- Email: [email protected]
Δ9-Tetrahydrocannabinol excites rat VTA dopamine neurons through activation of cannabinoid CB1 but not opioid receptors
Neuroscience Letters 226 (1997) 159–162
D9-Tetrahydrocannabinol excites rat VTA dopamine neurons through activation of cannabinoid CB1 but not opioid receptors Edward D. French* Department of Pharmacology, University of Arizona, College of Medicine, Tucson, AZ 85724-5050, USA Received 24 January 1997; revised version received 19 March 1997; accepted 1 April 1997
Abstract Behavioral, biochemical and recent electrophysiological data have increasingly implicated the involvement of dopamine in the central actions of cannabinoid compounds. However, the site and mechanism by which cannabinoids stimulate dopamine systems has been somewhat controversial. Central opioid systems have also been suggested to play a role in some cannabinoid-induced behaviors as evidenced by their attenuation in the presence of the opioid antagonist naloxone. However, recent studies using the cannabinoid receptor-selective antagonist SR141716A suggest that the central actions of psychoactive cannabinoids are mediated principally through activation of CB1 receptors. Using single cell electrophysiological recordings in the rat we assessed the effects of both SR141716A and naloxone on D9-tetrahydrocannabinol (THC)-induced activation of ventral tegmental dopamine neurons. While dopamine cell firing was dose-dependently increased following cumulative dosing with D9-THC it was partially or completely inhibited following pretreatment with 0.5 and 2 mg/kg SR141716A, respectively. However, 1 and 10 mg/kg naloxone failed to alter the response to D9-THC. These data provide the first evidence that D9-THC-induced changes in mesolimbic dopamine neuronal activity are mediated by the CB1 cannabinoid receptor, but a causal link for the involvement of opioid systems could not be established. 1997 Elsevier Science Ireland Ltd. Keywords: Cannabinoids; Electrophysiology; Ventral tegmental area
Marijuana is the most widely consumed illicit drug, a fact which could be used to argue that its chemical constituents act within the central nervous system (CNS) to produce positive reinforcement. Although the psychoactive ingredient in marijuana, D9-tetrahydrocannabinol (THC), may affect a variety of neurotransmitter systems through interactions with specific cannabinoid receptors, there is a compelling body of data linking D9-THC-dopamine interactions to some of the more global behavioral effects seen in animals following cannabinoid administration [2,10,15–17]. In fact D9-THC is presumed to mediate the reinforcing actions of marijuana through stimulation of dopamine neurotransmission in limbic areas of the brain [7]. Psychoactive cannabinoids increase extracellular concentrations of dopamine in the nucleus accumbens, an effect which is tetrodotoxin sensitive, calcium dependent and enhanced by haloperidol [4]; concordant with an impulse-dependent mechanism of * Tel.: +1 520 6264359; fax: +1 520 6264182; e-mail: [email protected]
action. The D9-THC-induced efflux of dopamine was also reported to be attenuated by naloxone, thereby implicating endogenous opioids in the dopaminergic effects of marijuana. However, it has been found that other D9-THCevoked behavioral changes are not sensitive to naloxone [19]. Recently we examined the effects of systemically administered cannabinoids on the activity of midbrain dopamine neurons within the rat [6]. Both D9-THC and the synthetic cannabinoid compound WIN 55,212–2 produced increased firing of dopamine neurons within the VTA and substantia nigra pars compacta. Furthermore, the inactivity of the nonpsychoactive cannabidiol and of the inactive enantiomer WIN 55,212–3 strongly suggested that the stimulatory effects we observed were cannabinoid receptor mediated. However, in order to validate this presumption the present study was designed to assess the D9-THC-induced stimulation of dopamine cell firing in the presence of the specific CB1 receptor antagonist SR141716A [13]. Moreover, to address the possible involvement of endogenous opioid sys-
