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GABAA receptors in VTA mediate the morphine-induced release of ascorbic acid in rat nucleus accumbens
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GABAA receptors in VTA mediate the morphine-induced release of ascorbic acid in rat nucleus accumbens Ji-Ye Sun, Jing-Yu Yang, Fang Wang, Yue Hou, Ying-Xu Dong, Chun-Fu Wu⁎ Department of Pharmacology, Shenyang Pharmaceutical University, 110016 Shenyang, PR China
A R T I C LE I N FO
AB S T R A C T
Article history:
Local perfusion of morphine produces increased levels of extracellular ascorbic acid (AA) in
Accepted 10 October 2010
the nucleus accumbens (NAc) of freely moving rats. However, the pathways that regulate
Available online 19 October 2010
morphine-induced AA release in the NAc are unclear. In the present study, we used high performance liquid chromatography with electrochemical detection (HPLC–ECD) to examine
Keywords:
the effects of intra-ventral tegmental area (VTA) administration of a GABAA agonist and
Nucleus accumbens
antagonist on morphine-induced increases in AA of the NAc. Also, using high performance
Morphine
liquid chromatography with fluorescent detection (HPLC–FD) and HPLC–ECD, the releases of
Microdialysis
γ-aminobutyric acid (GABA) and dopamine (DA) in the NAc induced by intra-VTA
Ascorbic acid
administration of a GABAA agonist and antagonist were also investigated. The results
GABAA receptor
obtained showed that morphine (1 mM), locally perfused into the NAc, significantly
Ventral tegmental area
increased AA release in the NAc and also GABA release. Intra-VTA infusion of bicuculline (150 ng/rat), a GABA receptor antagonist, not only abolished the enhanced extracellular AA and GABA levels produced by local perfusion of morphine but also decreased the basal release of extracellular GABA and increased the basal release of extracellular DA in the NAc. Muscimol (100 ng/rat), a GABA receptor agonist, affected the basal release of GABA and DA, but not the basal AA levels, or the morphine-induced changes in AA and GABA levels. These findings suggest that the GABAA receptors in the VTA play an important role in the modulation of morphine-induced AA release in the NAc, and the effect of morphine on AA release in the NAc is partially regulated by the GABAA receptor-mediated action of DA afferents from the VTA. © 2010 Elsevier B.V. All rights reserved.
1.
Introduction
The nucleus accumbens (NAc), an important brain structure involved in emotion, motivation, limbic-motor integration and, especially, behaviorally rewarding effects of opioid receptor agonists (Mogenson et al., 1982; Gu et al., 2005). It is markedly affected by morphine treatment and withdrawal and plays a crucial role in the reinforcing effects
of abused drugs (Pierce and Kalivas, 1997). In fact, the NAc is a region of convergence of midbrain GABAergic and dopaminergic pathways (Xi and Stein, 1998). The NAc receives extensive neuronal input from outside nuclei, such as glutaminergic input from the prefrontal cortex and hippocampus (Rada et al., 2003), and dopaminergic projection from the VTA (Grace and Bunney, 1979; Cheatwood et al., 2005).
⁎ Corresponding author. Fax: + 86 24 2384 3567. E-mail addresses: [email protected], [email protected] (C.-F. Wu). 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.10.029
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Ascorbic acid (AA), a potent neuromodulator in the central nervous system (CNS), plays a significant role in normal neuronal physiology (Rice, 2000). Increasing evidence has shown that the brain extracellular concentrations of AA change rapidly in response to drugs that affect neural activities. For example, it has been found that several typical addictive drugs, such as ethanol, morphine, amphetamine, and nicotine, which possess centrally stimulating properties, can increase AA release in the rat nucleus accumbens (NAc) (Brazell et al., 1990; Gu et al., 2005). AA can block the damphetamine-induced stereotype and enhance the antidopamine effect of haloperidol in rats (White et al., 1988). Pretreatment with AA can potentiate an ethanol-induced loss of the righting reflex in mice (Wu et al., 2000). These results suggest that AA release induced by an addictive drug might have a complex effect in the brain. Our previous studies have shown that, following administration to the NAc, morphine affected the release of AA. Moreover, the actions of morphine on AA levels following local infusion into the NAc and intraperitoneal injection have been found to be different from those of other reinforcing drugs (Gu et al., 2005, 2006; Dai et al., 2006). The NAc is greatly affected by morphine treatment and withdrawal and plays a crucial role in the reinforcing effects of morphine (Self, 2004). The mechanisms and the pathological implication of the morphine-induced increase in extracellular AA levels in the NAc are not yet well understood (Gu et al., 2005). In investigating the pathways that modulate morphineinduced AA release in NAc, the previous studies have found that neither the precortico-accumbens nor the hippocampalaccumbens projection affects local morphine-induced AA release in the NAc (Gu et al, 2005; Dai et al., 2006). Since the VTA is an important region involved in evolutionary and essential behaviors (Fields et al., 2007; Michaeli and Yaka, 2010) and the neuronal interaction between VTA and NAc is important in drug abuse, it has been proposed that VTA plays a role in morphine-induced AA release in the NAc. The NAc receives many afferents from outside nuclei, and it is also widely reported that GABAA receptors may play an important modulatory role in regulating the mesolimbic system in drug abuse (Xi et al., 1998). Pretreatment with the GABAA receptor antagonist, bicuculline, significantly reduced the effects of a μ opioid agonist on feeding behavior both in the NAc shell (Znamensky et al., 2001) and VTA (Echo et al., 2002). Moreover, several studies have shown that various neuronal systems, such as the glutamatergic, dopaminergic, cholinergic, and serotonergic systems, may be involved in central AA release (Grunewald, 1993; Liu et al., 2000). Therefore, it would be interesting to explore the possible functional link between VTA and NAc in terms of the GABAergic regulation in VTA on AA, as well as the release of other neurotransmitters in the NAc. The present study was designed to investigate the effects of microinjection of the GABAA receptor agonist, muscimol, and the antagonist, bicuculline, into the VTA on the morphine-induced changes in extracellular levels of AA in the rat NAc. To further clarify the regulatory roles of GABA receptors within the mesolimbic DA system, the release of DA and GABA in the NAc was also measured in freely behaving rats. The research was undertaken in the hope that the information on the role of neuron projection from the VTA to the NAc may
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shed light on the potential effects of morphine on the release of AA in the NAc.
2.
