kg ip) before an acute dose of morphine (20 mg/kg ip) in the nucleus accumbens, expressed as the percentage of baseline. Ascorbate significantly attenuated the effect of morphine on the levels of DOPAC (ANOVA, *P < 0.05).
dopamine release remains unclear, although a few possibilities exist. Some studies indicate that pharmacological stimulation of NMDA receptors of glutamate in the VTA promotes dopamine release in the nucleus accumbens [12,27], and high doses of ascorbate have been proposed to block NMDA receptors in vitro [17]. Therefore, high doses of ascorbate probably blocked the NMDA receptors in the VTA and reduced the release of dopamine in the nucleus accumbens. Other explanation for our results with high-dose ascorbate may be a blockade of dopamine receptors in the VTA. In vitro evidence suggests that ascorbate inhibits dopamine binding, and it appears to act like a dopamine receptor antagonist [9,13,24,28]. In the VTA, D2 autoreceptors stimulation by somatodendritically released dopamine directly modulates the excitability of dopamine cells and release of dopamine in the nucleus accumbens [3], while D1 receptors modulate the transmitter release of afferents to the
accumbens as well as the olfactory tubercle, amygdala and prefrontal cortex [30]. Most drugs of abuse have been shown to increase extracellular levels of dopamine in the nucleus accumbens [21]. In the present study, morphine administration induced an increase in dopamine’s acidic metabolite DOPAC in the nucleus accumbens, indicating the increase of intraneuronal synthesis and metabolism of dopamine [10]. This result is in agreement with previous findings [5,6,16,21 – 23]. Most studies have reported the increasing effects of one to three dosages at most of morphine on extracellular dopamine levels in the nucleus accumbens [5,6,21 –23]. Maisonneuve et al. (2001) studied the effects of morphine on extracellular dopamine levels in brain over a wide range of doses. This study showed that the dose –response curve for morphine’s effect on extracellular dopamine levels in the nucleus accumbens has a curvilinear shape while the dose – response curve for morphine effects on extracellular dopamine metabolites is a monotonic curve [16]. Previous localization studies have found opioid receptors on the GABAergic neurons in the VTA. When these GABAergic interneurons have been stimulated, the firing of the dopamine cells in the VTA is inhibited [30]. Previous studies suggest that the systemic administration of morphine inhibits these VTA GABA interneurons, which results in disinhibition of dopamine cells and release of dopamine is increased in the nucleus accumbens [4,30]. We also found that ascorbate alone had no effect on the extracellular level of DOPAC in the nucleus accumbens of naive rats. However, pretreatment with ascorbate attenuated the increase in the extracellular DOPAC level induced by acute morphine administration, indicating the modulation of mesolimbic dopaminergic pathway by ascorbate. The mechanisms by which high-dose ascorbate (500 – 1000 mg/kg) pretreatment antagonizes morphine-induced
Fig. 5. The placements of probes implanted in the nucleus accumbens of rats included in statistical analysis. Figure modified from the atlas of Paxinos and Watson [19]. Acbc: nucleus accumbens, core; AcbSh: nucleus accumbens, shell.
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dopaminergic cells [11]. Therefore, ascorbate might have modulated the release of dopamine in the nucleus accumbens by inhibiting D1 and D2 receptors in the VTA. Furthermore, some evidence indicates that ascorbate may interact with opioid peptides [8] and perhaps sigma receptors [20]. In conclusion, for the first time, our study showed that ascorbate pretreatment is capable of reducing morphineinduced dopamine release in the nucleus accumbens, suggesting that ascorbate may have a role in the modulation of mesolimbic rewarding pathway. Thus, it is probable that ascorbate might decrease the rewarding effect and psychic dependence of opioids. This hypothesis needs further behavioral experiments.