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tems in these dopaminergic stimulatory changes we also tested D9-THC in animals treated with the opioid receptor blocker naloxone. Adult male Sprague–Dawley rats (Harlan Sprague Dawley Inc.) weighing 250–350 g were used in all experiments. Chloral hydrate (350 mg/kg, i.p.) was used for the induction and maintenance of anesthesia throughout the recording period. This anesthetic had the advantage over ketamine which appears to attenuate the response of VTA neurons to the cannabinoids (unpublished observation). The preparation of the animals and methods for extracellular recordings from single dopamine neurons in the midbrain VTA have been detailed elsewhere [5]. Presumptive dopamine neurons were identified according to the following criteria: (1) biphasic (or triphasic) positive-negative waveform action potentials: (2) action potential duration .2 ms; (3) firing rates ,10 spikes/s; and (4) regular to bursting patterns of firing [3]. Once a neuron meeting these characteristics was found, the activity was monitored for approximately 5 min before injections were begun. Only one cell per animal was tested. Light microscopic verification of electrode placement was also made at the conclusion of the experiment [5]. All D9-THC injections were i.v. and separated by interinjection intervals of 2–4 min in a cumulative dosing paradigm of 0.125, 0.25, 0.5, 1, and 2 mg/kg resulting in a total cumulative dose of 3.875 mg/kg. SR141716A was administered i.p. as a bolus of either 0.5 or 2 mg/kg approximately 15 min prior to the first injection of D9-THC. Naloxone treatments of 1 or 10 mg/kg were also given i.p. 10 min before commencing the cannabinoid injections. D9-THC and SR141716A were prepared in the following vehicle (vol/vol): 10% Tween 80, 20% dimethyl sulfoxide (DMSO) and 70% distilled water. An aliquot of stock D9THC (100 mg/ml ethanol) was placed in a vial and evaporated to dryness under a stream of argon gas. DMSO was then added and vortexed, followed by Tween 80 and vortexed, and lastly water and vortexed. Naloxone HCl was dissolved in 165 mM NaCl. Solutions were prepared fresh for each experiment. SR141716A (N-piperidin-1-yl)5-(4-chlorophenyl)-1(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxiamide hydrochloride) was provided by Dr. Carmona-Rinaldi, Sanofi Research. D9-tetrahydrocannabinol and naloxone were purchased from Sigma Chemical Co. Cumulative dosing with i.v. D9-THC in vehicle-pretreated animals produced a dose-dependent increase in the firing rate of neurons identified as dopaminergic in nature (Fig. 1, top panel). Peak changes in rate averaged 55% at the maximum cumulative dose of 3.875 mg/kg (Fig. 2A). In contrast, pretreatment with SR141716A prevented D9THC-evoked excitation of VTA neurons (Fig. 1, bottom panel). Following 0.5 or 2 mg/kg SR141716A, the maximum change in firing rate in response to D9-THC was down to 30 and 6% over baseline, reductions of 45 and 90%, respectively (Fig. 2A). Basal firing rates of the three treatment groups (vehicle, 0.5 and 2 mg/kg SR141716A) were
not significantly different (3.1 ± 0.4, 3.6 ± 0.3 and 3.5 ± 0.6 spikes/s ± SEM). Following 2 mg/kg SR141716A alone, spontaneous firing rates declined by 9% which albeit a small change was significant when analyzed by the paired t-test. The DMSO/Tween/water vehicle alone was previously found to be ineffective [6]. The increase in firing rate elicited by D9-THC was also accompanied by increases in the percent of action potentials contained in bursts going from a basal level of 10–35% following 3.875 mg/kg D9-THC. SR141716A pretreatment also reduced the amount of burst firing to a maximum of 14% in the 0.5 mg/kg group and to 20% in the 2 mg/kg group. In contrast to the effects of SR141716A, naloxone pretreatment failed to blunt the response of VTA dopamine neurons to D9-THC. For example after 1 and 10 mg/kg naloxone, dopamine cell firing rates in response to D9THC increased to 58.5 and 51% above baseline, respectively (Fig. 2B); not significantly different from controls (two-way ANOVA). Basal firing rates were also not affected by naloxone. The results of the present study are the first to show that D9-THC, the psychoactive ingredient in marijuana, stimulates VTA dopamine neuronal activity through activation of
Fig. 1. Single dopamine neurons representative of responses to D9-THC following pretreatment with vehicle or the CB1 receptor antagonist SR141716A. Numbers on the ordinate refer to number of spikes/s converted from spikes collected in 10 s bins. Abscissa is time with the horizontal line below top panel denoting a period of 5 min. Open and closed circles show time of pretreatment with 1 ml/kg i.p. vehicle (V) or 2 mg/kg i.p. SR141716A. Control injections of i.v. saline (S) and D9-THC were given at the arrowheads. Doses of THC were as follows: 0.125, 0.25, 0.5, 1 and 2 mg/kg.
