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The effect of morphine sensitization on extracellular concentrations of GABA in dorsal hippocampus of male rats
European Journal of Pharmacology 669 (2011) 66–70
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and Analgesia
The effect of morphine sensitization on extracellular concentrations of GABA in dorsal hippocampus of male rats Maryam Farahmandfar a, Mohammad-Reza Zarrindast b, Mehdi Kadivar c, Seyed Morteza Karimian d, Nasser Naghdi a,⁎ a
Department of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, Iran Department of Pharmacology, Tehran University of Medical Sciences, Tehran, Iran Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran d Department of Physiology, Tehran University of Medical Sciences, Tehran, Iran b c
a r t i c l e
i n f o
Article history: Received 1 March 2011 Received in revised form 26 June 2011 Accepted 30 July 2011 Available online 22 August 2011 Keywords: Morphine Behavioral sensitization GABA Hippocampus Microdialysis
a b s t r a c t Repeated, intermittent exposure to drugs of abuse, such as morphine results in response enhancements to subsequent drug treatments, a phenomenon referred to as behavioral sensitization. As persistent neuronal sensitization may contribute to the long-lasting consequences of drug abuse, characterizing the neurochemical mechanisms of sensitization is providing insights into addiction. Although it has been shown that GABAergic systems in the CA1 region of dorsal hippocampus are involved in morphine sensitization, the alteration of extracellular level of GABA in this area in morphine sensitization has not been investigated. In the present study, using the in vivo microdialysis technique, we investigated the effect of morphine sensitization on extracellular GABA concentration in CA1 region of dorsal hippocampus of freely moving rats. Sensitization was induced by subcutaneous (s.c.) injection of morphine, once daily for 3 days followed by 5 days free of the opioid treatment. The results showed that extracellular GABA concentration in CA1 was decreased following acute administration of morphine in non-sensitized rats. However, morphine-induced behavioral sensitization significantly increased the extracellular GABA concentration in this area. The enhancement of GABA in morphine sensitized rats was inhibited by administration of naloxone 30 min before each of three daily doses of morphine. These results suggest an adaptation of the GABAergic neuronal transmission in dorsal hippocampus induced by morphine sensitization and it is implied that opioid receptors may play an important role in this effect. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Behavioral sensitization defined as the progressive and persistent enhancement of behavioral responses elicited by repeated administration of drugs (Kuribara, 1995; Shippenberg et al., 1996). Druginduced behavioral sensitization may be the result of multiple adaptive neuronal responses, some of which include permanent changes to synaptic transmission (Wolf, 1998). Extensive prior evidence indicates that the dopaminergic system is a key element in the acquisition and expression of morphine-induced behavioral sensitization (Jeziorski and White, 1995; Serrano et al., 2002). However, the nature of this phenomenon cannot be explained exclusively on the basis of changes in dopaminergic neurotransmission (Tjon et al., 1995). γ-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system and has a widespread distribution in the mammalian brain. It has been suggested that GABAergic and opioidergic systems are interconnected through μ-opioid receptors (Xi ⁎ Corresponding author at: Department of Physiology and Pharmacology, Pasteur Institute of Iran, P.O. Box 1316943551, Tehran, Iran. Tel./fax: +98 21 6646 5132. E-mail address: [email protected] (N. Naghdi). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.07.050
and Stein, 2002). There are some studies indicating that acute morphine treatment decreases extracellular concentration of GABA in ventral tegmental area (Sotomayor et al., 2005) and periaqueductal gray matter (Stillera et al., 1996), whereas chronic morphine treatment increases the level of GABA in prefrontal cortex (Gao et al., 2007). Morphine treatment has also been shown to result in decreased GABA release (Capogna et al., 1993; Vaughan et al., 1997), increased GABA uptake, and elevated GABA transporter (i.e., GAT-1) expression in the hippocampus (Hu et al., 2003). It has been demonstrated that agonists of both GABAA (Zarrindast et al., 2007) and GABAB (Bartoletti et al., 2007) receptors prevent the acquisition or development phase of opioid sensitization. Some studies showed that behavioral sensitization to opioids by direct modulation of GABA release (Schoffelmeer et al., 2001) or indirect effects on the other neurotransmitters (De Rover et al., 2005) can alter the GABAergic synaptic transmission in some of the brain regions such as nucleus accumbens (Schoffelmeer et al., 2001) and striatum (Schoffelmeer et al., 1997). It has been shown that dorsal hippocampus densely innervates the nucleus accumbens, which mediates the expression of behavioral sensitization (Degoulet et al., 2008). Zarrindast et al. (2008) reported that GABAergic receptors in the CA1 region of dorsal hippocampus
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have an important role in the reversal effect of morphine sensitization on impairment of memory induced by morphine. However, the effects of morphine sensitization on the extracellular level of GABA in this area have not been investigated. On the other hand, our previous study revealed that morphine-induced impairment of spatial memory is reversed by morphine sensitization (Farahmandfar et al., 2010). As the CA1 has an essential role in spatial memory and navigation (Manns et al., 2003), we hypothesized that changes in GABA concentration in this area may be one of the potential factors responsible for our previous results. In the present study, we investigated the effect of morphine on extracellular GABA concentration in CA1 region of dorsal hippocampus of non-sensitized and morphine-sensitized rats by using microdialysis via high performance liquid chromatography fluorescence detection method. 