SYNAPSE 69:103–114 (2015)
Cannabinoid CB1 Receptors Activation and Coactivation With D2 Receptors Modulate GABAergic Neurotransmission in the Globus Pallidus and Increase Motor Asymmetry 1 2 1 ~ GUADALUPE MUNOZ-ARENAS, FRANCISCO PAZ-BERMUDEZ, ANA BAEZ-CORDERO, 3 2 BRENDA GONZALEZ-HERN 2 AND CABALLERO-FLORAN, RENE ANDEZ, BENJAMIN FLORAN, 1* I. DANIEL LIMON 1 Laboratorio de Neurofarmacologıa, Facultad de Ciencias Quımicas, and Posgrado en Ciencias Quımicas, Benemerita Universidad Aut onoma de Puebla, Puebla, 72570, Mexico 2 Departamento de Fisiologıa, Biofısica y Neurociencias, Centro de Investigaci on y de Estudios Avanzados del Instituto Politecnico Nacional, Mexico 3 Facultad de Ciencias Biol ogicas, Universidad Aut onoma de Nuevo Le on, Mexico
KEY WORDS
globus pallidus; cannabinoid CB1 receptor; dopaminergic D2 receptor; GABA uptake; GABA release; motor asymmetry
ABSTRACT The cannabinoid CB1 (CB1R) and dopaminergic D2 (D2R) receptors modify GABAergic transmission in the globus pallidus. Although dopaminergic denervation produces changes in the expression and supersensitization of these receptors, the consequences of these changes on GABAergic neurotransmission are unknown. The aim of this study was to show the effects of CB1R and D2R activation and coactivation on the uptake and release of [3H]GABA in the globus pallidus of hemiparkinsonian rats as well as their effects on motor behavior. The activation of CB1R blocked GABA uptake and decreased GABA release in the globus pallidus in the dopamine denervated side, whereas the co-activation of CB1R-D2R increased GABA release and had no effect on GABA uptake. A microinjection of the CB1R agonist ACEA into the globus pallidus ipsilaterally to a 6-OHDA lesion potentiated turning behavior that was induced by methamphetamine. However, a microinjection of the D2R agonist quinpirole did not modify this behavior, and a microinjection of a mixture of CB1R and D2R agonists significantly potentiated turning behavior. The behavioral effects produced after the activation of the CB1R and the co-activation of CB1R and D2R can be explained by increased GABAergic neurotransmission produced by a block of GABA uptake and an increase in the release of GABA in the globus pallidus, respectively. Synapse 69:103–114, 2015. VC 2014 Wiley Periodicals, Inc.
INTRODUCTION The basal ganglia (BG) modulate movement through direct and indirect pathways (Obeso and Lanciego, 2011). Segregated into the direct and indirect pathways, dopaminergic D1 (D1R) and D2 receptors, respectively, regulate the activity of striatonigral and striatopallidal neurons, synergizing both pathways during the execution of movement (Cui et al., 2013). In a similar manner, the action of dopamine on pallidal and nigral terminals is important for the control of movement (Galvan and Wichmann, 2008). D1R are Golf protein coupled receptors that are located at striato-nigral terminals and whose activation increases GABA release (Floran et al., Ó 2014 WILEY PERIODICALS, INC.
1990). D2Rs are Gi/o protein coupled receptors that are located in striatopallidal neurons and their activation inhibits GABA release (Flor an et al., 1997). However, other receptors, such as CB1R, are also Contract grant sponsor: CONACYT, Mexico; Contract grant number: 257137 (to M.A.G.), 152326 (to B.F.), and 169023 and VIEP-BUAP-2012–2013 (to I.D.L.) *Correspondence to: Daniel Limon, Departamento de Farmacia, Laboratorio de Neurofarmacologıa and Posgrado en Ciencias Quımicas. Universidad Autonoma de Puebla, C.U., Col. San Manuel, 72570 Puebla, Mexico. E-mail: [email protected] Received 28 April 2014; Revised 29 November 2014; Accepted 3 December 2014 DOI: 10.1002/syn.21796 Published online 17 (wileyonlinelibrary.com).