Results
2.1. Effects of muscimol and bicuculline on morphine-induced AA release in the NAc Local infusion of muscimol (100 ng) and bicuculline (150 ng) bilateral to the VTA had no effect on the dialysate concentrations of AA in the NAc [F(5,29) = 0.32, P > 0.05; F(5,29) = 0.28, P > 0.05] (Fig. 1). Morphine (1 mM), locally perfused into the NAc, significantly increased the AA release in the NAc (Fig. 2) (Gu et al., 2006). However, bilateral infusion of muscimol (100 ng) and bicuculline (150 ng) into the VTA had a different effect on the morphine-induced release of AA. As shown in Fig. 2, muscimol has no effect on the action of AA induced by morphine [F(5,38) = 0.95, P > 0.05]. However, the increased release of AA induced by morphine was inhibited by bicuculline [F(5,43) = 29.62, P < 0.001].
2.2. Effects of muscimol and bicuculline on basal and morphine-induced release of GABA in the NAc To determine the effects of GABAA receptors on the functional link between the VTA and NAc, extracellular GABA concentrations in the NAc were determined following bilateral infusion of muscimol (100 ng) and bicuculline (150 ng) into the VTA. Muscimol significantly increased GABA release in the NAc [F (5,27) = 34.46, P < 0.001] while bicuculline significantly reduced it [F(5,36) = 12.05, P < 0.001] (Fig. 3). Morphine (1 mM) significantly increased GABA release in the Nac while bilateral infusion of muscimol (100 ng) into the VTA did not affect the morphine-induced release of GABA [F (3,25) = 0.25, P > 0.05]. However, bicuculline (150 ng) inhibited the increased GABA release induced by morphine [F(5,96) = 117.36, P < 0.001] (Fig. 4).
Fig. 1 – Effect of AA release in the NAc induced by bilateral VTA infusion of bicuculline and muscimol in rats. Results are means ± SEM of the data obtained in six rats per group and expressed as the percentage changes in the corresponding baselines.
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Fig. 2 – Effect of AA release in the NAc induced by morphine after bilateral VTA infusion of bicuculline and muscimol in rats. Results are means ± SEM of the data obtained in six rats per group and expressed as the percentage changes in the corresponding baselines. *P < 0.05, **P < 0.01; Mor + bicuculline group compared with Mor group.
2.3. Effects of muscimol and bicuculline on the release of DA in the NAc DA levels in the NAc were measured following administration of muscimol (100 ng) and bicuculline (150 ng). Muscimol markedly reduced DA release [F(5,65) = 8.63, P < 0.05] while bicuculline markedly increased it [F(5,85) = 17.26, P < 0.001] in the NAc (Fig. 5).
3.
Discussion
Previous studies have shown that local perfusion of morphine into the NAc of rats significantly increases the release of AA in
Fig. 3 – Effect of GABA release in the NAc induced by bilateral VTA infusion of bicuculline and muscimol in rats. Results are means ± SEM of the data obtained in six rats per group and expressed as the percentage changes in the corresponding baselines. *P < 0.05, **P < 0.01; bicuculline and muscimol group compared with saline group.
Fig. 4 – Effect of GABA release in the NAc induced by morphine after bilateral VTA infusion of bicuculline and muscimol in rats. Results are means ± SEM of the data obtained in six rats per group and expressed as the percentage changes in the corresponding baselines. *P < 0.05, ** P < 0.01; Mor + bicuculline group compared with Mor group.
this brain region (Dai et al., 2006; Gu et al., 2006). However, undercutting the prefrontal cortex, which could cut down both the cortico-striatal and cortico-accumbens pathways, does not affect drug-induced AA release in the NAc (Gu et al., 2005). Further studies have shown that damage to the integrity of the hippocampal-accumbens glutamatergic pathway had no effect on AA release in the NAc induced by local perfusion of morphine (Dai et al., 2006). The present study shows that VTA administration of a GABAA receptor agonist and antagonist did not affect the basal release of AA in the NAc. However, the GABAA receptor antagonist, bicuculline, prevented the increasing effect of morphine on AA release in the NAc. This is the first observation that the local action of morphine on AA
Fig. 5 – Effect of DA release in the NAc induced by bilateral VTA infusion of bicuculline and muscimol in rats. Results are means ± SEM of the data obtained in six rats per group and expressed as the percentage changes in the corresponding baselines. *P < 0.05, **P < 0.01, ***P < 0.001; bicuculline and muscimol group compared with saline group.
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release in NAc could be affected by the GABAergic system originating from the VTA. Studies have demonstrated that there is a functional link between AA release and the GABAergic system. For instance, GABA infusion into the substantia nigra pars reticulata inhibits AA release in the ipsilateral striatum of the rat. In contrast, intranigral application of a GABA antagonist has the opposite effect (Christensen et al., 2000). GABA induces the release of AA from the rat striatum, and the releasing action of GABA is mimicked by the GABA agonist, muscimol (Bigelow et al., 1984). Results from the present study show that morphine can increase the release of GABA in the NAc. However, this effect can also be inhibited by bicuculline administration in the VTA. These results provide further support for the hypothesis that AA release is GABA-receptor mediated and synaptically localized (Bigelow et al., 1984), although studies have shown that the release of AA undergoes a hetero-exchange mechanism of glutamate and ascorbate (Rebec, 1997). The role of the mesolimbic dopaminergic system in the rewarding effects is well known (Spanagel and Welss, 1999). Many studies have suggested that the mesolimbic dopaminergic system that projects from the VTA to the NAc is critical for the initiation of opioid reinforcement (Koob, 1992; Wise, 1998; Hyman and Malenka, 2001; Robinson and Berridge, 2003; Sahraei et al., 2005). Studies have shown that morphine activates VTA dopamine neurons and enhances dopamine release in the NAc via inhibition of GABA neurons (Johnson and North, 1992). Intra-VTA administration of a GABAA receptor agonist and antagonist can have different effects on the expression of morphine positive reinforcement. It has been reported that GABAA receptors located in the VTA affected the expression of morphine-induced conditioned place preference (CPP) in rats. Intra-VTA administration of the GABAA receptor agonist, muscimol, increased the expression of CPP induced by morphine. A reduction in the expression of morphine-induced CPP was observed after intra-VTA injection of the GABAA antagonist, bicuculline (Sahraei et al., 2005). Activation of pre-synaptic GABAA receptors leads to a decreased GABA release from GABAergic interneurons and there is a subsequent increase in dopaminergic neuronal activity. On the other hand, activation of postsynaptic GABAA receptors may reduce the activity of dopaminergic neurons within the VTA. This phenomenon was considered as the main cause of the effects of muscimol and bicuculline on morphine-induced seeking behavior. The present study noted that bicuculline antagonized the action of morphine on AA release in the NAc. Therefore, it appears that the results of morphine observed in the present study could be modulated by the action on the dopaminergic projection from the VTA to the NAc. The present study shows that intra-VTA application of bicuculline significantly enhances DA release in the NAc, and muscimol has the opposite effect. Dopamine afferents coordinate the activity of NAc neurons that, for the major part, consist of GABA producing medium-sized spiny neurons (Pierce and Kalivas, 1997). GABAA receptors in the VTA appear to be located on both DA neurons (Johnson and North, 1992; Xi and Stein, 1998) and non-DA (GABAergic) neurons (Churchill et al., 1992). The prevailing view that has emerged since these studies is that this effect of muscimol reflects an indirect disinhibition of DA