2. Materials and methods 2.1. Drugs and animals Male Wistar rats (230 ± 20 g) were housed four per cage and maintained on a 12 h light–dark cycle in an air conditioned constant temperature (23 ± 1 °C) room. Food and water were made available ad libitum. All experiments were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, USA) revised 1996. Morphine sulfate (Temad, Tehran, Iran) and naloxone hydrochloride (Tolid —Daru, Tehran, Iran) were dissolved in sterile 0.9% saline just before the experiment and were injected subcutaneously (s.c.). o-Phthalaldehyde (OPA) was purchased from Sigma-Aldrich and other reagents and solvents were of HPLC-quality and were obtained from Sigma-Aldrich or Merck (Germany). 2.2. Drug treatment Six animals were used in each of seven experimental groups. The schedule of drug administration was based on our previous studies in order to induce behavioral sensitization (Farahmandfar et al., 2010; Farahmandfar et al., 2011; Zarrindast et al., 2006). In experiments where animals received three injections, the control groups also received three saline injections. 2.2.1. Experiment 1: Effects of acute administration of morphine on the extracellular level of GABA in CA1 of non-sensitized male rats In this experiment, two groups of animals received subcutaneous (s.c.) administration of saline (1 ml/kg, s.c.) or morphine (5 mg/kg, s.c.), an effective dose of morphine on spatial memory impairment (Farahmandfar et al., 2010), following a 60-min collection of GABA basal level. 2.2.2. Experiment 2: Effects of morphine sensitization on the extracellular level of GABA in CA1 of male rats Two groups of animals received saline (1 ml/kg, s.c.), once daily for 3 days. After 5 days with no drug treatment, following a 60-min collection of GABA basal level, the animals received a challenge of saline (1 ml/kg, s.c.) [Saline/Saline] or morphine (5 mg/kg, s.c.) [Saline/Morphine]. Another group received for 3 days, once daily, morphine (20 mg/kg, s.c.) and after 5 days (no drug treatment), microdialysis experiments were performed and a challenge of morphine (5 mg/kg, s.c) [Morphine/Morphine] was injected following collection of basal level of GABA. 2.2.3. Experiment 3: Effect of morphine sensitization on the extracellular level of GABA in CA1 of male rats in the presence or absence of naloxone In this experiment, two groups of animals received once daily injections of saline (1 ml/kg, s.c) or the opioid receptor antagonist, naloxone (2 mg/kg, s.c.), 30 min prior to s.c. injections of morphine (20 mg/kg/day×3 days). After 5 days (no drug treatment), microdialysis
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experiments were performed and animals received a challenge of morphine (5 mg/kg, s.c.), following a 60-min collection of GABA basal level. 2.3. Surgery and brain dialysis For the implantation of microdialysis guide cannula (MAB 4.15.IC, Microbiotech/se AB, Stockholm, Sweden), rats were anesthetized with a mixture of ketamine hydrochloride (Sigma,Germany) and xylazine (ketamine 60 mg/kg, xylazine 12 mg/kg, i.p.) and mounted in a stereotaxic frame (Stoelting, USA). The guide cannula was inserted into the hippocampal CA1 area with the following coordinates: 3.8 mm posterior to the bregma, ±2.2 laterals to midline and 2.7 ventral to the skull surface (Paxinos and Watson, 1986). After surgery, at least a sevenday recovery period was supposed before any other intervention. On the experiment day, microdialysis probe (MAB 4.15.1.Cu, b6000 kDa, 1 mm membrane length, Microbiotech/se AB, Stockholm, Sweden) was inserted into the guide cannula and were connected to the microdialysis pump (WPI, SP 210, syringe pump) and perfused with artificial cerebrospinal fluid (ACSF: 114 mM NaCl, 3 mM KCl, 1 mM CaCl2, 2 mM MgSo4, 1.25 mM NaH2PO4, 26 mM NaHCO3, 1 mM NaOH, 10 mM glucose and pH = 7.4) at a flow rate of 2 μl/min. Following a 20-min equilibration period, six consecutive 10-min samples were collected for determination of basal GABA level. Subsequently, saline or morphine was administered and dialysate samples were collected every 10 min for 1 h and were put on ice and instantly placed in − 80 °C. 2.4. GABA determination GABA was measured by reverse-phase high-pressure liquid chromatography (Column: Shim-pack VP-ODS, 250 L×4.6 mm, 5 μm; Pump: LC-10 AD, Shimadzu, Japan) coupled to fluorescence detector (RF-10 AXL, Shimadzu, Japan, set at wavelength: EX.=340 nm, EM.=475 nm), following pre-column derivatization with o-phthaldialdehyde (OPA) according to the method described by de Freitas Silva et al. (2009). The mobile phase consisted of 0.05 M sodium acetate, tetrahydrofuran and methanol (50:1:49, v/v) adjusted to pH 4.0. The mobile phase was filtered through Millipore 0.45 μm Durapore membrane filters and vacuum degassed prior to use. Chromatographic analyses were performed at 25 ± 2 °C. Compounds were eluted isocratically over a 9 min runtime at a flow rate of 1 mL/min. 2.5. Histological analysis Following microdialysis experiments, animals were sacrificed by decapitation and brains were removed. For histological examination of guide cannula and microdialysis probe placement in CA1, 100 μm thick sections were taken, mounted on slides, stained with cresyl violet and the guide cannula and probe track were examined for each rat. Only rats with correct placement were included in data analysis. 2.6. Statistical analysis Basal values of extracellular GABA were the means of six consecutive samples and were expressed as nmol/L. The data of basal concentrations of GABA in each experiment were analyzed by Student's ttest in comparison with respective control group. The changes of GABA concentration after different treatments were analyzed by repeated measures two-way analysis of variance (ANOVA) with the drug treatments as between group factors and time as within group factors. Results showing significant overall changes were subjected to post hoc Tukey's test. The data were expressed as percentage of basal values. A probability level of P b 0.05 was regarded as statistically significant.
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3.1. Effect of acute morphine and morphine sensitization in the presence or absence of naloxone on the basal levels of GABA in CA1 Mean basal levels of GABA in non-sensitized, morphine sensitized and morphine-sensitized rats in the presence or absence of naloxone were assessed. As shown in Fig. 1, three above mentioned groups showed no difference in the mean basal levels of GABA in CA1 as compared to their respective control groups [(P = 0.25), (P = 0.47) and (P = 0.68)], respectively (Fig. 1).