E.D. French / Neuroscience Letters 226 (1997) 159–162
Fig. 2. Cumulative dose-response effects of D9-THC on VTA dopamine neurons in presence of SR141716A (A) or naloxone (B). Injections of THC were given as described in Fig. 1 legend. SR and naloxone pretreatments were i.p. and given approximately 15 and 10 min, respectively, prior to the commencement of cannabinoid administration. Percent changes in firing rate were calculated on the activity in 500 spikes prior to each drug injection over activity in 500 spikes in the post-saline period prior to the first D9-THC injection.
CB1 cannabinoid receptors. This conclusion is based upon the observation that D9-THC-induced excitation of dopamine neuronal firing is attenuated in animals pretreated with the CB1-receptor selective antagonist SR141716A. This finding further validates the results of a recent study where D9-THC and the active cannabimimetic WIN 55,212–2 elicited increases in VTA firing rates, while the non-psychoactive cannabidiol and the inactive enantiomer WIN 55,212–3 failed to do so [6]. Thus, it would appear that the activation of cannabinoid CB1 receptors mediates the excitatory effects of D9-THC on mesolimbic dopamine neurons, even though the site or ionic mechanism(s) that mediate this stimulatory effect remains unknown. The doses of SR141716A used here to antagonize the effects of D9-THC on VTA firing are similar to doses used in other studies. For example, in the rat the AD50 for blocking D9-THC impairment in radial arm maze performance
161
was 2.4 mg/kg [9], while that required for attenuating D9THC-induced antinociception was 2 mg/kg [12]. In a WIN 55,212–2 discrimination task the ED50 for SR141716A antagonism was 1.6 mg/kg [11]. Moreover, the ED50 for inhibiting [3H]CP55,940 in an ex vivo binding study was found to be 1.1 mg/kg i.p., and 0.39 mg/kg to inhibit the specific binding of [3H]SR141716A [14,15]. Thus, it would be reasonable to conclude that the 0.5 and 2 mg/kg doses used here sufficiently blocked central CB1 receptors. Furthermore, the partial blockade of D9-THC-induced increases in VTA firing with the smaller 0.5 mg/kg dose of SR141716A would support the competitive nature of this antagonism. The fact that SR141716A did produce a small but significant decrease in firing rate raises the possibility that endogenous cannabinoids may play a tonic role in regulating VTA baseline neuronal activity. The present findings also strongly suggest that D9-THC’s effects on dopamine neuronal activity are not mediated through central opioid receptors. Earlier behavioral studies showing cross tolerance between morphine and D9-THC [1], and naloxone blockade of D9-THC-induced antinociception had implicated a role for endogenous opioid systems in the central actions of cannabinoids [20]. These findings received additional support from the fact that naloxone administration produced opiate-like withdrawal signs in animals perinatally exposed to D9-THC [18]. Also, high concentrations of cannabinoids displaced radiolabeled dihydromorphine and naloxone binding from m receptors and DPDPE from d receptors [1,18]. However, more recent studies have shown that naloxone by various routes of administration and up to doses as high as 60 mg/kg was not able to block or reverse cannabinoid-induced antinociception [19]. Furthermore, D9-THC-induced inhibition of adenylate cyclase was insensitive to naloxone [19]. Nevertheless, the findings of Gardner et al. [8] that low dose naloxone attenuated the D9-THC augmentation of intracranial self-stimulation did suggest that cannabinoid modulation of reward circuits of the brain involved endogenous