55
cell activity in VTA (Doherty and Gratton, 2007). Activation of the GABAA receptors localized on DA cells should directly hyperpolarize DA neurons and decrease both VTA somatodendritic and efferent terminal DA release in the NAc (Xi and Stain, 1998). It has been proposed in previous studies that VTA GABAA receptors mediate regulation of meso-NAc DA function (Xi and Stein, 1998). Muscimol, activating post-synaptic GABAA receptors, may reduce the activity of dopaminergic neurons within the VTA. This function is similar to GABA activity, and as a result, there is reduced DA release at the target sites including NAc (Sahraei et al., 2005). It was also observed that the GABAA agonist, muscimol, increased GABA release in the NAc, but the antagonist, bicuculline, had the opposite effect. Moreover, bicuculline could block the increased release of GABA induced by morphine in the NAc. It has been reported that bicuculline dose-dependently potentiates the NAc DA stress response, but muscimol does not (Doherty and Gratton, 2007). This effect of bicuculline would be consistent with the idea that the activity of DA neurons is also regulated by a direct GABAA receptormediated action of VTA GABA afferents (Steffensen et al., 2008). Accordingly, it appears that bicuculline inhibits the activity of GABAergic neurons in the VTA and increases the activity of dopaminergic afferents to the NAc, preventing the synthesis and release of GABA. Finally, the action of morphine on the release of AA and GABA in the NAc is blocked. In light of these results, the simplest explanation for the potential effects on morphine-induced AA release is that this dose of bicuculline inactivated GABAA receptors located on GABA interneurons, inhibited their activity and then, in turn, disinhibited the activity of DA cells, which finally was related to the effects of blocking the AA release induced by morphine in the NAc (Fig.6). Supporting data have also emerged from postmortem studies in humans. In the NAc of schizophrenic patients, the dopamine concentration is increased while that of GABA is reduced (Bird et al., 1977; Perry et al., 1979; Mackay et al., 1982). Inhibition of GABAA receptors in the VTA should attenuate AA release in the NAc. This suggests that increased NAc GABAergic activity and decreased dopaminergic activity during bicuculline treatment with VTA may block the release of extracellular AA induced by morphine-perfusion into the NAc. In conclusion, the data reported in the present study show that application of a GABAA receptor antagonist in the VTA could inhibit the effects of morphine on the release of AA and GABA in the NAc. Based on the current findings, a new hypothesis is proposed, namely, that the GABAergic system in the VTA may be involved in the morphine-induced release of AA and GABA in the NAc, although much evidence is needed in support of this. These results also indicate that this action of morphine in the NAc is regulated by the GABAA receptormediated action of DA afferents from the VTA.
4.
Experimental procedures
4.1.
Animals
Male Sprague–Dawley rats weighing 180–220 g were used. All animals were supplied by the Experimental Animal Centre of Shenyang Pharmaceutical University and kept with standard
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Fig. 6 – Diagram showing the possible mechanisms through which AA and GABA release may be regulated by the DAergic inputs (blue afferents) from the VTA to the NAc (Mora et al., 2008). The perfusion of GABA antagonist bicuculline locally into the VTA would result in an inhibition of GABAergic neurons (red afferents) in the VTA. Activation of the DAergic afferents to the NAc increases DA release, decreases the GABA release, and then prevents the morphine-induced release of AA in the NAc.
light–dark cycle (light on at 06:30 a.m.). Rats were housed in cages with food and water available ad libitum. All experiments and procedures were carried out according to the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of China.
4.2.
Drugs
Morphine hydrochloride was purchased from Shenyang First Pharmaceutical Factory (Shenyang, China). Ringer's solution (ACSF) was composed of 147 mM NaCl, 2.2 mM CaCl2 and 4 mM KCl (Dai et al., 2006; Gu et al., 2006). Muscimol and bicuculline, purchased from Sigma Chemicals (St. Louis, MO, USA), were dissolved in ACSF. Morphine was dissolved in ACSF for the perfusion with the concentrations used in previous experiments (Gu et al., 2006). The o-phthalaldehyde solution was prepared as follows: 9 mg OPA was dissolved in 167 μl methanol then 1.5 ml 0.4 M sodium borate and 7 μl 2mercaptoethanol were added and mixed well. The solution was stored at 4 °C for one week and protected from light.
4.3.
Surgical procedure and microdialysis in NAc
The procedure used to prepare and implant the dialysis probe was as previously described (Wu et al., 1988). The rats were anesthetized with chloral hydrate (350 mg/kg, i.p.) and Hospal AN 69 dialysis fibers (310 μm o.d., 200 μm i.d., Dasco, Bologna, Italy) were implanted transversally through the NAc (coordinates: A: + 1.7 mm from bregma, V: −7.5 mm from occipital bone (Gu et al., 2005). A dialysis tube was covered with SuperEpoxy glue over its whole length except for the zones (2.0 mm wide) which were to be positioned in the NAc (Liu et al., 2000). This kind of probe has a total dialysis length of 4.0 mm and can
collect the neurochemicals of interest from both sides of the NAc. The animals were individually housed in plastic cages and left to recover for about 24 h after surgery. Surgical procedures for implantation of intra-VTA cannula were also performed under anesthesia. A 26-gauge stainless-steel guide cannula was directed into the VTA (A/P −6.0 mm, M/L 1.0 mm, D/V − 7.8 mm) bilaterally according to a rat brain atlas (Ikemoto et al., 1998; Paxinos and Watson, 1997) and was fixed to the skull by dental acrylic. The cannula was secured to the skull surface with dental cement. When the dental cement had hardened, a dummy cannula, cut to the same dimensions as the guide cannula, was inserted to seal the top of the guide cannula to prevent clogging and minimize possible infection. The rats were allowed 1 day for recovery. Injection was made by inserting a 33-gauge stainless-steel injector tube into the guide cannula. The injector tube was attached to PE-10 tubing fitted to a 10-μl Hamilton Syringe. One microliter of solution was thereafter infused into the lateral cerebral ventricle over 2 min. Brain dialysis was performed about 24 h after the probe implantation in freely moving rats. ACSF was pumped through the dialysis probe at a constant rate of 2 μl/min. After an hour, the dialysis samples were collected and analyzed every 20 min. Test solutions were continuously pumped through the dialysis probe when the output of AA, GABA or DA became stable in the last three samples with a variation of no more than 10%. After the basal release of the neurochemicals became stable, morphine (1 mM) was administrated to the NAc by local perfusion for 3 h. The bicuculline and muscimol were injected into the VTA 1 h after the morphine administration. At the end of the experiments, the position of the dialysis fiber was verified by visual examination, and the datum was discarded if the fiber was positioned incorrectly (Liu et al., 2000; Gu et al., 2005). The rats with a bilateral VTA cannula received the bilateral microinjections following 24 h of food deprivation; the details of the injections were as follows: bicuculline at a total dose of 150 ng (n=6, 75 ng/0.5 μl each side) and muscimol at total dose of 100 ng (n=6, 50 ng/0.5 μl each side).