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present in these striatopallidal neurons (Chaves-Kirsten et al., 2013; Herkenham et al., 1991; Julian et al., 2003). CB1R are also coupled Gi/o proteins that inhibit GABA release (Gonzalez et al., 2009; K€ofalvi et al., 2005) and they also contribute to the control of motor behavior, as was shown in the striatum and lez et al., 2006; Kelsey the globus pallidus (Gonza et al., 2009; Souilhac et al., 1995). Localization of D2R and CB1R in the same presynaptic terminal of striatum and globus pallidus has been demonstrated by autoradiographic techniques in the rat (Herkenham et al., 1991; Martın et el., 2008), suggesting an interaction in the modulation of GABAergic neurotransmission. Functional evidence for the effect of the CB1R/D2R relationship on GABAergic transmission in the pallidum has been reported by Gonzalez et al. (2009). They found that the coactivation of these receptors produces an increase in GABA release by normal rats, possibly by a similar increase in cAMP formation (Glass and Felder, 1997; Jarrahian et al., 2004; Khan and Lee, 2013). Additionally, it has been shown that CB1R activation modulates GABA uptake in the globus pallidus of intact rats (Maneuf et al., 1996); however, the role of CB1R/D2R coactivation in GABA uptake is unknown. Knowledge of the control of GABAergic neurotransmission by CB1R and D2R in the striato-pallidal pathway is important as the globus pallidus occupies a key point in BG circuitry (Galvan et al., 2005). In Parkinson’s disease, the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) produces an imbalance in the GABAergic neurotransmission of the direct and indirect pathways (Blum et al., 2001; Obeso and Lanciego, 2011). It also leads to a series of neurochemical changes that result in increased expression (Antonini et al., 1997) and supersensitivity (Deumens et al., 2002) of the D2R in the caudate-putamen of Parkinson patients. Furthermore, an increase in both the mRNA of CB1R (Romero et al., 2000; Martın et al., 2008) and agonist binding in the caudate nucleus (Lastres-Becker et al., 2001) was found. Finally, an increase in endocannabinoid levels, such as those of 2-arachidonyl glycerol (2AG) (Di Marzo et al., 2000), are characteristic changes in CB1R activity during parkinsonism.
Abbreviations D2R CB1R ACEA AM251 6-OHDA
BG MFB
Synapse
dopaminergic D2 receptors cannabinoid CB1 receptors Arachidonyl-2-chloroethylamide N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide 2,4,5-Trihydroxyphenethylamine hydrochloride, 2,5Dihydroxytyramine hydrochloride, 2-(2,4,5-Trihydroxyphenyl)ethylamine hydrochloride basal ganglia medial forebrain bundle
The effects of the interaction of CB1 and D2 receptors in pallidal GABAergic neurotransmission (i.e., the uptake and release of GABA) in parkinsonism is not known. Furthermore, it is also unknown how these changes contribute to the hyperactivity of the indirect pathway (Di Marzo et al., 2000) that underlines hypokinesia (Fernandez-Espejo et al., 2005). To contribute to the understanding of the changes and effects of CB1Rs and D2Rs and their interaction on GABAergic transmission in the globus pallidus after dopaminergic denervation, we studied the activation and coactivation of CB1R and D2R on the uptake and release of [3H]GABA in pallidal slices of hemiparkinsonian rats. We further studied the effects of this neurotransmission on motor behavior by measuring the modifications of turning behavior induced by methamphetamine microinjecting, CB1R and D2R ligands in the globus pallidus of hemiparkinsonian rats. MATERIAL AND METHODS Drugs Arachidonyl-2-chloroethylamide (ACEA) and N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4methyl-1H-pyrazole-3-carboxamide (AM251) were purchased from Tocris Cookson Inc. (Ballwin, MO). Amino oxyacetic acid, 3-piperidinecarboxylic acid (Nipecotic