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Time (min) As shown in Fig. 2, effect of acute administration of morphine on the extracellular level of GABA in CA1 of non-sensitized rats was determined. The results showed that acute morphine 5 mg/kg decreased extracellular GABA levels by nearly 40% of baseline at 20 and 40 min after morphine administration (Fig. 2). Repeated measures ANOVA revealed significant effects of group [F (1,10) =33.18; Pb 0.001], time [F(6,60) =3.29; Pb 0.05] and a significant interaction of group ×time [F (6,60) =5.59; Pb 0.05]. Two-way ANOVA followed by Tukey's test revealed that acute administration of morphine caused a significant decrease in the extracellular level of GABA on each time point in CA1 as compared to saline group [(Pb 0.05)] (Fig. 2). 3.3. Effect of morphine sensitization on extracellular GABA concentration in CA1 The effects of repeated administration of saline or morphine with a challenge of morphine on extracellular GABA concentration are shown in Fig. 3. Repeated measures ANOVA revealed significant effects of group [F(1,10) = 123.6; P b 0.001], time [F(6,60) = 3.22; P b 0.01] and a significant interaction of group × time [F(6,60) = 25.1; P b 0.01]. Post hoc Tukey's test showed that a challenge of morphine (5 mg/kg) leads to a significant decrease in GABA concentration on each time point in the CA1 area in saline pretreated group (Saline/Morphine) as compared with the control group (Saline/Saline) [Pb 0.001]. However, challenge dose of morphine (5 mg/kg) caused a significant increase in GABA level on each time point in the CA1 in morphine (20 mg/kg) pretreated group (Morphine/Morphine) as compared with the saline pretreated group (Saline/Morphine) [Pb 0.001] (Fig. 3).
Fig. 2. Effect of acute injection of saline (1 ml/kg, s.c) and morphine (5 mg/kg, s.c) on basal extracellular level of GABA in CA1 of non-sensitized rats. Results are expressed as means ± SEM of the percent of basal values (n = 6). *P b 0.05 significantly different from respective basal values. #P b 0.05 significantly different from the corresponding value of saline group.
with naloxone is shown in Fig. 4. The results showed that the challenge dose of morphine (5 mg/kg) in naloxone with morphine pretreated group (Naloxone+ Morphine/Morphine) significantly decreased extracellular GABA levels by nearly 40% of baseline at 30 and 40 min after administration. Repeated measures ANOVA revealed significant effects of group [F(1,10)= 103.1; P b 0.001], time [F(6,60) = 3.2; P b 0.05] and a significant interaction of group× time [F(6,60)= 23.7; P b 0.001]. Post hoc Tukey's test showed a significant decrease in GABA concentration in CA1 on each time point in animals pretreated with morphine in combination with naloxone (Naloxone+Morphine/Morphine) as compared to the control group (Saline+Morphine/Morphine) [(Pb 0.001)] (Fig. 4). 4. Discussion In the present experiments, we evaluated the effects of morphine administration on the extracellular GABA concentration in the CA1 area of non-sensitized and morphine-sensitized rats by using microdialysis in freely moving animals and HPLC-FLD method. Recent evidence has indicated a specific involvement of the GABAergic system in the mechanisms underlying addictive behavior (Vaughan et al., 1997; Xi and Stein, 2002). It has been reported that activation of the 180
3.4. Effect of naloxone on extracellular GABA concentration in CA1 of morphine-sensitized rats
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Fig. 1. The basal levels of GABA in non-sensitized (A), morphine sensitized (B) and morphine-sensitized rats in combination with naloxone (C) groups in comparison with their respective control groups. The basal contents of GABA in the samples were the mean ± S.E.M. of the averages of the six baseline samples in six rats.
Fig. 3. Effect of saline or morphine (5 mg/kg, s.c) challenge on basal extracellular level of GABA in CA1 of rats pretreated with saline (Saline/Saline and Saline/Morphine ) or morphine (20 mg/kg) [Morphine/Morphine ]. Results are expressed as means ± SEM of the percent of basal values (n = 6). *P b 0.05 and **P b 0.01 significantly different from the respective basal values. #P b 0.05 and ##P b 0.01 significantly different from the corresponding value of Saline/Morphine group.
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GABA (% of mean of the baseline)
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Time (min) Fig. 4. Effect of morphine (5 mg/kg s.c) challenge on basal extracellular level of GABA in CA1 of saline (1 ml/kg, s.c)+ morphine (20 mg/kg s.c) [Saline + Morphine/Morphine ] and naloxone (2 mg/kg s.c)+ morphine (20 mg/kg s.c) [Naloxone + Morphine/ Morphine ] pre-treated rats. Results are expressed as means± SEM of the percent of basal values (n= 6). *Pb 0.05 significantly different from respective basal values. # P b 0.05, ##P b 0.01 and ###P b 0.001 significantly different from the corresponding value of Saline+ Morphine/Morphine group.