opioids. This supposition was also supported by the finding that mesolimbic dopamine release following D9-THC was sensitive to low doses of naloxone [4]. However, the present results clearly show that the ability of D9-THC to activate VTA dopamine neurons is not altered by opiate receptor blockade, even at doses considerably higher than those used elsewhere [7], or by an order of magnitude greater than that needed to block m, d and k opioid receptors. The low dose naloxone used here has been shown to effectively block morphine-induced stimulation of VTA dopamine neurons [5]. Thus, if the effects of D9-THC on ICSS and limbic dopamine release occur through activation of midbrain dopamine neurons, endogenous opioid systems do not appear to be involved in this stimulatory action of D9-THC. In conclusion, the present data show that increased mesolimbic dopamine neuronal activity following D9-THC occurs via activation of central CB1 cannabinoid receptors. Since VTA-mesolimbic pathways have been found to play a
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pivotal role in the reinforcing properties of a number of psychoactive drugs the limbic dopamine response to CB1 cannabinoid receptor stimulation could underlie the abuse liability properties of marijuana. This study was funded by grant DA09025 from the National Institute on Drug Abuse. [1] Bloom, A.S. and Dewey, W.L., A comparison of some pharmacological actions of morphine and D9-tetrahydrocannabinol in the mouse, Psychopharmacology, 57 (1978) 243–248. [2] Bloom, A.S. and Hillard, C.J., Cannabinoids, neurotransmitters, receptors, and brain membranes. In D.J. Harvey (Ed.), Marijuana ’84, IRL Press, Oxford, 1985, pp. 217–231. [3] Bunney, B.S., Walters, J.R., Roth, R.H. and Aghajanian, G.K., Dopaminergic neurons: effects of antipsychotic drugs and amphetamine on single cell activity, J. Pharmacol. Exp. Ther., 185 (1973) 560– 571. [4] Chen, J., Paredes, W., Li, J., Smith, D., Lowinson, J. and Gardner, E.L., D9-Tetrahydrocannabinol produces naloxone-blockable enhancement of presynaptic basal dopamine efflux in nucleus accumbens of conscious, freely-moving rats as measured by intracerebral microdialysis, Psychopharmacology, 102 (1990) 156–162. [5] French, E.D., Competitive NMDA receptor antagonists attenuate phencyclidine-induced excitations of A10 dopamine neurons, Eur. J. Pharmacol., 271 (1992) 1–7. [6] French, E.D., Dillon, K. and Wu, X., Cannabinoids excite dopamine neurons in the ventral tegmentum and substantia nigra, NeuroReport, 8 (1997) 649–652. [7] Gardner, E.L. and Lowinson, J.H., Marijuana’s interaction with brain reward systems: update 1991, Pharmacol. Biochem. Behav., 40 (1991) 571–580. [8] Gardner, E.L., Paredes, W., Smith, D. and Zukin, R.S., Facilitation of brain stimulation reward by D9-tetrahydrocannabinol is mediated by an endogenous opioid mechanism, Adv. Biosci., 75 (1989) 671–674. [9] Lichtman, A.H. and Martin, B.R., D9-THC impairs spatial memory through a cannabinoid receptor mechanism, Psychopharmacology, 126 (1996) 125–131. [10] Ng Cheong Ton, J.M., Gerhardt, G., Friedemann, M., Etgen, A.M., Rose, G.M., Sharpless, N.S. and Gardner, E.L., The effects of D9-