acid), trans-(-)-(4aR)-4,4a,5,6,7,8,8a,9-Octahydro-5-propyl-1H-pyrazolo[3,4-g]quinoline monohydrochloride (Quinpirole), methamphetamine, and 6-hydroxydopamine hydrochloride (6-OHDA) were obtained from Sigma (St. Louis, MO). Animals The GABA uptake and GABA release experiments were carried out in the Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional using male Wistar rats (230–250 g) that were maintained and handled according to the institution’s Animal Care Committee guidelines. The behavioral experiments used male Wistar rats (230–250 g) that were purchased from the breeding colony of the Claude Bernard Bioterium at the Benemerita Universidad Autonoma de Puebla (BUAP). All procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals from the “Norma Mexicana 062.” Animals were individually housed in a temperature and humidity-controlled environment with a 12 h:12 h light:dark cycle with free access to food and water. All efforts were made to minimize both the number of animals used as well as the stress and discomfort experienced by the animals during the experimental procedures. Unilateral 6-OHDA lesion The rats were anaesthetized with chloral hydrate (300 mg/kg i.p.), placed on a David Kopf stereotaxic frame, and injected unilaterally with 6-hydroxydopamine
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Fig. 1. A. Protocol of behavioral and neurochemical probes. B. Cannula position in the globus pallidus of the rats. Top row: diagrammatic representation of local injections in the globus pallidus. Bottom row: representative coronal brain slices of animals employed
in behavioral probes. Anteroposterior with reference in bregma is indicated in diagrams. Plates are taken from Paxinos and Watson’s atlas (1998). The thickness of the section was 300 mm and the dots indicate the site of injection in the nucleus.
(6-OHDA; 16 mg/2 mL of saline containing 0.1% ascorbic acid, perfusion rate 0.2 mL/min) in the medial forebrain bundle (MFB) at coordinates (AP 21.8, L 22.4, V 27.0 mm) in accordance with Paxinos and Watson’s atlas (1998). Rats were pretreated with desipramine (10 mg/kg i.p.) 40 min before surgery to prevent noradrenergicneuron damage. To ensure that the degree of the lesion
was compatible with a depletion of more than 90% of DA content, the animals were injected with methamphetamine (5 mg/kg i.p.) 8 days after surgery and tested for turning behavior (see Turning behavior section). Only animals that made 500 or more ipsilateral turns in 80 min after injection were included in the experiments (Hudson et al., 1993; Rangel-Barajas et al., 2008). Synapse
~ G. MUNOZ-ARENAS ET AL.
106 Neurochemical probes Slice preparation
For each experiment of GABA uptake and GABA release, eight intact or hemiparkinsonian rats were sacrificed by decapitation. Their brains were then removed and submerged in oxygenated ice-cold artificial cerebrospinal fluid (ACSF) with a composition (in mM) of NaCl 118.25, KCl 1.75, MgSO4 1, KH2PO4 1.25, NaHCO3 25, CaCl2 2, and D-glucose 10. The solutions were gassed continuously with O2–CO2 [95:5 (v/v)] at 4 C and with a pH adjusted to 7.4. The brains were then glued with cyanoacrylate to a metal cube mounted on a Petri dish filled with ice-cold ACSF. Three coronal slices [AP: 20.8 to 21.3 mm from bregma, according to Paxinos and Watson’s stereotaxic atlas (1998); 300 mm thick] containing the largest extent of globus pallidus (two per slice, and pooling separately the pallidum from the ipsilateral and contralateral sides in hemiparkinsonian rats) were then obtained with a vibroslicer (Campden Instruments Ltd., Leicester, UK). Pallidal punches (1 mm diameter) were obtained under a stereoscopic microscope and by using a tissue chopper to ensure a uniform size. Pallidal punches of eight rats (six per rat) were pooled in a container with ice-cold ACSF until the end of the dissection. Three to five experiments were conducted with four replicates in each condition.