central GABAergic neurotransmission is closely connected with mesolimbic dopaminergic neurotransmission during addiction processes (Johnson and North, 1992). The mesolimbic dopaminergic system may have an important role in the development of behavioral sensitization to psychostimulants and opioids (Jeziorski and White, 1995; Serrano et al., 2002). Moreover, mesolimbic dopamine release and dopamine cell firing have been inhibited by the GABA projection and GABA inter-neurons within the ventral tegmental area (Enrico et al., 1998; Steffensen et al., 1998; Westerink et al., 1998). Current studies strongly suggest that GABAergic and glutamatergic cells in both the ventral tegmental area and the limbic forebrain are also important in the physical and psychological dependence on morphine, or behavioral sensitization in animal models (Madhavan et al., 2010; Wolf, 2002; Zarrindast et al., 2008). Hippocampus as a part of limbic formation plays a key role in the formation of synaptic plasticity and neural adaptation in the CNS and has numerous opiate receptors and GABAergic neurons (Cohen et al., 1992). It has been suggested that connections of dorsal hippocampus to the nucleus accumbens and ventral tegmental area may play an important role in behavioral sensitization and drug-seeking behavior (Jentsch and Taylor, 1999). However, the effect of this structure and its GABAergic neurotransmission on the morphine sensitization has been less understood. According to our previous study, single administration of morphine (2.5, 5 and 7.5 mg/kg), 30 min before training of Morris water maze task, decreased the spatial memory acquisition in non-sensitized rats with a maximum effect of 5 mg/kg of morphine (Farahmandfar et al., 2010). The results of the present experiments showed that acute administration of morphine (5 mg/kg) significantly decreased extracellular GABA levels in the CA1 to nearly 40% of baseline at 20 and 40 min after morphine administration. This result is a direct verification that spatial learning deficit after acute injection of morphine is significantly correlated with the reduction of GABA concentration in dorsal hippocampus. Our data is also in agreement with the previous results which have reported that acute injection of morphine decreased the extracellular level of GABA in some other brain regions such as ventral tegmental area (Sotomayor et al., 2005) and periaqueductal gray matter (Stiller et al., 1996). Furthermore, repeated exposure to drugs of abuse is thought to cause persistent behavioral sensitization and associated adaptations of neurotransmissions, which can play an important role in certain aspects of drug addiction (Everitt et al., 2001). A number of studies have demonstrated that morphine sensitization can alter the level of dopamine or
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glutamate concentration in some brain regions (Cadoni and Di Chiara, 1999; Hao et al., 2007; Mikkola et al., 2000). There are few studies considering the effect of morphine sensitization on the extracellular level of GABA in addiction-associated regions. In one study by Ojanen et al. (2007), it has been reported that morphine sensitization had no effect on the extracellular level of GABA in the ventral tegmental area in alcohol-preferring AA rats. In the present study, our finding indicates that morphine sensitization induced by pre-treatment with morphine, once daily for 3 days followed by 5 days free of the opioid treatment, can significantly increase the extracellular level of GABA in CA1 of rats at 30 and 50 min after injection of morphine challenge dose. These data suggest that reduction of extracellular GABA concentration induced by acute morphine can be restored in morphine-sensitized rats. The circuitry involved in behavioral sensitization is complex, because sensitization represents a cascade of events involving different transmitter systems and different brain regions such as nucleus accumbens, ventral tegmental area, the basolateral amygdala, the prefrontal cortex and the hippocampus (Wolf, 2003). The effects of GABAergic systems on morphineinduced behavioral sensitization were investigated in several studies (Bartoletti et al., 2007; Li et al., 2004; Narita et al., 2003). It has been shown that hippocampal GABAergic receptors are involved in the reversal effect of morphine sensitization on memory impairment induced by acute morphine (Zarrindast et al., 2008). On the other hand, our previous observations showed that impairment of spatial memory acquisition induced by pre-training injection of morphine (5 mg/kg) was significantly reduced in morphine (20 mg/kg)-sensitized rats in Morris water maze task (Farahmandfar et al., 2010). As hippocampal GABAergic system has an essential role in learning and memory especially spatial memory and navigation (Manns et al., 2003), we can suggest that alteration of GABA levels in CA1 of dorsal hippocampus following morphine sensitization may be one of the responsible factor for the effect of this phenomenon on the spatial memory paradigm. The present data also indicated that injection of naloxone, 30 min before morphine administration during 3 days of sensitization, prevented the increase of GABA concentration induced by morphine sensitization. Thus, the involvement of μ-opioid receptors in the sensitization processes and alteration of dorsal hippocampal GABA concentration by morphine seems likely. In conclusion, the results of the present study provide an evidence of interaction between morphine and GABAergic system in the CA1 region of dorsal hippocampus and implied an adaptation of GABAergic neurotransmission induced by morphine sensitization that μ-opioid receptors may play an important role in this process.
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Schoffelmeer, A.N., Wardeh, G., Vanderschuren, L.J., 2001. Morphine acutely and persistently attenuates nonvesicular GABA release in rat nucleus accumbens. Synapse 42, 87–94. Serrano, A., Aguilar, M.A., Manzanedo, C., Rodriguez-Arias, M., Minarro, J., 2002. Effects of DA D1 and D2 antagonists on the sensitisation to the motor effects of morphine in mice. Prog. Neuropsychopharmacol. Biol. Psychiatry 26, 1263–1271. Shippenberg, T.S., Heidbreder, C., Lefevour, A., 1996. Sensitization to the conditioned rewarding effects of morphine: pharmacology and temporal characteristics. Eur. J. Pharmacol. 299, 33–39. Sotomayor, R., Forray, M.I., Gysling, K., 2005. Acute morphine administration increases extracellular DA levels in the rat lateral septum by decreasing the GABAergic inhibitory tone in the ventral tegmental area. J. Neurosci. Res. 81, 132–139. Steffensen, S.C., Svingos, A.L., Pickel, V.M., Henriksen, S.J., 1998. Electrophysiological characterization of GABAergic neurons in the ventral tegmental area. J. Neurosci. 