[14] [15]
[16]
[17]
[18]
[19] [20]
References [1] M.A. Bozarth, R.A. Wise, Intracranial self-administration of morphine into the ventral tegmental area in rats, Life Sci. 28 (1981) 551 – 555. [2] C.L. Broekkamp, A.G. Phillips, Facilitation of self-administration behavior following intracerebral microinjections of opioids into the ventral tegmental area, Pharmacol. Biochem. Behav. 11 (1979) 289 – 295. [3] B.T. Chen, M.E. Rice, Synaptic regulation of somatodendritic dopamine release by glutamate and GABA differs between substantia nigra and ventral tegmental area, J. Neurochem. 81 (2002) 158 – 169. [4] D.P. Devine, P. Leone, D. Pocock, R.A. Wise, Differential involvement of ventral tegmental mu, delta and kappa opioid receptors in modulation of basal mesolimbic dopamine release: in vivo microdialysis study, J. Pharmacol. Exp. Ther. 266 (1993) 1236 – 1246. [5] G. Di chiara, A. Imperato, Opposite effects of mu and kappa opiate agonists on dopamine release in the nucleus accumbens and in the dorsal caudate of freely moving rats, J. Pharmacol. Exp. Ther. 244 (1988) 1067 – 1080. [6] A. Di Giannuario, S. Pieretti, A. Catalani, A. Loizzo, Orphanin FQ reduces morphine-induced dopamine release in the nucleus accumbens: a microdialysis study in rats, Neurosci. Lett. 272 (1999) 183 – 186. [7] R.L. Dorris, R.E. Dill, Potentiation of haloperidol induced catalepsy by ascorbic acid in rats and nonhuman primates, Pharmacol. Biochem. Behav. 24 (1986) 781 – 783. [8] C.E. Dunlap, F.M. Lesile, Effect of ascorbate on the toxicity of morphine in mice, Neuropharmacology 24 (1985) 797 – 804. [9] R.E. Heikkila, F.S. Kabbat, L. Manzino, Differential inhibitory effects of ascorbic acid on the binding of dopamine agonists and antagonists to neostriatal membrane preparations: correlations with behavioral effects, Res. Commun. Chem. Pathol. Pharmacol. 34 (1981) 409 – 421. [10] A. Honkanen, T.P. Piepponen, L. Ahtee, Morphine-stimulated metabolism of striatal and limbic dopamine is dissimilarly sensitized in rats upon withdrawal from chronic morphine treatment, Neurosci. Lett. 180 (1994) 119 – 122. [11] P.W. Kalivas, Neurotransmitter regulation of dopamine neurons in the VTA, Brain Res. Rev. 18 (1993) 75 – 113. [12] P.W. Kalivas, P. Duffy, J. Barrow, Regulation of the mesocorticolimbic system by glutamic acid receptor subtypes, J. Pharmacol. Exp. Ther. 251 (1998) 378 – 387. [13] S.O. Kayaalp, N.H. Neff, Differentiation by ascorbic acid of dopamine
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Research Report
Ascorbate reduces morphine-induced extracellular DOPAC level in the nucleus accumbens: A microdialysis study in rats Z. Rajaei a,*, H. Alaei a, A. Nasimi a, H. Amini b, A. Ahmadiani b b
a Department of Physiology, Isfahan University of Medical Sciences, Isfahan, Iran Neuroscience Research Center, Shaheed Beheshti University of Medical Sciences, Tehran, Iran
Accepted 7 June 2005 Available online 26 July 2005
Abstract Most drugs of abuse increase dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) release in the shell of the nucleus accumbens. The effects of ascorbate, which is known to modulate dopamine neurotransmission, on the extracellular level of DOPAC in the nucleus accumbens of naive rats and of rats treated acutely with morphine were studied by using in vivo microdialysis and high performance liquid chromatography with electrochemical detection (HPLC-ECD). Acute morphine (20 mg/kg ip) treatment increased the level of DOPAC in the nucleus accumbens to approximately 170% of basal level. Acute treatment with ascorbate (500 mg/kg ip) alone did not alter nucleus accumbens’ DOPAC level, but pretreatment with ascorbate (500 mg/kg ip) 30 min before morphine administration attenuated the effects of acute morphine on the level of DOPAC. These results suggest that ascorbate modulates the mesolimbic dopaminergic pathway. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitters/modulators/transporters and receptors Topic: Opioids/anatomy, physiology and behavior—catecholamines Keywords: Ascorbate; DOPAC; Morphine; Microdialysis; Nucleus accumbens
1. Introduction Opioid agents such as morphine and heroin are readily self-administered by many species, including humans [16]. Injected or smocked, heroin produces a Frush_ within seconds or minutes. This pleasurable effect is caused by the interaction of heroin’s metabolites, 6-monoacetyl morphine and morphine, with opioid receptors, leading to immediate and specific changes in neurotransmission [16]. Dopaminergic as well as non-dopaminergic systems, including opioid, glutamate and GABA, have been postulated to underlie the rewarding effect of morphine [31]. The mesolimbic dopaminergic system, which originates in the ventral tegmental area (VTA) and projects to the nucleus accumbens, appears to be importantly involved [14].