4.4.
Analytical procedure
Each dialysate sample (40 μl) was divided into two fractions. Twenty microliters was immediately injected directly into the HPLC–ECD for determination of the AA content. A reverse phase column (C-18, 5 μm, Agilent) was used with a mobile phase composed of 155.6 mM NaCl and EDTA-Na2 0.54 mM with tetrabutylammonium bromide 1.5 mM as an ion pairing reagent. The mobile phase was pumped using an LC-10A pump (SHIMADZU, Japan) at a flow rate of 1.0 ml/min. The detector (L-ECD-6A, SHIMADZU, Japan) was set at +0.60 V (Liu et al., 2000). The remaining 20 μl was frozen at −20 °C until required for HPLC–FD (excitation wavelength: 340 nm, emission wavelength: 450 nm, SHIMADZU, Japan) for GABA, following precolumn derivatization with o-phthalaldehyde (Hao et al., 2005), with some modifications. Gradient elution was used to separate the mixture of amino acids (Table 1). Mobile phase A consisted of 0.1 M sodium acetate buffer, pH 6.7 while mobile phase B contained 98% methanol and 2% tetrahydrofuran (Tiff). The flow rate of the pump was set at 1.0 ml/min. All the mobile phases were passed through a
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Table 1 – Gradient program of HPLC (mm). Time
A%
B%
0.00 7.00 12.00 13.00 20.00
65 35 35 65 65
35 65 65 35 35
0.22 μm filter. The column temperature was maintained at 37 °C (Hao et al., 2005). Another group of animals was used to collect the dialysate for DA determination using HPLC–ECD with the following conditions: a reverse phase column (C-18, 5 μm, Agilent) with a mobile phase composed of 85 mM citrazinic acid, 100 mM sodium acetate anhydrous, 0.2 mM Na2EDTA, and 1.2 mM sodium 1-octanesulfonate adjusted to pH 3.7.
4.5.
Histological verification of the injection site
In pilot studies, at the end of the experiments, the anatomical accuracy of the needle placements was verified by injecting 5 μl trypan blue into the VTA. Then, the head was fixed in 10% formalin for 24 h with the injecting tube in situ before section. The location of the tip of the injecting tube was verified and all the tips of the injecting tube were in the VTA of rat in the present study.
4.6.
Statistical analysis
Statistical analysis was carried out using SPSS 13.0 software for Windows (SPSS Inc., Chicago, IL, USA). All values were expressed as mean ± S.E.M. The levels of AA, GABA and DA were expressed as the percentage change compared with the respective basal value that was the mean of three consecutive samples before drug administration within a variance of 10%, and for each time point, a one-way ANOVA was used. The level of significance was set at P < 0.05.
Acknowledgments This research is partly supported by the Project of Key Laboratory for New Drug Screening, Key Laboratory for Pharmacodynamics, of Liaoning Province, and by the National Key Scientific Project for New Drug Discovery and Development, (2009ZX09301-012), 2009-2010, P. R. China.
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activation of presynaptic G-protein coupled inwardly-rectifying potassium channels. Neuroscience 165, 1159–1169. Mogenson, G.J., Jones, D.L., Yim, C.Y., 1982. From motivation to action: Functional interface between the limbic system and the motor system. Prog. Neurobiol. 14, 69–97. Mora, F., Segovia, S., del Arco, A., 2008. Glutamate-domapineGABA interactions in the aging basal ganglia. Brain Res. Rev. 58, 340–353. Paxinos, G., Watson, C., 1997. The Rat Brain in Stereotaxic Coordinates, compact third edition. Academic Press. Perry, T.L., Buchanan, J., Kish, S.J., Hansen, S., 1979. Gamma-aminobutyric-acid deficiency in brain of schizophrenic patients. Lancet 313, 237–239. Pierce, R.Ch., Kalivas, P.W., 1997. A circuitry model of the expression of behavioral sensitization to amphetamine-like psychostimulants. Brain Res. Rev. 25, 192–216. Rada, P., Moreno, S.A., Tucci, S., Gonzalez, L.E., Harrison, T., Chau, D.T., Hoebel, B.G., Hernandez, L., 2003. Glutamate release in the nucleus accumbens is involved in behavioral depression during the porsolt swim test. Neuroscience 119, 557–765. Rebec, G.V., 1997. Ascorbate: An antioxidant and extracellular neuromodulator. In: Connor, J.R. (Ed.), Metals and Oxidative Damage in Neurological Disorders. Plenum Press, New York, pp. 149–173. Rice, M.E., 2000. Ascorbate regulation and its neuroprotective role in the brain. Trends Neurosci. 23, 209–216. Robinson, T.E., Berridge, K.C., 2003. Addiction. Annu. Rev. Psychol. 54, 25–53. Sahraei, H., Amiri, Y.A., Haeri-Rohani, A., Sepehri, H., Salimi, S.H., Pourmotabbed, A., Ghoshooni, H., Zahirodin, A., Zardooz, H., 2005. Different effects of GABAergic receptors located in the ventral tegmental area on the expression of morphine-induced conditioned place preference in rat. Eur. J. Pharmacol. 524, 95–101.