18, 8003–8015. Stiller, C.O., Bergquist, J., Beck, O., Ekman, R., Brodin, E., 1996. Local administration of morphine decreases the extracellular level of GABA in the periaqueductal gray matter of freely moving rats. Neurosci. Lett. 209, 165–168. Stillera, C.O., Bergquistb, J., Beckc, O., Ekmanb, R., Brodina, E., 1996. Local administration of morphine decreases the extracellular level of GABA in the periaqueductal gray matter of freely moving rats. Neurosci. Lett. 209, 165–168. Tjon, G.H., De Vries, T.J., Nestby, P., Wardeh, G., Mulder, A.H., Schoffelmeer, A.N., 1995. Intermittent and chronic morphine treatment induces long-lasting changes in delta-opioid receptor-regulated acetylcholine release in rat striatum and nucleus accumbens. Eur. J. Pharmacol. 283, 169–176. Vaughan, C.W., Ingram, S.L., Connor, M.A., Christie, M.J., 1997. How opioids inhibit GABA-mediated neurotransmission. Nature 390, 611–614. Westerink, B.H., Enrico, P., Feimann, J., De Vries, J.B., 1998. The pharmacology of mesocortical dopamine neurons: a dual-probe microdialysis study in the ventral tegmental area and prefrontal cortex of the rat brain. J. Pharmacol. Exp. Ther. 285, 143–154. Wolf, M.E., 1998. The role of excitatory amino acids in behavioral sensitization to psychomotor stimulants. Prog. Neurobiol. 54, 679–720. Wolf, M.E., 2002. Addiction: making the connection between behavioral change and neuronal plasticity in specific pathways. Mol. Interv. 2, 146–157. Wolf, M.E., 2003. LTP may trigger addiction. Mol. Interv. 3, 248–252. Xi, Z.X., Stein, E.A., 2002. GABAergic mechanisms of opiate reinforcement. Alcohol Alcohol. 37, 485–494. Zarrindast, M.R., Farahmandfar, M., Rostami, P., Rezayof, A., 2006. The influence of central administration of dopaminergic and cholinergic agents on morphine-induced amnesia in morphine-sensitized mice. J. Psychopharmacol. 20, 59–66. Zarrindast, M.R., Heidari-Darvishani, A., Rezayof, A., Fathi-Azarbaijani, F., Jafari-Sabet, M., Hajizadeh-Moghaddam, A., 2007. Morphine-induced sensitization in mice: changes in locomotor activity by prior scheduled exposure to GABAA receptor agents. Behav. Pharmacol. 18, 303–310. Zarrindast, M.R., Hoghooghi, V., Rezayof, A., 2008. Inhibition of morphine-induced amnesia in morphine-sensitized mice: involvement of dorsal hippocampal GABAergic receptors. Neuropharmacology 54, 569–576.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and Analgesia
The effect of morphine sensitization on extracellular concentrations of GABA in dorsal hippocampus of male rats Maryam Farahmandfar a, Mohammad-Reza Zarrindast b, Mehdi Kadivar c, Seyed Morteza Karimian d, Nasser Naghdi a,⁎ a
Department of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, Iran Department of Pharmacology, Tehran University of Medical Sciences, Tehran, Iran Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran d Department of Physiology, Tehran University of Medical Sciences, Tehran, Iran b c
a r t i c l e
i n f o
Article history: Received 1 March 2011 Received in revised form 26 June 2011 Accepted 30 July 2011 Available online 22 August 2011 Keywords: Morphine Behavioral sensitization GABA Hippocampus Microdialysis
a b s t r a c t Repeated, intermittent exposure to drugs of abuse, such as morphine results in response enhancements to subsequent drug treatments, a phenomenon referred to as behavioral sensitization. As persistent neuronal sensitization may contribute to the long-lasting consequences of drug abuse, characterizing the neurochemical mechanisms of sensitization is providing insights into addiction. Although it has been shown that GABAergic systems in the CA1 region of dorsal hippocampus are involved in morphine sensitization, the alteration of extracellular level of GABA in this area in morphine sensitization has not been investigated. In the present study, using the in vivo microdialysis technique, we investigated the effect of morphine sensitization on extracellular GABA concentration in CA1 region of dorsal hippocampus of freely moving rats. Sensitization was induced by subcutaneous (s.c.) injection of morphine, once daily for 3 days followed by 5 days free of the opioid treatment. The results showed that extracellular GABA concentration in CA1 was decreased following acute administration of morphine in non-sensitized rats. However, morphine-induced behavioral sensitization significantly increased the extracellular GABA concentration in this area. The enhancement of GABA in morphine sensitized rats was inhibited by administration of naloxone 30 min before each of three daily doses of morphine. These results suggest an adaptation of the GABAergic neuronal transmission in dorsal hippocampus induced by morphine sensitization and it is implied that opioid receptors may play an important role in this effect. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Behavioral sensitization defined as the progressive and persistent enhancement of behavioral responses elicited by repeated administration of drugs (Kuribara, 1995; Shippenberg et al., 1996). Druginduced behavioral sensitization may be the result of multiple adaptive neuronal responses, some of which include permanent changes to synaptic transmission (Wolf, 1998). Extensive prior evidence indicates that the dopaminergic system is a key element in the acquisition and expression of morphine-induced behavioral sensitization (Jeziorski and White, 1995; Serrano et al., 2002). However, the nature of this phenomenon cannot be explained exclusively on the basis of changes in dopaminergic neurotransmission (Tjon et al., 1995). γ-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system and has a widespread distribution in the mammalian brain. It has been suggested that GABAergic and opioidergic systems are interconnected through μ-opioid receptors (Xi ⁎ Corresponding author at: Department of Physiology and Pharmacology, Pasteur Institute of Iran, P.O. Box 1316943551, Tehran, Iran. Tel./fax: +98 21 6646 5132. E-mail address: [email protected] (N. Naghdi). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.07.050
and Stein, 2002). There are some studies indicating that acute morphine treatment decreases extracellular concentration of GABA in ventral tegmental area (Sotomayor et al., 2005) and periaqueductal gray matter (Stillera et al., 1996), whereas chronic morphine treatment increases the level of GABA in prefrontal cortex (Gao et al., 2007). Morphine treatment has also been shown to result in decreased GABA release (Capogna et al., 1993; Vaughan et al., 1997), increased GABA uptake, and elevated GABA transporter (i.e., GAT-1) expression in the hippocampus (Hu et al., 2003). It has been demonstrated that agonists of both GABAA (Zarrindast et al., 2007) and GABAB (Bartoletti et al., 2007) receptors prevent the acquisition or development phase of opioid sensitization. Some studies showed that behavioral sensitization to opioids by direct modulation of GABA release (Schoffelmeer et al., 2001) or indirect effects on the other neurotransmitters (De Rover et al., 2005) can alter the GABAergic synaptic transmission in some of the brain regions such as nucleus accumbens (Schoffelmeer et al., 2001) and striatum (Schoffelmeer et al., 1997). It has been shown that dorsal hippocampus densely innervates the nucleus accumbens, which mediates the expression of behavioral sensitization (Degoulet et al., 2008). Zarrindast et al. (2008) reported that GABAergic receptors in the CA1 region of dorsal hippocampus