[3H]GABA uptake The time sequences of the neurochemical tests are shown in Figure 1A. [3H]GABA uptake was assessed using a method taken from Gonzalez et al. (2006). Briefly, pallidal punches of 1 mm diameter, two per chamber, were stabilized for 30 min in ACSF at 37 C. When the stabilization was complete, ACSF was replaced with fresh ASCF at 37 C, which contained [3H]GABA (86 Ci/mmol; Amersham Biosciences) in a final concentration of 0.5 mM for another 30 min. During both the stabilization and [3H]GABA incubations, drugs were present in the ACSF. At the end of the experimental period, the slices were rinsed three times with ACSF at 4 C and sonicated with 1.0 mL of 0.1 N HCl for 1 h. The radioactivity was determined by scintillation counting. The [3H]GABA uptake was calculated as fmol/mg of wet tissue and then expressed as a percentage compared with the control group. The experiments were repeated between three and five times, with four replicates per experiment. To determine whether the effects described might be influenced by a change in the specific activity produced by endogenous GABA, we determined, in one group of experiments, whether changing the specific activity of the uptake solution modified our results. Synapse
A 10-fold dilution of the label did not significantly modify the uptake data. [3H]GABA release The methods described by Floran et al. (2004) were used to measure the release of [3H]GABA and to analyze the data. Once microdissected, the pallidal punches from the obtained pool were incubated for 30 min at 37 C in ACSF and were then transferred to and incubated for 30 min in 2 mL of an ACSF solution that contained 8 nM [3H]GABA and 10 mM aminooxyacetic acid (a GABA transaminase inhibitor to prevent degradation of the label). Finally, the excess radiolabel was removed with two washes of ACSF that contained 10 mM nipecotic acid (a GABA-uptake inhibitor used to prevent the reuptake of the radiolabel) and 10 mM aminooxyacetic acid. Both compounds were present in the perfusion solution for the rest of the experiments. The slices were spread between the chambers (two or three per chamber) of a superfusion system with an 80 mL volume for each chamber and 20 chambers in parallel were perfused with the solution at a rate of 0.5 mL/min for 1 h. The basal release of [3H]GABA was measured by collecting four fractions of the superfusate at 4-min intervals (2 mL per fraction) before release was stimulated by replacing the perfusing solution to a solution of 20 mM K1 (101.25 mM NaCl, 18.75 mM KCl, 1 mM MgSO4, 1.25 mM KH2PO4, 25 mM NaHCO3, 2 mM CaCl2, and 10 mM D-glucose). Six more fractions were collected in the high-K1 medium. All drugs were added to the medium at fraction 2 (i.e., before changing the superfusion to the high-K1 medium) to explore the effects on basal release. To determine the total amount of tritium remaining in the tissue, the slices were collected, treated with 1 mL of 1 M HCl, and allowed to stand for 1 h before the addition of the scintillator. The [3H]GABA release was initially expressed as a fraction of the total amount of tritium remaining in the tissue. The effect of drugs on the basal release of [3H]GABA was assessed by comparing the fractional release in fraction 2 (immediately before exposure of the tissue to the drug) and fraction four (immediately before exposure to 20 mM of K1), using the paired Student’s t-test. Drug- and treatment-induced changes in the release of [3H]GABA were assessed by comparing the area under the appropriate release curves between the first and last fractions collected after the change to high K1, based on the assumption that the basal release of [3H]GABA would remain unchanged from the level measured in the fraction immediately preceding the K1 stimulation. In a previous article (Garcia et al., 1997), we found that the fall in basal release was less than 1%. In experiments in which this was tested, the basal release in fraction