Self, D.W., 2004. Regulation of drug-taking and -seeking behaviors by neuroadaptations in the mesolimbic dopamine system. Neuropharmacology 47, 242–255. Spanagel, R., Welss, F., 1999. The dopamine hypothesis of reward: past and current status. Trends Neurosci. 22, 521–527. Steffensen, S.C., Taylor, S.R., Horton, M.L., Barber, E.N., Lyle, L.T., Stobbs, S.H., Allison, D.W., 2008. Cocaine disinhibits dopamine neurons in the ventral tegmental area via use-dependent blockade of GABA neuron voltage-sensitive sodium channels. Eur. J. Neurosci. 28, 2028–2040. White, L.K., Carpenter, M., Block, M., Basse-Tomusk, A., Gardiner, T.W., Rebec, G.V., 1988. Ascorbate antagonizes the behavioral effects of amphetamine by a central mechanism. Psychopharmacology 94, 284–287. Wise, R.A., 1998. Drug-activation of brain reward pathways. Drug Alcohol Depend. 51, 13–22. Wu, C.F., Bertorelli, R., Sacconi, M., Pepeu, G., Consolo, S., 1988. Decrease of brain acetylcholine release in aging freely-moving detected by microdialysis. Neurobiol. Aging 9, 357–361. Wu, C.F., Zhang, H.L., Liu, W., 2000. Potentiation of ethanol-induced loss of the righting reflex by ascorbic acid in mice: interaction with dopamine antagonists. Pharmacol. Biochem. Behav. 66, 413–418. Xi, Z.X., Stein, E.A., 1998. Nucleus accumbens dopamine release modulation by mesolimbic GABAA receptors-an in vivo electrochemical study. Brain Res. 798, 156–165. Xi, Z.X., Fuller, S.A., Stein, E.A., 1998. Dopamine release in the nucleus accumbens during heroin self-administration is modulated by k opioid receptors: an in vivo fast-cyclic voltammetry study. J. Pharmacol. Exp. Ther. 284, 151–161. Znamensky, V., Echo, J.A., Lamonte, N., Christian, G., Ragnauth, A., Bodnar, R.J., 2001. γ-Aminobutyric acid receptor subtype antagonists differentially alter opioid-induced feeding in the shell region of the nucleus accumbens in rats. Brain Res. 906, 84–91.
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Research Report
GABAA receptors in VTA mediate the morphine-induced release of ascorbic acid in rat nucleus accumbens Ji-Ye Sun, Jing-Yu Yang, Fang Wang, Yue Hou, Ying-Xu Dong, Chun-Fu Wu⁎ Department of Pharmacology, Shenyang Pharmaceutical University, 110016 Shenyang, PR China
A R T I C LE I N FO
AB S T R A C T
Article history:
Local perfusion of morphine produces increased levels of extracellular ascorbic acid (AA) in
Accepted 10 October 2010
the nucleus accumbens (NAc) of freely moving rats. However, the pathways that regulate
Available online 19 October 2010
morphine-induced AA release in the NAc are unclear. In the present study, we used high performance liquid chromatography with electrochemical detection (HPLC–ECD) to examine
Keywords:
the effects of intra-ventral tegmental area (VTA) administration of a GABAA agonist and
Nucleus accumbens
antagonist on morphine-induced increases in AA of the NAc. Also, using high performance
Morphine
liquid chromatography with fluorescent detection (HPLC–FD) and HPLC–ECD, the releases of
Microdialysis
γ-aminobutyric acid (GABA) and dopamine (DA) in the NAc induced by intra-VTA
Ascorbic acid
administration of a GABAA agonist and antagonist were also investigated. The results
GABAA receptor
obtained showed that morphine (1 mM), locally perfused into the NAc, significantly
Ventral tegmental area
increased AA release in the NAc and also GABA release. Intra-VTA infusion of bicuculline (150 ng/rat), a GABA receptor antagonist, not only abolished the enhanced extracellular AA and GABA levels produced by local perfusion of morphine but also decreased the basal release of extracellular GABA and increased the basal release of extracellular DA in the NAc. Muscimol (100 ng/rat), a GABA receptor agonist, affected the basal release of GABA and DA, but not the basal AA levels, or the morphine-induced changes in AA and GABA levels. These findings suggest that the GABAA receptors in the VTA play an important role in the modulation of morphine-induced AA release in the NAc, and the effect of morphine on AA release in the NAc is partially regulated by the GABAA receptor-mediated action of DA afferents from the VTA. © 2010 Elsevier B.V. All rights reserved.
1.
Introduction
The nucleus accumbens (NAc), an important brain structure involved in emotion, motivation, limbic-motor integration and, especially, behaviorally rewarding effects of opioid receptor agonists (Mogenson et al., 1982; Gu et al., 2005). It is markedly affected by morphine treatment and withdrawal and plays a crucial role in the reinforcing effects
of abused drugs (Pierce and Kalivas, 1997). In fact, the NAc is a region of convergence of midbrain GABAergic and dopaminergic pathways (Xi and Stein, 1998). The NAc receives extensive neuronal input from outside nuclei, such as glutaminergic input from the prefrontal cortex and hippocampus (Rada et al., 2003), and dopaminergic projection from the VTA (Grace and Bunney, 1979; Cheatwood et al., 2005).