* Corresponding author. Fax: +98 311 6688597. E-mail address: [email protected] (Z. Rajaei). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.06.040
Laboratory animals self-administer morphine into the VTA [1], and morphine infusions into the VTA lower brain stimulation reward thresholds [2]. Morphine, like other drugs that have abuse liability, increases extracellular dopamine level in the nucleus accumbens when administered systematically or infused directly into the VTA [5,15]. Ascorbate, the endogenous form of vitamin C, is found in high concentration with a heterogeneous distribution in the mammalian brain [18]. Ascorbate is released from glutamatergic neurons as part of the glutamate reuptake process, in which the high-affinity glutamate transporter exchanges ascorbate for glutamate [24]. Experimental evidence indicates that the ascorbate is capable of modulating the effects of dopamine in the mammalian brain. In many respects, it appears to act like a dopamine receptor antagonist. This conclusion first emerged from the dopamine receptor binding experiments that commonly included ascorbate in the assay as an antioxidant [24]. However, numerous laboratories reported that ascorbate actually inhibited the
Z. Rajaei et al. / Brain Research 1053 (2005) 62 – 66
binding of both dopamine agonists and antagonists in a dose-dependent manner [9,13,28]. Ample behavioral evidence support an antidopaminergic action of ascorbate, including the findings that ascorbate potentiates the catalepsy induced by the antipsychotic, haloperidol [7,25], and that the systemic and intraneostriatal administration of ascorbate blocks amphetamine-induced hyperactivity [25,29]. These results suggest that ascorbate inhibits dopamine neurotransmission. The aim of this study was to investigate whether ascorbate might prevent the increase of the dopamine system activity induced by morphine in the nucleus accumbens. Since DOPAC is indicating intraneuronal synthesis and metabolism of dopamine [10], therefore, the present study investigated the effect of ascorbate (administered ip) on the extracellular DOPAC level in the nucleus accumbens using in vivo microdialysis in rats. Additionally, the effect of ascorbate on the DOPAC level in the nucleus accumbens in response to a single morphine injection was also assessed.
2. Materials and methods 2.1. Drugs Morphine sulfate (Temad Company, Iran; 20 mg/kg ip) and ascorbate (BDH England; 500 mg/kg ip) were dissolved in normal saline.
63
NaH2PO4, 7.2 mM glucose, pH 7.4) [26], at a flow rate of 2 Al/min, using a syringe pump (model 100, Stoelting). After a 60 min washout period, dialysis samples were collected for 20 min periods successively in vials containing 10 Al of 0.5 M acetic acid to minimize decomposition. Up to six baseline samples were collected prior to drug injections, and samples were collected for 2 h following the drug injection. The microdialysis samples were stored at 70 -C until analyzed. 2.4. Experimental groups Experimental groups consisted of (1) saline group that was injected with saline following the collection of the baseline samples. (2) ascorbate group that was injected with ascorbate (500 mg/kg ip) following the collection of the baseline samples. (3) saline + morphine group that was injected with saline 10 min after the collection of the fourth baseline sample and, 30 min later, received an injection of morphine (20 mg/kg ip). (4) ascorbate + morphine group that was injected with ascorbate (500 mg/kg) 10 min after the collection of the fourth baseline sample and, 30 min later, received an injection of morphine (20 mg/kg ip). In all groups, the effects of the drug were monitored for 2 h. 2.5. Histology