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Fig. 2. CB1 receptors activation, decreases the [3H]GABA uptake in slices of the globus pallidus. A. Dose–response curve of ACEA on CB1 receptors. B. Effect of ACEA, AM251, quinpirole, and coadministration of ACEA and quinpirole on the [3H]GABA uptake in the globus pallidus. Effect of ACEA in hemiparkinsonian rats, (C) without a lesion and (D) denervated on the [3H]GABA uptake. Values
are mean 6 SE determined in three independent experiments, four replicates in each experiment (n 5 8 rats per experiment). Data were analyzed with a one-way ANOVA and post-test Tukey test (**P
Cannabinoid CB1 Receptors Activation and Coactivation With D2 Receptors Modulate GABAergic Neurotransmission in the Globus Pallidus and Increase Motor Asymmetry 1 2 1 ~ GUADALUPE MUNOZ-ARENAS, FRANCISCO PAZ-BERMUDEZ, ANA BAEZ-CORDERO, 3 2 BRENDA GONZALEZ-HERN 2 AND CABALLERO-FLORAN, RENE ANDEZ, BENJAMIN FLORAN, 1* I. DANIEL LIMON 1 Laboratorio de Neurofarmacologıa, Facultad de Ciencias Quımicas, and Posgrado en Ciencias Quımicas, Benemerita Universidad Aut onoma de Puebla, Puebla, 72570, Mexico 2 Departamento de Fisiologıa, Biofısica y Neurociencias, Centro de Investigaci on y de Estudios Avanzados del Instituto Politecnico Nacional, Mexico 3 Facultad de Ciencias Biol ogicas, Universidad Aut onoma de Nuevo Le on, Mexico
KEY WORDS
globus pallidus; cannabinoid CB1 receptor; dopaminergic D2 receptor; GABA uptake; GABA release; motor asymmetry
ABSTRACT The cannabinoid CB1 (CB1R) and dopaminergic D2 (D2R) receptors modify GABAergic transmission in the globus pallidus. Although dopaminergic denervation produces changes in the expression and supersensitization of these receptors, the consequences of these changes on GABAergic neurotransmission are unknown. The aim of this study was to show the effects of CB1R and D2R activation and coactivation on the uptake and release of [3H]GABA in the globus pallidus of hemiparkinsonian rats as well as their effects on motor behavior. The activation of CB1R blocked GABA uptake and decreased GABA release in the globus pallidus in the dopamine denervated side, whereas the co-activation of CB1R-D2R increased GABA release and had no effect on GABA uptake. A microinjection of the CB1R agonist ACEA into the globus pallidus ipsilaterally to a 6-OHDA lesion potentiated turning behavior that was induced by methamphetamine. However, a microinjection of the D2R agonist quinpirole did not modify this behavior, and a microinjection of a mixture of CB1R and D2R agonists significantly potentiated turning behavior. The behavioral effects produced after the activation of the CB1R and the co-activation of CB1R and D2R can be explained by increased GABAergic neurotransmission produced by a block of GABA uptake and an increase in the release of GABA in the globus pallidus, respectively. Synapse 69:103–114, 2015. VC 2014 Wiley Periodicals, Inc.
INTRODUCTION The basal ganglia (BG) modulate movement through direct and indirect pathways (Obeso and Lanciego, 2011). Segregated into the direct and indirect pathways, dopaminergic D1 (D1R) and D2 receptors, respectively, regulate the activity of striatonigral and striatopallidal neurons, synergizing both pathways during the execution of movement (Cui et al., 2013). In a similar manner, the action of dopamine on pallidal and nigral terminals is important for the control of movement (Galvan and Wichmann, 2008). D1R are Golf protein coupled receptors that are located at striato-nigral terminals and whose activation increases GABA release (Floran et al., Ó 2014 WILEY PERIODICALS, INC.
1990). D2Rs are Gi/o protein coupled receptors that are located in striatopallidal neurons and their activation inhibits GABA release (Flor an et al., 1997). However, other receptors, such as CB1R, are also Contract grant sponsor: CONACYT, Mexico; Contract grant number: 257137 (to M.A.G.), 152326 (to B.F.), and 169023 and VIEP-BUAP-2012–2013 (to I.D.L.) *Correspondence to: Daniel Limon, Departamento de Farmacia, Laboratorio de Neurofarmacologıa and Posgrado en Ciencias Quımicas. Universidad Autonoma de Puebla, C.U., Col. San Manuel, 72570 Puebla, Mexico. E-mail: [email protected] Received 28 April 2014; Revised 29 November 2014; Accepted 3 December 2014 DOI: 10.1002/syn.21796 Published online 17 (wileyonlinelibrary.com).