⁎ Corresponding author. Fax: + 86 24 2384 3567. E-mail addresses: [email protected], [email protected] (C.-F. Wu). 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.10.029
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Ascorbic acid (AA), a potent neuromodulator in the central nervous system (CNS), plays a significant role in normal neuronal physiology (Rice, 2000). Increasing evidence has shown that the brain extracellular concentrations of AA change rapidly in response to drugs that affect neural activities. For example, it has been found that several typical addictive drugs, such as ethanol, morphine, amphetamine, and nicotine, which possess centrally stimulating properties, can increase AA release in the rat nucleus accumbens (NAc) (Brazell et al., 1990; Gu et al., 2005). AA can block the damphetamine-induced stereotype and enhance the antidopamine effect of haloperidol in rats (White et al., 1988). Pretreatment with AA can potentiate an ethanol-induced loss of the righting reflex in mice (Wu et al., 2000). These results suggest that AA release induced by an addictive drug might have a complex effect in the brain. Our previous studies have shown that, following administration to the NAc, morphine affected the release of AA. Moreover, the actions of morphine on AA levels following local infusion into the NAc and intraperitoneal injection have been found to be different from those of other reinforcing drugs (Gu et al., 2005, 2006; Dai et al., 2006). The NAc is greatly affected by morphine treatment and withdrawal and plays a crucial role in the reinforcing effects of morphine (Self, 2004). The mechanisms and the pathological implication of the morphine-induced increase in extracellular AA levels in the NAc are not yet well understood (Gu et al., 2005). In investigating the pathways that modulate morphineinduced AA release in NAc, the previous studies have found that neither the precortico-accumbens nor the hippocampalaccumbens projection affects local morphine-induced AA release in the NAc (Gu et al, 2005; Dai et al., 2006). Since the VTA is an important region involved in evolutionary and essential behaviors (Fields et al., 2007; Michaeli and Yaka, 2010) and the neuronal interaction between VTA and NAc is important in drug abuse, it has been proposed that VTA plays a role in morphine-induced AA release in the NAc. The NAc receives many afferents from outside nuclei, and it is also widely reported that GABAA receptors may play an important modulatory role in regulating the mesolimbic system in drug abuse (Xi et al., 1998). Pretreatment with the GABAA receptor antagonist, bicuculline, significantly reduced the effects of a μ opioid agonist on feeding behavior both in the NAc shell (Znamensky et al., 2001) and VTA (Echo et al., 2002). Moreover, several studies have shown that various neuronal systems, such as the glutamatergic, dopaminergic, cholinergic, and serotonergic systems, may be involved in central AA release (Grunewald, 1993; Liu et al., 2000). Therefore, it would be interesting to explore the possible functional link between VTA and NAc in terms of the GABAergic regulation in VTA on AA, as well as the release of other neurotransmitters in the NAc. The present study was designed to investigate the effects of microinjection of the GABAA receptor agonist, muscimol, and the antagonist, bicuculline, into the VTA on the morphine-induced changes in extracellular levels of AA in the rat NAc. To further clarify the regulatory roles of GABA receptors within the mesolimbic DA system, the release of DA and GABA in the NAc was also measured in freely behaving rats. The research was undertaken in the hope that the information on the role of neuron projection from the VTA to the NAc may
53
shed light on the potential effects of morphine on the release of AA in the NAc.
2.
Results
2.1. Effects of muscimol and bicuculline on morphine-induced AA release in the NAc Local infusion of muscimol (100 ng) and bicuculline (150 ng) bilateral to the VTA had no effect on the dialysate concentrations of AA in the NAc [F(5,29) = 0.32, P > 0.05; F(5,29) = 0.28, P > 0.05] (Fig. 1). Morphine (1 mM), locally perfused into the NAc, significantly increased the AA release in the NAc (Fig. 2) (Gu et al., 2006). However, bilateral infusion of muscimol (100 ng) and bicuculline (150 ng) into the VTA had a different effect on the morphine-induced release of AA. As shown in Fig. 2, muscimol has no effect on the action of AA induced by morphine [F(5,38) = 0.95, P > 0.05]. However, the increased release of AA induced by morphine was inhibited by bicuculline [F(5,43) = 29.62, P < 0.001].
2.2. Effects of muscimol and bicuculline on basal and morphine-induced release of GABA in the NAc To determine the effects of GABAA receptors on the functional link between the VTA and NAc, extracellular GABA concentrations in the NAc were determined following bilateral infusion of muscimol (100 ng) and bicuculline (150 ng) into the VTA. Muscimol significantly increased GABA release in the NAc [F (5,27) = 34.46, P < 0.001] while bicuculline significantly reduced it [F(5,36) = 12.05, P < 0.001] (Fig. 3). Morphine (1 mM) significantly increased GABA release in the Nac while bilateral infusion of muscimol (100 ng) into the VTA did not affect the morphine-induced release of GABA [F (3,25) = 0.25, P > 0.05]. However, bicuculline (150 ng) inhibited the increased GABA release induced by morphine [F(5,96) = 117.36, P < 0.001] (Fig. 4).
Fig. 1 – Effect of AA release in the NAc induced by bilateral VTA infusion of bicuculline and muscimol in rats. Results are means ± SEM of the data obtained in six rats per group and expressed as the percentage changes in the corresponding baselines.
54
BR A IN RE S EA RCH 1 3 68 ( 20 1 1 ) 5 2 –58
Fig. 2 – Effect of AA release in the NAc induced by morphine after bilateral VTA infusion of bicuculline and muscimol in rats. Results are means ± SEM of the data obtained in six rats per group and expressed as the percentage changes in the corresponding baselines. *P < 0.05, **P < 0.01; Mor + bicuculline group compared with Mor group.
2.3. Effects of muscimol and bicuculline on the release of DA in the NAc DA levels in the NAc were measured following administration of muscimol (100 ng) and bicuculline (150 ng). Muscimol markedly reduced DA release [F(5,65) = 8.63, P < 0.05] while bicuculline markedly increased it [F(5,85) = 17.26, P < 0.001] in the NAc (Fig. 5).
3.
Discussion
Previous studies have shown that local perfusion of morphine into the NAc of rats significantly increases the release of AA in
Fig. 3 – Effect of GABA release in the NAc induced by bilateral VTA infusion of bicuculline and muscimol in rats. Results are means ± SEM of the data obtained in six rats per group and expressed as the percentage changes in the corresponding baselines. *P < 0.05, **P < 0.01; bicuculline and muscimol group compared with saline group.
Fig. 4 – Effect of GABA release in the NAc induced by morphine after bilateral VTA infusion of bicuculline and muscimol in rats. Results are means ± SEM of the data obtained in six rats per group and expressed as the percentage changes in the corresponding baselines. *P < 0.05, ** P < 0.01; Mor + bicuculline group compared with Mor group.
this brain region (Dai et al., 2006; Gu et al., 2006). However, undercutting the prefrontal cortex, which could cut down both the cortico-striatal and cortico-accumbens pathways, does not affect drug-induced AA release in the NAc (Gu et al., 2005). Further studies have shown that damage to the integrity of the hippocampal-accumbens glutamatergic pathway had no effect on AA release in the NAc induced by local perfusion of morphine (Dai et al., 2006). The present study shows that VTA administration of a GABAA receptor agonist and antagonist did not affect the basal release of AA in the NAc. However, the GABAA receptor antagonist, bicuculline, prevented the increasing effect of morphine on AA release in the NAc. This is the first observation that the local action of morphine on AA
Fig. 5 – Effect of DA release in the NAc induced by bilateral VTA infusion of bicuculline and muscimol in rats. Results are means ± SEM of the data obtained in six rats per group and expressed as the percentage changes in the corresponding baselines. *P < 0.05, **P < 0.01, ***P < 0.001; bicuculline and muscimol group compared with saline group.