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have an important role in the reversal effect of morphine sensitization on impairment of memory induced by morphine. However, the effects of morphine sensitization on the extracellular level of GABA in this area have not been investigated. On the other hand, our previous study revealed that morphine-induced impairment of spatial memory is reversed by morphine sensitization (Farahmandfar et al., 2010). As the CA1 has an essential role in spatial memory and navigation (Manns et al., 2003), we hypothesized that changes in GABA concentration in this area may be one of the potential factors responsible for our previous results. In the present study, we investigated the effect of morphine on extracellular GABA concentration in CA1 region of dorsal hippocampus of non-sensitized and morphine-sensitized rats by using microdialysis via high performance liquid chromatography fluorescence detection method. 2. Materials and methods 2.1. Drugs and animals Male Wistar rats (230 ± 20 g) were housed four per cage and maintained on a 12 h light–dark cycle in an air conditioned constant temperature (23 ± 1 °C) room. Food and water were made available ad libitum. All experiments were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, USA) revised 1996. Morphine sulfate (Temad, Tehran, Iran) and naloxone hydrochloride (Tolid —Daru, Tehran, Iran) were dissolved in sterile 0.9% saline just before the experiment and were injected subcutaneously (s.c.). o-Phthalaldehyde (OPA) was purchased from Sigma-Aldrich and other reagents and solvents were of HPLC-quality and were obtained from Sigma-Aldrich or Merck (Germany). 2.2. Drug treatment Six animals were used in each of seven experimental groups. The schedule of drug administration was based on our previous studies in order to induce behavioral sensitization (Farahmandfar et al., 2010; Farahmandfar et al., 2011; Zarrindast et al., 2006). In experiments where animals received three injections, the control groups also received three saline injections. 2.2.1. Experiment 1: Effects of acute administration of morphine on the extracellular level of GABA in CA1 of non-sensitized male rats In this experiment, two groups of animals received subcutaneous (s.c.) administration of saline (1 ml/kg, s.c.) or morphine (5 mg/kg, s.c.), an effective dose of morphine on spatial memory impairment (Farahmandfar et al., 2010), following a 60-min collection of GABA basal level. 2.2.2. Experiment 2: Effects of morphine sensitization on the extracellular level of GABA in CA1 of male rats Two groups of animals received saline (1 ml/kg, s.c.), once daily for 3 days. After 5 days with no drug treatment, following a 60-min collection of GABA basal level, the animals received a challenge of saline (1 ml/kg, s.c.) [Saline/Saline] or morphine (5 mg/kg, s.c.) [Saline/Morphine]. Another group received for 3 days, once daily, morphine (20 mg/kg, s.c.) and after 5 days (no drug treatment), microdialysis experiments were performed and a challenge of morphine (5 mg/kg, s.c) [Morphine/Morphine] was injected following collection of basal level of GABA. 2.2.3. Experiment 3: Effect of morphine sensitization on the extracellular level of GABA in CA1 of male rats in the presence or absence of naloxone In this experiment, two groups of animals received once daily injections of saline (1 ml/kg, s.c) or the opioid receptor antagonist, naloxone (2 mg/kg, s.c.), 30 min prior to s.c. injections of morphine (20 mg/kg/day×3 days). After 5 days (no drug treatment), microdialysis
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experiments were performed and animals received a challenge of morphine (5 mg/kg, s.c.), following a 60-min collection of GABA basal level. 2.3. Surgery and brain dialysis For the implantation of microdialysis guide cannula (MAB 4.15.IC, Microbiotech/se AB, Stockholm, Sweden), rats were anesthetized with a mixture of ketamine hydrochloride (Sigma,Germany) and xylazine (ketamine 60 mg/kg, xylazine 12 mg/kg, i.p.) and mounted in a stereotaxic frame (Stoelting, USA). The guide cannula was inserted into the hippocampal CA1 area with the following coordinates: 3.8 mm posterior to the bregma, ±2.2 laterals to midline and 2.7 ventral to the skull surface (Paxinos and Watson, 1986). After surgery, at least a sevenday recovery period was supposed before any other intervention. On the experiment day, microdialysis probe (MAB 4.15.1.Cu, b6000 kDa, 1 mm membrane length, Microbiotech/se AB, Stockholm, Sweden) was inserted into the guide cannula and were connected to the microdialysis pump (WPI, SP 210, syringe pump) and perfused with artificial cerebrospinal fluid (ACSF: 114 mM NaCl, 3 mM KCl, 1 mM CaCl2, 2 mM MgSo4, 1.25 mM NaH2PO4, 26 mM NaHCO3, 1 mM NaOH, 10 mM glucose and pH = 7.4) at a flow rate of 2 μl/min. Following a 20-min equilibration period, six consecutive 10-min samples were collected for determination of basal GABA level. Subsequently, saline or morphine was administered and dialysate samples were collected every 10 min for 1 h and were put on ice and instantly placed in − 80 °C. 2.4. GABA determination GABA was measured by reverse-phase high-pressure liquid chromatography (Column: Shim-pack VP-ODS, 250 L×4.6 mm, 5 μm; Pump: LC-10 AD, Shimadzu, Japan) coupled to fluorescence detector (RF-10 AXL, Shimadzu, Japan, set at wavelength: EX.=340 nm, EM.=475 nm), following pre-column derivatization with o-phthaldialdehyde (OPA) according to the method described by de Freitas Silva et al. (2009). The mobile phase consisted of 0.05 M sodium acetate, tetrahydrofuran and methanol (50:1:49, v/v) adjusted to pH 4.0. The mobile phase was filtered through Millipore 0.45 μm Durapore membrane filters and vacuum degassed prior to use. Chromatographic analyses were performed at 25 ± 2 °C. Compounds were eluted isocratically over a 9 min runtime at a flow rate of 1 mL/min. 2.5. Histological analysis Following microdialysis experiments, animals were sacrificed by decapitation and brains were removed. For histological examination of guide cannula and microdialysis probe placement in CA1, 100 μm thick sections were taken, mounted on slides, stained with cresyl violet and the guide cannula and probe track were examined for each rat. Only rats with correct placement were included in data analysis. 2.6. Statistical analysis Basal values of extracellular GABA were the means of six consecutive samples and were expressed as nmol/L. The data of basal concentrations of GABA in each experiment were analyzed by Student's ttest in comparison with respective control group. The changes of GABA concentration after different treatments were analyzed by repeated measures two-way analysis of variance (ANOVA) with the drug treatments as between group factors and time as within group factors. Results showing significant overall changes were subjected to post hoc Tukey's test. The data were expressed as percentage of basal values. A probability level of P b 0.05 was regarded as statistically significant.