2.2. Subjects and surgery Experiments were performed on 21 male Wistar rats (240 –280 g). They were housed in groups of four per cage in a colony room controlled for temperature with a normal 12 h light/12 h dark cycle. Food and water were available ad libitum. Animals were anesthetized with chloral hydrate (450 mg/ kg, ip), and supplementary doses were administered if needed. Body temperature was maintained at 37 T 0.1 -C with a temperature control unit (Letica, Spain). The rats were implanted stereotaxically with a guide cannula unilaterally, with tips aimed over the nucleus accumbens. The coordinates were chosen such that the tip of the dialysis probes (220 Am outer diameters and a 1 mm active membrane, AgnTho’s, Lidingo, Sweden) was located in the medial portion of the shell of the accumbens (Ap = 1.7 mm, L = 0.8 mm from bregma, DV = 7.5 mm from the surface of skull) [19]. The cannula was fixed firmly to the skull with dental cement. 2.3. In vivo microdialysis The microdialysis probe was inserted through the guide cannula and was continuously perfused with artificial cerebrospinal fluid (140 mM Nacl, 3 mM KCl, 2.4 mM CaCl2, 1 mM MgCl2, 1.2 mM Na2HPO4, 0.27 mM
Upon completion of a microdialysis experiment, the animal was sacrificed by a high dose of the anesthetic then transcardially perfused with 100 ml of saline followed by 100 ml of 10% formalin. The brain was removed and stored in 10% formalin for at least 24 h. Brains were sectioned coronally at 30 Am by a freezing microtome (Leica, Germany). Sections were mounted on gelatin-coated slides and stained with cresyl violet. The tracks left by the probes were identified using a light microscope, and their exact positions determined by reference to a rat brain atlas [19]. Only the dialysates of animals whose probe were within the shell of the nucleus accumbens were analyzed. 2.6. Catecholamine assay Dialysate samples were analyzed for DOPAC by high performance liquid chromatography (HPLC) with electrochemical detection. The HPLC system consisted of a Shimadzu C18 column (250 4.6 mm 5Am), a Shimadzu solvent delivery system (LC 10 ADvp) and a Shimadzu electrochemical detector (LC-ECD 6 A) with a working electrode set at a potential of 0.75 V. The mobile phase consisted of 0.07 M sodium acetate, 0.1 mM sodium octyl sulfate, 0.1 mM EDTA in 10% HPLC grade acetonitrile, adjusted to pH 3.7 with acetic acid. The flow rate was set at 1 ml/min.
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2.7. Statistical analysis Baseline values for DOPAC (mean of the value of the six 20-min samples T SEM), expressed as pmol/10 Al. The data were expressed as percentage of baseline values. The values at different intervals were compared both within each experimental group and between 4 experimental groups using repeated measures ANOVA. A Tukey test for post-hoc comparisons was performed when appropriate. A P < 0.05 was considered as significant.
3. Results 3.1. Acute effect of ascorbate Baseline values for DOPAC did not significantly differ between the two groups of saline (2.061 T 0.31 pmol/10 Al) and ascorbate (2.3 T 0.31 pmol/10 Al). As shown in Figs. 1 and 2, two-way ANOVA with repeated measures revealed that extracellular levels of DOPAC in the nucleus accumbens did not significantly change due to treatment with saline or ascorbate (500 mg/kg). 3.2. Effect of ascorbate in rats treated acutely with morphine The baseline values of DOPAC did not significantly differ between the two groups of saline + morphine (2.85 T 0.38 pmol/10 Al) and ascorbate + morphine (2.36 T 0.39 pmol/10 Al). As shown in Figs. 3 and 4, one-way ANOVA with repeated measures revealed that acute treatment with morphine (20 mg/kg ip) significantly increased the extracellular levels of DOPAC ( P < 0.05) in the nucleus accumbens. Two-way ANOVA with repeated measures revealed that pretreatment with 500 mg/kg ascorbate significantly attenuated the effect of morphine on the
Fig. 1. The extracellular levels (mean T SEM) of DOPAC in the nucleus accumbens, expressed as the percentage of baseline, prior to and following saline (n = 5) or ascorbate (500 mg/kg ip; n = 6) administration at time 0 (arrow). There was no significant change due to saline or ascorbate (ANOVA, P > 0.05).
Fig. 2. Average (TSEM) of the DOPAC changes over 2 h induced by saline and ascorbate (500 mg/kg ip) in the nucleus accumbens, expressed as percentage of baseline. There was no significant changes due to saline or ascorbate (ANOVA, P > 0.05).
extracellular levels of DOPAC ( P < 0.05) in the nucleus accumbens. 3.3. Locations of the accumbal dialysis probes All animals that were included in the biochemical analysis had appropriate probe placements within the shell region of the nucleus accumbens. Representative of the accumbal probe placements are shown in Fig. 5.