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release in NAc could be affected by the GABAergic system originating from the VTA. Studies have demonstrated that there is a functional link between AA release and the GABAergic system. For instance, GABA infusion into the substantia nigra pars reticulata inhibits AA release in the ipsilateral striatum of the rat. In contrast, intranigral application of a GABA antagonist has the opposite effect (Christensen et al., 2000). GABA induces the release of AA from the rat striatum, and the releasing action of GABA is mimicked by the GABA agonist, muscimol (Bigelow et al., 1984). Results from the present study show that morphine can increase the release of GABA in the NAc. However, this effect can also be inhibited by bicuculline administration in the VTA. These results provide further support for the hypothesis that AA release is GABA-receptor mediated and synaptically localized (Bigelow et al., 1984), although studies have shown that the release of AA undergoes a hetero-exchange mechanism of glutamate and ascorbate (Rebec, 1997). The role of the mesolimbic dopaminergic system in the rewarding effects is well known (Spanagel and Welss, 1999). Many studies have suggested that the mesolimbic dopaminergic system that projects from the VTA to the NAc is critical for the initiation of opioid reinforcement (Koob, 1992; Wise, 1998; Hyman and Malenka, 2001; Robinson and Berridge, 2003; Sahraei et al., 2005). Studies have shown that morphine activates VTA dopamine neurons and enhances dopamine release in the NAc via inhibition of GABA neurons (Johnson and North, 1992). Intra-VTA administration of a GABAA receptor agonist and antagonist can have different effects on the expression of morphine positive reinforcement. It has been reported that GABAA receptors located in the VTA affected the expression of morphine-induced conditioned place preference (CPP) in rats. Intra-VTA administration of the GABAA receptor agonist, muscimol, increased the expression of CPP induced by morphine. A reduction in the expression of morphine-induced CPP was observed after intra-VTA injection of the GABAA antagonist, bicuculline (Sahraei et al., 2005). Activation of pre-synaptic GABAA receptors leads to a decreased GABA release from GABAergic interneurons and there is a subsequent increase in dopaminergic neuronal activity. On the other hand, activation of postsynaptic GABAA receptors may reduce the activity of dopaminergic neurons within the VTA. This phenomenon was considered as the main cause of the effects of muscimol and bicuculline on morphine-induced seeking behavior. The present study noted that bicuculline antagonized the action of morphine on AA release in the NAc. Therefore, it appears that the results of morphine observed in the present study could be modulated by the action on the dopaminergic projection from the VTA to the NAc. The present study shows that intra-VTA application of bicuculline significantly enhances DA release in the NAc, and muscimol has the opposite effect. Dopamine afferents coordinate the activity of NAc neurons that, for the major part, consist of GABA producing medium-sized spiny neurons (Pierce and Kalivas, 1997). GABAA receptors in the VTA appear to be located on both DA neurons (Johnson and North, 1992; Xi and Stein, 1998) and non-DA (GABAergic) neurons (Churchill et al., 1992). The prevailing view that has emerged since these studies is that this effect of muscimol reflects an indirect disinhibition of DA
55
cell activity in VTA (Doherty and Gratton, 2007). Activation of the GABAA receptors localized on DA cells should directly hyperpolarize DA neurons and decrease both VTA somatodendritic and efferent terminal DA release in the NAc (Xi and Stain, 1998). It has been proposed in previous studies that VTA GABAA receptors mediate regulation of meso-NAc DA function (Xi and Stein, 1998). Muscimol, activating post-synaptic GABAA receptors, may reduce the activity of dopaminergic neurons within the VTA. This function is similar to GABA activity, and as a result, there is reduced DA release at the target sites including NAc (Sahraei et al., 2005). It was also observed that the GABAA agonist, muscimol, increased GABA release in the NAc, but the antagonist, bicuculline, had the opposite effect. Moreover, bicuculline could block the increased release of GABA induced by morphine in the NAc. It has been reported that bicuculline dose-dependently potentiates the NAc DA stress response, but muscimol does not (Doherty and Gratton, 2007). This effect of bicuculline would be consistent with the idea that the activity of DA neurons is also regulated by a direct GABAA receptormediated action of VTA GABA afferents (Steffensen et al., 2008). Accordingly, it appears that bicuculline inhibits the activity of GABAergic neurons in the VTA and increases the activity of dopaminergic afferents to the NAc, preventing the synthesis and release of GABA. Finally, the action of morphine on the release of AA and GABA in the NAc is blocked. In light of these results, the simplest explanation for the potential effects on morphine-induced AA release is that this dose of bicuculline inactivated GABAA receptors located on GABA interneurons, inhibited their activity and then, in turn, disinhibited the activity of DA cells, which finally was related to the effects of blocking the AA release induced by morphine in the NAc (Fig.6). Supporting data have also emerged from postmortem studies in humans. In the NAc of schizophrenic patients, the dopamine concentration is increased while that of GABA is reduced (Bird et al., 1977; Perry et al., 1979; Mackay et al., 1982). Inhibition of GABAA receptors in the VTA should attenuate AA release in the NAc. This suggests that increased NAc GABAergic activity and decreased dopaminergic activity during bicuculline treatment with VTA may block the release of extracellular AA induced by morphine-perfusion into the NAc. In conclusion, the data reported in the present study show that application of a GABAA receptor antagonist in the VTA could inhibit the effects of morphine on the release of AA and GABA in the NAc. Based on the current findings, a new hypothesis is proposed, namely, that the GABAergic system in the VTA may be involved in the morphine-induced release of AA and GABA in the NAc, although much evidence is needed in support of this. These results also indicate that this action of morphine in the NAc is regulated by the GABAA receptormediated action of DA afferents from the VTA.
4.
Experimental procedures
4.1.
Animals
Male Sprague–Dawley rats weighing 180–220 g were used. All animals were supplied by the Experimental Animal Centre of Shenyang Pharmaceutical University and kept with standard
56
BR A IN RE S EA RCH 1 3 68 ( 20 1 1 ) 5 2 –58
Fig. 6 – Diagram showing the possible mechanisms through which AA and GABA release may be regulated by the DAergic inputs (blue afferents) from the VTA to the NAc (Mora et al., 2008). The perfusion of GABA antagonist bicuculline locally into the VTA would result in an inhibition of GABAergic neurons (red afferents) in the VTA. Activation of the DAergic afferents to the NAc increases DA release, decreases the GABA release, and then prevents the morphine-induced release of AA in the NAc.
light–dark cycle (light on at 06:30 a.m.). Rats were housed in cages with food and water available ad libitum. All experiments and procedures were carried out according to the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of China.
4.2.