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3.1. Effect of acute morphine and morphine sensitization in the presence or absence of naloxone on the basal levels of GABA in CA1 Mean basal levels of GABA in non-sensitized, morphine sensitized and morphine-sensitized rats in the presence or absence of naloxone were assessed. As shown in Fig. 1, three above mentioned groups showed no difference in the mean basal levels of GABA in CA1 as compared to their respective control groups [(P = 0.25), (P = 0.47) and (P = 0.68)], respectively (Fig. 1).
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Time (min) As shown in Fig. 2, effect of acute administration of morphine on the extracellular level of GABA in CA1 of non-sensitized rats was determined. The results showed that acute morphine 5 mg/kg decreased extracellular GABA levels by nearly 40% of baseline at 20 and 40 min after morphine administration (Fig. 2). Repeated measures ANOVA revealed significant effects of group [F (1,10) =33.18; Pb 0.001], time [F(6,60) =3.29; Pb 0.05] and a significant interaction of group ×time [F (6,60) =5.59; Pb 0.05]. Two-way ANOVA followed by Tukey's test revealed that acute administration of morphine caused a significant decrease in the extracellular level of GABA on each time point in CA1 as compared to saline group [(Pb 0.05)] (Fig. 2). 3.3. Effect of morphine sensitization on extracellular GABA concentration in CA1 The effects of repeated administration of saline or morphine with a challenge of morphine on extracellular GABA concentration are shown in Fig. 3. Repeated measures ANOVA revealed significant effects of group [F(1,10) = 123.6; P b 0.001], time [F(6,60) = 3.22; P b 0.01] and a significant interaction of group × time [F(6,60) = 25.1; P b 0.01]. Post hoc Tukey's test showed that a challenge of morphine (5 mg/kg) leads to a significant decrease in GABA concentration on each time point in the CA1 area in saline pretreated group (Saline/Morphine) as compared with the control group (Saline/Saline) [Pb 0.001]. However, challenge dose of morphine (5 mg/kg) caused a significant increase in GABA level on each time point in the CA1 in morphine (20 mg/kg) pretreated group (Morphine/Morphine) as compared with the saline pretreated group (Saline/Morphine) [Pb 0.001] (Fig. 3).
Fig. 2. Effect of acute injection of saline (1 ml/kg, s.c) and morphine (5 mg/kg, s.c) on basal extracellular level of GABA in CA1 of non-sensitized rats. Results are expressed as means ± SEM of the percent of basal values (n = 6). *P b 0.05 significantly different from respective basal values. #P b 0.05 significantly different from the corresponding value of saline group.
with naloxone is shown in Fig. 4. The results showed that the challenge dose of morphine (5 mg/kg) in naloxone with morphine pretreated group (Naloxone+ Morphine/Morphine) significantly decreased extracellular GABA levels by nearly 40% of baseline at 30 and 40 min after administration. Repeated measures ANOVA revealed significant effects of group [F(1,10)= 103.1; P b 0.001], time [F(6,60) = 3.2; P b 0.05] and a significant interaction of group× time [F(6,60)= 23.7; P b 0.001]. Post hoc Tukey's test showed a significant decrease in GABA concentration in CA1 on each time point in animals pretreated with morphine in combination with naloxone (Naloxone+Morphine/Morphine) as compared to the control group (Saline+Morphine/Morphine) [(Pb 0.001)] (Fig. 4). 4. Discussion In the present experiments, we evaluated the effects of morphine administration on the extracellular GABA concentration in the CA1 area of non-sensitized and morphine-sensitized rats by using microdialysis in freely moving animals and HPLC-FLD method. Recent evidence has indicated a specific involvement of the GABAergic system in the mechanisms underlying addictive behavior (Vaughan et al., 1997; Xi and Stein, 2002). It has been reported that activation of the 180
3.4. Effect of naloxone on extracellular GABA concentration in CA1 of morphine-sensitized rats
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Fig. 1. The basal levels of GABA in non-sensitized (A), morphine sensitized (B) and morphine-sensitized rats in combination with naloxone (C) groups in comparison with their respective control groups. The basal contents of GABA in the samples were the mean ± S.E.M. of the averages of the six baseline samples in six rats.