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present in these striatopallidal neurons (Chaves-Kirsten et al., 2013; Herkenham et al., 1991; Julian et al., 2003). CB1R are also coupled Gi/o proteins that inhibit GABA release (Gonzalez et al., 2009; K€ofalvi et al., 2005) and they also contribute to the control of motor behavior, as was shown in the striatum and lez et al., 2006; Kelsey the globus pallidus (Gonza et al., 2009; Souilhac et al., 1995). Localization of D2R and CB1R in the same presynaptic terminal of striatum and globus pallidus has been demonstrated by autoradiographic techniques in the rat (Herkenham et al., 1991; Martın et el., 2008), suggesting an interaction in the modulation of GABAergic neurotransmission. Functional evidence for the effect of the CB1R/D2R relationship on GABAergic transmission in the pallidum has been reported by Gonzalez et al. (2009). They found that the coactivation of these receptors produces an increase in GABA release by normal rats, possibly by a similar increase in cAMP formation (Glass and Felder, 1997; Jarrahian et al., 2004; Khan and Lee, 2013). Additionally, it has been shown that CB1R activation modulates GABA uptake in the globus pallidus of intact rats (Maneuf et al., 1996); however, the role of CB1R/D2R coactivation in GABA uptake is unknown. Knowledge of the control of GABAergic neurotransmission by CB1R and D2R in the striato-pallidal pathway is important as the globus pallidus occupies a key point in BG circuitry (Galvan et al., 2005). In Parkinson’s disease, the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) produces an imbalance in the GABAergic neurotransmission of the direct and indirect pathways (Blum et al., 2001; Obeso and Lanciego, 2011). It also leads to a series of neurochemical changes that result in increased expression (Antonini et al., 1997) and supersensitivity (Deumens et al., 2002) of the D2R in the caudate-putamen of Parkinson patients. Furthermore, an increase in both the mRNA of CB1R (Romero et al., 2000; Martın et al., 2008) and agonist binding in the caudate nucleus (Lastres-Becker et al., 2001) was found. Finally, an increase in endocannabinoid levels, such as those of 2-arachidonyl glycerol (2AG) (Di Marzo et al., 2000), are characteristic changes in CB1R activity during parkinsonism.
Abbreviations D2R CB1R ACEA AM251 6-OHDA
BG MFB
Synapse
dopaminergic D2 receptors cannabinoid CB1 receptors Arachidonyl-2-chloroethylamide N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide 2,4,5-Trihydroxyphenethylamine hydrochloride, 2,5Dihydroxytyramine hydrochloride, 2-(2,4,5-Trihydroxyphenyl)ethylamine hydrochloride basal ganglia medial forebrain bundle
The effects of the interaction of CB1 and D2 receptors in pallidal GABAergic neurotransmission (i.e., the uptake and release of GABA) in parkinsonism is not known. Furthermore, it is also unknown how these changes contribute to the hyperactivity of the indirect pathway (Di Marzo et al., 2000) that underlines hypokinesia (Fernandez-Espejo et al., 2005). To contribute to the understanding of the changes and effects of CB1Rs and D2Rs and their interaction on GABAergic transmission in the globus pallidus after dopaminergic denervation, we studied the activation and coactivation of CB1R and D2R on the uptake and release of [3H]GABA in pallidal slices of hemiparkinsonian rats. We further studied the effects of this neurotransmission on motor behavior by measuring the modifications of turning behavior induced by methamphetamine microinjecting, CB1R and D2R ligands in the globus pallidus of hemiparkinsonian rats. MATERIAL AND METHODS Drugs Arachidonyl-2-chloroethylamide (ACEA) and N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4methyl-1H-pyrazole-3-carboxamide (AM251) were purchased from Tocris Cookson Inc. (Ballwin, MO). Amino oxyacetic acid, 3-piperidinecarboxylic acid (Nipecotic