Drugs
Morphine hydrochloride was purchased from Shenyang First Pharmaceutical Factory (Shenyang, China). Ringer's solution (ACSF) was composed of 147 mM NaCl, 2.2 mM CaCl2 and 4 mM KCl (Dai et al., 2006; Gu et al., 2006). Muscimol and bicuculline, purchased from Sigma Chemicals (St. Louis, MO, USA), were dissolved in ACSF. Morphine was dissolved in ACSF for the perfusion with the concentrations used in previous experiments (Gu et al., 2006). The o-phthalaldehyde solution was prepared as follows: 9 mg OPA was dissolved in 167 μl methanol then 1.5 ml 0.4 M sodium borate and 7 μl 2mercaptoethanol were added and mixed well. The solution was stored at 4 °C for one week and protected from light.
4.3.
Surgical procedure and microdialysis in NAc
The procedure used to prepare and implant the dialysis probe was as previously described (Wu et al., 1988). The rats were anesthetized with chloral hydrate (350 mg/kg, i.p.) and Hospal AN 69 dialysis fibers (310 μm o.d., 200 μm i.d., Dasco, Bologna, Italy) were implanted transversally through the NAc (coordinates: A: + 1.7 mm from bregma, V: −7.5 mm from occipital bone (Gu et al., 2005). A dialysis tube was covered with SuperEpoxy glue over its whole length except for the zones (2.0 mm wide) which were to be positioned in the NAc (Liu et al., 2000). This kind of probe has a total dialysis length of 4.0 mm and can
collect the neurochemicals of interest from both sides of the NAc. The animals were individually housed in plastic cages and left to recover for about 24 h after surgery. Surgical procedures for implantation of intra-VTA cannula were also performed under anesthesia. A 26-gauge stainless-steel guide cannula was directed into the VTA (A/P −6.0 mm, M/L 1.0 mm, D/V − 7.8 mm) bilaterally according to a rat brain atlas (Ikemoto et al., 1998; Paxinos and Watson, 1997) and was fixed to the skull by dental acrylic. The cannula was secured to the skull surface with dental cement. When the dental cement had hardened, a dummy cannula, cut to the same dimensions as the guide cannula, was inserted to seal the top of the guide cannula to prevent clogging and minimize possible infection. The rats were allowed 1 day for recovery. Injection was made by inserting a 33-gauge stainless-steel injector tube into the guide cannula. The injector tube was attached to PE-10 tubing fitted to a 10-μl Hamilton Syringe. One microliter of solution was thereafter infused into the lateral cerebral ventricle over 2 min. Brain dialysis was performed about 24 h after the probe implantation in freely moving rats. ACSF was pumped through the dialysis probe at a constant rate of 2 μl/min. After an hour, the dialysis samples were collected and analyzed every 20 min. Test solutions were continuously pumped through the dialysis probe when the output of AA, GABA or DA became stable in the last three samples with a variation of no more than 10%. After the basal release of the neurochemicals became stable, morphine (1 mM) was administrated to the NAc by local perfusion for 3 h. The bicuculline and muscimol were injected into the VTA 1 h after the morphine administration. At the end of the experiments, the position of the dialysis fiber was verified by visual examination, and the datum was discarded if the fiber was positioned incorrectly (Liu et al., 2000; Gu et al., 2005). The rats with a bilateral VTA cannula received the bilateral microinjections following 24 h of food deprivation; the details of the injections were as follows: bicuculline at a total dose of 150 ng (n=6, 75 ng/0.5 μl each side) and muscimol at total dose of 100 ng (n=6, 50 ng/0.5 μl each side).
4.4.
Analytical procedure
Each dialysate sample (40 μl) was divided into two fractions. Twenty microliters was immediately injected directly into the HPLC–ECD for determination of the AA content. A reverse phase column (C-18, 5 μm, Agilent) was used with a mobile phase composed of 155.6 mM NaCl and EDTA-Na2 0.54 mM with tetrabutylammonium bromide 1.5 mM as an ion pairing reagent. The mobile phase was pumped using an LC-10A pump (SHIMADZU, Japan) at a flow rate of 1.0 ml/min. The detector (L-ECD-6A, SHIMADZU, Japan) was set at +0.60 V (Liu et al., 2000). The remaining 20 μl was frozen at −20 °C until required for HPLC–FD (excitation wavelength: 340 nm, emission wavelength: 450 nm, SHIMADZU, Japan) for GABA, following precolumn derivatization with o-phthalaldehyde (Hao et al., 2005), with some modifications. Gradient elution was used to separate the mixture of amino acids (Table 1). Mobile phase A consisted of 0.1 M sodium acetate buffer, pH 6.7 while mobile phase B contained 98% methanol and 2% tetrahydrofuran (Tiff). The flow rate of the pump was set at 1.0 ml/min. All the mobile phases were passed through a
BR A IN RE S E A RCH 1 3 68 ( 20 1 1 ) 5 2 –5 8
Table 1 – Gradient program of HPLC (mm). Time
A%
B%
0.00 7.00 12.00 13.00 20.00
65 35 35 65 65
35 65 65 35 35
0.22 μm filter. The column temperature was maintained at 37 °C (Hao et al., 2005). Another group of animals was used to collect the dialysate for DA determination using HPLC–ECD with the following conditions: a reverse phase column (C-18, 5 μm, Agilent) with a mobile phase composed of 85 mM citrazinic acid, 100 mM sodium acetate anhydrous, 0.2 mM Na2EDTA, and 1.2 mM sodium 1-octanesulfonate adjusted to pH 3.7.
4.5.
Histological verification of the injection site
In pilot studies, at the end of the experiments, the anatomical accuracy of the needle placements was verified by injecting 5 μl trypan blue into the VTA. Then, the head was fixed in 10% formalin for 24 h with the injecting tube in situ before section. The location of the tip of the injecting tube was verified and all the tips of the injecting tube were in the VTA of rat in the present study.
4.6.
Statistical analysis
Statistical analysis was carried out using SPSS 13.0 software for Windows (SPSS Inc., Chicago, IL, USA). All values were expressed as mean ± S.E.M. The levels of AA, GABA and DA were expressed as the percentage change compared with the respective basal value that was the mean of three consecutive samples before drug administration within a variance of 10%, and for each time point, a one-way ANOVA was used. The level of significance was set at P < 0.05.
Acknowledgments This research is partly supported by the Project of Key Laboratory for New Drug Screening, Key Laboratory for Pharmacodynamics, of Liaoning Province, and by the National Key Scientific Project for New Drug Discovery and Development, (2009ZX09301-012), 2009-2010, P. R. China.
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