4. Discussion The mesolimbic pathway is often thought of as the ‘‘reward pathway’’ and connects the VTA with the nucleus
Fig. 3. The extracellular levels (mean T SEM) of DOPAC in the nucleus accumbens, expressed as the percentage of baseline, prior to and following saline (n = 5) or ascorbate (500 mg/kg ip; n = 5) pretreatment before an acute dose of morphine (20 mg/kg ip). Saline or ascorbate administered at time 30 (first arrow), 20 mg/kg morphine administered at time 0 (second arrow). Ascorbate significantly attenuated the effect of morphine on the levels of DOPAC (ANOVA, *P < 0.05).
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Fig. 4. Average (TSEM) of the DOPAC changes over 2 h induced by saline or ascorbate pretreatment (500 mg/kg ip) before an acute dose of morphine (20 mg/kg ip) in the nucleus accumbens, expressed as the percentage of baseline. Ascorbate significantly attenuated the effect of morphine on the levels of DOPAC (ANOVA, *P < 0.05).
dopamine release remains unclear, although a few possibilities exist. Some studies indicate that pharmacological stimulation of NMDA receptors of glutamate in the VTA promotes dopamine release in the nucleus accumbens [12,27], and high doses of ascorbate have been proposed to block NMDA receptors in vitro [17]. Therefore, high doses of ascorbate probably blocked the NMDA receptors in the VTA and reduced the release of dopamine in the nucleus accumbens. Other explanation for our results with high-dose ascorbate may be a blockade of dopamine receptors in the VTA. In vitro evidence suggests that ascorbate inhibits dopamine binding, and it appears to act like a dopamine receptor antagonist [9,13,24,28]. In the VTA, D2 autoreceptors stimulation by somatodendritically released dopamine directly modulates the excitability of dopamine cells and release of dopamine in the nucleus accumbens [3], while D1 receptors modulate the transmitter release of afferents to the
accumbens as well as the olfactory tubercle, amygdala and prefrontal cortex [30]. Most drugs of abuse have been shown to increase extracellular levels of dopamine in the nucleus accumbens [21]. In the present study, morphine administration induced an increase in dopamine’s acidic metabolite DOPAC in the nucleus accumbens, indicating the increase of intraneuronal synthesis and metabolism of dopamine [10]. This result is in agreement with previous findings [5,6,16,21 – 23]. Most studies have reported the increasing effects of one to three dosages at most of morphine on extracellular dopamine levels in the nucleus accumbens [5,6,21 –23]. Maisonneuve et al. (2001) studied the effects of morphine on extracellular dopamine levels in brain over a wide range of doses. This study showed that the dose –response curve for morphine’s effect on extracellular dopamine levels in the nucleus accumbens has a curvilinear shape while the dose – response curve for morphine effects on extracellular dopamine metabolites is a monotonic curve [16]. Previous localization studies have found opioid receptors on the GABAergic neurons in the VTA. When these GABAergic interneurons have been stimulated, the firing of the dopamine cells in the VTA is inhibited [30]. Previous studies suggest that the systemic administration of morphine inhibits these VTA GABA interneurons, which results in disinhibition of dopamine cells and release of dopamine is increased in the nucleus accumbens [4,30]. We also found that ascorbate alone had no effect on the extracellular level of DOPAC in the nucleus accumbens of naive rats. However, pretreatment with ascorbate attenuated the increase in the extracellular DOPAC level induced by acute morphine administration, indicating the modulation of mesolimbic dopaminergic pathway by ascorbate. The mechanisms by which high-dose ascorbate (500 – 1000 mg/kg) pretreatment antagonizes morphine-induced
Fig. 5. The placements of probes implanted in the nucleus accumbens of rats included in statistical analysis. Figure modified from the atlas of Paxinos and Watson [19]. Acbc: nucleus accumbens, core; AcbSh: nucleus accumbens, shell.
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dopaminergic cells [11]. Therefore, ascorbate might have modulated the release of dopamine in the nucleus accumbens by inhibiting D1 and D2 receptors in the VTA. Furthermore, some evidence indicates that ascorbate may interact with opioid peptides [8] and perhaps sigma receptors [20]. In conclusion, for the first time, our study showed that ascorbate pretreatment is capable of reducing morphineinduced dopamine release in the nucleus accumbens, suggesting that ascorbate may have a role in the modulation of mesolimbic rewarding pathway. Thus, it is probable that ascorbate might decrease the rewarding effect and psychic dependence of opioids. This hypothesis needs further behavioral experiments.
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[16]
[17]
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