acid), trans-(-)-(4aR)-4,4a,5,6,7,8,8a,9-Octahydro-5-propyl-1H-pyrazolo[3,4-g]quinoline monohydrochloride (Quinpirole), methamphetamine, and 6-hydroxydopamine hydrochloride (6-OHDA) were obtained from Sigma (St. Louis, MO). Animals The GABA uptake and GABA release experiments were carried out in the Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional using male Wistar rats (230–250 g) that were maintained and handled according to the institution’s Animal Care Committee guidelines. The behavioral experiments used male Wistar rats (230–250 g) that were purchased from the breeding colony of the Claude Bernard Bioterium at the Benemerita Universidad Autonoma de Puebla (BUAP). All procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals from the “Norma Mexicana 062.” Animals were individually housed in a temperature and humidity-controlled environment with a 12 h:12 h light:dark cycle with free access to food and water. All efforts were made to minimize both the number of animals used as well as the stress and discomfort experienced by the animals during the experimental procedures. Unilateral 6-OHDA lesion The rats were anaesthetized with chloral hydrate (300 mg/kg i.p.), placed on a David Kopf stereotaxic frame, and injected unilaterally with 6-hydroxydopamine
ACTIVATION AND COACTIVATION OF CB1R AND D2R
105
Fig. 1. A. Protocol of behavioral and neurochemical probes. B. Cannula position in the globus pallidus of the rats. Top row: diagrammatic representation of local injections in the globus pallidus. Bottom row: representative coronal brain slices of animals employed
in behavioral probes. Anteroposterior with reference in bregma is indicated in diagrams. Plates are taken from Paxinos and Watson’s atlas (1998). The thickness of the section was 300 mm and the dots indicate the site of injection in the nucleus.
(6-OHDA; 16 mg/2 mL of saline containing 0.1% ascorbic acid, perfusion rate 0.2 mL/min) in the medial forebrain bundle (MFB) at coordinates (AP 21.8, L 22.4, V 27.0 mm) in accordance with Paxinos and Watson’s atlas (1998). Rats were pretreated with desipramine (10 mg/kg i.p.) 40 min before surgery to prevent noradrenergicneuron damage. To ensure that the degree of the lesion
was compatible with a depletion of more than 90% of DA content, the animals were injected with methamphetamine (5 mg/kg i.p.) 8 days after surgery and tested for turning behavior (see Turning behavior section). Only animals that made 500 or more ipsilateral turns in 80 min after injection were included in the experiments (Hudson et al., 1993; Rangel-Barajas et al., 2008). Synapse
~ G. MUNOZ-ARENAS ET AL.
106 Neurochemical probes Slice preparation
For each experiment of GABA uptake and GABA release, eight intact or hemiparkinsonian rats were sacrificed by decapitation. Their brains were then removed and submerged in oxygenated ice-cold artificial cerebrospinal fluid (ACSF) with a composition (in mM) of NaCl 118.25, KCl 1.75, MgSO4 1, KH2PO4 1.25, NaHCO3 25, CaCl2 2, and D-glucose 10. The solutions were gassed continuously with O2–CO2 [95:5 (v/v)] at 4 C and with a pH adjusted to 7.4. The brains were then glued with cyanoacrylate to a metal cube mounted on a Petri dish filled with ice-cold ACSF. Three coronal slices [AP: 20.8 to 21.3 mm from bregma, according to Paxinos and Watson’s stereotaxic atlas (1998); 300 mm thick] containing the largest extent of globus pallidus (two per slice, and pooling separately the pallidum from the ipsilateral and contralateral sides in hemiparkinsonian rats) were then obtained with a vibroslicer (Campden Instruments Ltd., Leicester, UK). Pallidal punches (1 mm diameter) were obtained under a stereoscopic microscope and by using a tissue chopper to ensure a uniform size. Pallidal punches of eight rats (six per rat) were pooled in a container with ice-cold ACSF until the end of the dissection. Three to five experiments were conducted with four replicates in each condition.