Fig. 3. Effect of saline or morphine (5 mg/kg, s.c) challenge on basal extracellular level of GABA in CA1 of rats pretreated with saline (Saline/Saline and Saline/Morphine ) or morphine (20 mg/kg) [Morphine/Morphine ]. Results are expressed as means ± SEM of the percent of basal values (n = 6). *P b 0.05 and **P b 0.01 significantly different from the respective basal values. #P b 0.05 and ##P b 0.01 significantly different from the corresponding value of Saline/Morphine group.
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Time (min) Fig. 4. Effect of morphine (5 mg/kg s.c) challenge on basal extracellular level of GABA in CA1 of saline (1 ml/kg, s.c)+ morphine (20 mg/kg s.c) [Saline + Morphine/Morphine ] and naloxone (2 mg/kg s.c)+ morphine (20 mg/kg s.c) [Naloxone + Morphine/ Morphine ] pre-treated rats. Results are expressed as means± SEM of the percent of basal values (n= 6). *Pb 0.05 significantly different from respective basal values. # P b 0.05, ##P b 0.01 and ###P b 0.001 significantly different from the corresponding value of Saline+ Morphine/Morphine group.
central GABAergic neurotransmission is closely connected with mesolimbic dopaminergic neurotransmission during addiction processes (Johnson and North, 1992). The mesolimbic dopaminergic system may have an important role in the development of behavioral sensitization to psychostimulants and opioids (Jeziorski and White, 1995; Serrano et al., 2002). Moreover, mesolimbic dopamine release and dopamine cell firing have been inhibited by the GABA projection and GABA inter-neurons within the ventral tegmental area (Enrico et al., 1998; Steffensen et al., 1998; Westerink et al., 1998). Current studies strongly suggest that GABAergic and glutamatergic cells in both the ventral tegmental area and the limbic forebrain are also important in the physical and psychological dependence on morphine, or behavioral sensitization in animal models (Madhavan et al., 2010; Wolf, 2002; Zarrindast et al., 2008). Hippocampus as a part of limbic formation plays a key role in the formation of synaptic plasticity and neural adaptation in the CNS and has numerous opiate receptors and GABAergic neurons (Cohen et al., 1992). It has been suggested that connections of dorsal hippocampus to the nucleus accumbens and ventral tegmental area may play an important role in behavioral sensitization and drug-seeking behavior (Jentsch and Taylor, 1999). However, the effect of this structure and its GABAergic neurotransmission on the morphine sensitization has been less understood. According to our previous study, single administration of morphine (2.5, 5 and 7.5 mg/kg), 30 min before training of Morris water maze task, decreased the spatial memory acquisition in non-sensitized rats with a maximum effect of 5 mg/kg of morphine (Farahmandfar et al., 2010). The results of the present experiments showed that acute administration of morphine (5 mg/kg) significantly decreased extracellular GABA levels in the CA1 to nearly 40% of baseline at 20 and 40 min after morphine administration. This result is a direct verification that spatial learning deficit after acute injection of morphine is significantly correlated with the reduction of GABA concentration in dorsal hippocampus. Our data is also in agreement with the previous results which have reported that acute injection of morphine decreased the extracellular level of GABA in some other brain regions such as ventral tegmental area (Sotomayor et al., 2005) and periaqueductal gray matter (Stiller et al., 1996). Furthermore, repeated exposure to drugs of abuse is thought to cause persistent behavioral sensitization and associated adaptations of neurotransmissions, which can play an important role in certain aspects of drug addiction (Everitt et al., 2001). A number of studies have demonstrated that morphine sensitization can alter the level of dopamine or
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glutamate concentration in some brain regions (Cadoni and Di Chiara, 1999; Hao et al., 2007; Mikkola et al., 2000). There are few studies considering the effect of morphine sensitization on the extracellular level of GABA in addiction-associated regions. In one study by Ojanen et al. (2007), it has been reported that morphine sensitization had no effect on the extracellular level of GABA in the ventral tegmental area in alcohol-preferring AA rats. In the present study, our finding indicates that morphine sensitization induced by pre-treatment with morphine, once daily for 3 days followed by 5 days free of the opioid treatment, can significantly increase the extracellular level of GABA in CA1 of rats at 30 and 50 min after injection of morphine challenge dose. These data suggest that reduction of extracellular GABA concentration induced by acute morphine can be restored in morphine-sensitized rats. The circuitry involved in behavioral sensitization is complex, because sensitization represents a cascade of events involving different transmitter systems and different brain regions such as nucleus accumbens, ventral tegmental area, the basolateral amygdala, the prefrontal cortex and the hippocampus (Wolf, 2003). The effects of GABAergic systems on morphineinduced behavioral sensitization were investigated in several studies (Bartoletti et al., 2007; Li et al., 2004; Narita et al., 2003). It has been shown that hippocampal GABAergic receptors are involved in the reversal effect of morphine sensitization on memory impairment induced by acute morphine (Zarrindast et al., 2008). On the other hand, our previous observations showed that impairment of spatial memory acquisition induced by pre-training injection of morphine (5 mg/kg) was significantly reduced in morphine (20 mg/kg)-sensitized rats in Morris water maze task (Farahmandfar et al., 2010). As hippocampal GABAergic system has an essential role in learning and memory especially spatial memory and navigation (Manns et al., 2003), we can suggest that alteration of GABA levels in CA1 of dorsal hippocampus following morphine sensitization may be one of the responsible factor for the effect of this phenomenon on the spatial memory paradigm. The present data also indicated that injection of naloxone, 30 min before morphine administration during 3 days of sensitization, prevented the increase of GABA concentration induced by morphine sensitization. Thus, the involvement of μ-opioid receptors in the sensitization processes and alteration of dorsal hippocampal GABA concentration by morphine seems likely. In conclusion, the results of the present study provide an evidence of interaction between morphine and GABAergic system in the CA1 region of dorsal hippocampus and implied an adaptation of GABAergic neurotransmission induced by morphine sensitization that μ-opioid receptors may play an important role in this process.
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