[3H]GABA uptake The time sequences of the neurochemical tests are shown in Figure 1A. [3H]GABA uptake was assessed using a method taken from Gonzalez et al. (2006). Briefly, pallidal punches of 1 mm diameter, two per chamber, were stabilized for 30 min in ACSF at 37 C. When the stabilization was complete, ACSF was replaced with fresh ASCF at 37 C, which contained [3H]GABA (86 Ci/mmol; Amersham Biosciences) in a final concentration of 0.5 mM for another 30 min. During both the stabilization and [3H]GABA incubations, drugs were present in the ACSF. At the end of the experimental period, the slices were rinsed three times with ACSF at 4 C and sonicated with 1.0 mL of 0.1 N HCl for 1 h. The radioactivity was determined by scintillation counting. The [3H]GABA uptake was calculated as fmol/mg of wet tissue and then expressed as a percentage compared with the control group. The experiments were repeated between three and five times, with four replicates per experiment. To determine whether the effects described might be influenced by a change in the specific activity produced by endogenous GABA, we determined, in one group of experiments, whether changing the specific activity of the uptake solution modified our results. Synapse
A 10-fold dilution of the label did not significantly modify the uptake data. [3H]GABA release The methods described by Floran et al. (2004) were used to measure the release of [3H]GABA and to analyze the data. Once microdissected, the pallidal punches from the obtained pool were incubated for 30 min at 37 C in ACSF and were then transferred to and incubated for 30 min in 2 mL of an ACSF solution that contained 8 nM [3H]GABA and 10 mM aminooxyacetic acid (a GABA transaminase inhibitor to prevent degradation of the label). Finally, the excess radiolabel was removed with two washes of ACSF that contained 10 mM nipecotic acid (a GABA-uptake inhibitor used to prevent the reuptake of the radiolabel) and 10 mM aminooxyacetic acid. Both compounds were present in the perfusion solution for the rest of the experiments. The slices were spread between the chambers (two or three per chamber) of a superfusion system with an 80 mL volume for each chamber and 20 chambers in parallel were perfused with the solution at a rate of 0.5 mL/min for 1 h. The basal release of [3H]GABA was measured by collecting four fractions of the superfusate at 4-min intervals (2 mL per fraction) before release was stimulated by replacing the perfusing solution to a solution of 20 mM K1 (101.25 mM NaCl, 18.75 mM KCl, 1 mM MgSO4, 1.25 mM KH2PO4, 25 mM NaHCO3, 2 mM CaCl2, and 10 mM D-glucose). Six more fractions were collected in the high-K1 medium. All drugs were added to the medium at fraction 2 (i.e., before changing the superfusion to the high-K1 medium) to explore the effects on basal release. To determine the total amount of tritium remaining in the tissue, the slices were collected, treated with 1 mL of 1 M HCl, and allowed to stand for 1 h before the addition of the scintillator. The [3H]GABA release was initially expressed as a fraction of the total amount of tritium remaining in the tissue. The effect of drugs on the basal release of [3H]GABA was assessed by comparing the fractional release in fraction 2 (immediately before exposure of the tissue to the drug) and fraction four (immediately before exposure to 20 mM of K1), using the paired Student’s t-test. Drug- and treatment-induced changes in the release of [3H]GABA were assessed by comparing the area under the appropriate release curves between the first and last fractions collected after the change to high K1, based on the assumption that the basal release of [3H]GABA would remain unchanged from the level measured in the fraction immediately preceding the K1 stimulation. In a previous article (Garcia et al., 1997), we found that the fall in basal release was less than 1%. In experiments in which this was tested, the basal release in fraction
ACTIVATION AND COACTIVATION OF CB1R AND D2R
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Fig. 2. CB1 receptors activation, decreases the [3H]GABA uptake in slices of the globus pallidus. A. Dose–response curve of ACEA on CB1 receptors. B. Effect of ACEA, AM251, quinpirole, and coadministration of ACEA and quinpirole on the [3H]GABA uptake in the globus pallidus. Effect of ACEA in hemiparkinsonian rats, (C) without a lesion and (D) denervated on the [3H]GABA uptake. Values
are mean 6 SE determined in three independent experiments, four replicates in each experiment (n 5 8 rats per experiment). Data were analyzed with a one-way ANOVA and post-test Tukey test (**P