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tetrahydrocannabinol on potassium-evoked release of dopamine in the rat caudate nucleus: an in vivo electrochemical and in vivo microdialysis study, Brain Res., 451 (1988) 59–68. Perio, A., Rinalid-Carmona, M., Maruani, J., Barth, F., Le Fur, G. and Soubrie, P., Central mediation of the cannabinoid cue: activity of a selective CB1 antagonist, SR141716A, Behav. Pharmacol., 7 (1996) 65–71. Reche, I., Fuentes, J.A. and Ruiz-Gayo, M., A role for central cannabinoid and opioid systems in peripheral D9-tetrahydrocannabinolinduced analgesia in mice, Eur. J. Pharmacol., 301 (1996) 75–81. Rinaldi-Carmona, M., Barth, F., Heaulme, M., Shire, D., Calandra, B., Congy, C., Martinez, S., Maruani, J., Neliat, G., Caput, D., Ferrara, P., Soubrie, P., Breliere, J.C. and Le Fur, G., SR141716A, a potent and selective antagonist of the brain cannabinoid receptor, FEBS Lett., 350 (1994) 240–244. Rinaldi-Carmona, M., Barth, F., Heaulme, M., Alonso, R., Shire, D., Congy, C., Soubrie, P., Breliere, J.C. and Le Fur, G., Biochemical and pharmacological characterisation of SR141716A, the first potent and selective brain cannabinoid receptor antagonist, Life Sci., 56 (1995) 1941–1947. Rinalid-Carmona, M., Pialot, F., Congy, C., Redon, E., Barth, F., Bachy, A., Breliere, J.C., Soubrie, P. and Le Fur, G., Characterization and distribution of binding sites for [3H]-SR 141716A, a selective brain (CB1) cannabinoid receptor antagonist, in rodent brain, Life Sci., 58 (1996) 1239–1247. Sakurai-Yamashita, Y., Kataoka, Y., Fujiwara, M., Mine, K. and Ueki, S., D9-Tetrahydrocannabinol facilitates striatal dopaminergic transmission, Pharmacol. Biochem. Behav., 33 (1989) 397–400. Souilhac, J., Poncelet, M., Rinalid-Carmona, M., Le Fur, G. and Soubrie, P., Intrastriatal injection of cannabinoid receptor agonists induced turning behavior in mice, Pharmacol. Biochem. Behav., 51 (1995) 3–7. Vayse, P.J.J., Gardner, E.L. and Zukin, R.S., Modulation of rat brain opioid receptors by cannabinoids, J. Pharmacol. Exp. Ther., 241 (1987) 534–539. Welch, S.P., Blockade of cannabinoid-induced antinociception by norbinaltorphimine, but not N,N-diallyl-tyrosine-Aib-phenylalanineleucine, ICI 174,864 or naloxone in mice, J. Pharmacol. Exp. Ther., 265 (1993) 633–640. Wilson, R.S. and May, E.L., Analgesic properties of the tretrahydrocannabinols, metabolites and analogs, J. Med. Chem., 18 (1975) 700–705.
D9-Tetrahydrocannabinol excites rat VTA dopamine neurons through activation of cannabinoid CB1 but not opioid receptors Edward D. French* Department of Pharmacology, University of Arizona, College of Medicine, Tucson, AZ 85724-5050, USA Received 24 January 1997; revised version received 19 March 1997; accepted 1 April 1997
Abstract Behavioral, biochemical and recent electrophysiological data have increasingly implicated the involvement of dopamine in the central actions of cannabinoid compounds. However, the site and mechanism by which cannabinoids stimulate dopamine systems has been somewhat controversial. Central opioid systems have also been suggested to play a role in some cannabinoid-induced behaviors as evidenced by their attenuation in the presence of the opioid antagonist naloxone. However, recent studies using the cannabinoid receptor-selective antagonist SR141716A suggest that the central actions of psychoactive cannabinoids are mediated principally through activation of CB1 receptors. Using single cell electrophysiological recordings in the rat we assessed the effects of both SR141716A and naloxone on D9-tetrahydrocannabinol (THC)-induced activation of ventral tegmental dopamine neurons. While dopamine cell firing was dose-dependently increased following cumulative dosing with D9-THC it was partially or completely inhibited following pretreatment with 0.5 and 2 mg/kg SR141716A, respectively. However, 1 and 10 mg/kg naloxone failed to alter the response to D9-THC. These data provide the first evidence that D9-THC-induced changes in mesolimbic dopamine neuronal activity are mediated by the CB1 cannabinoid receptor, but a causal link for the involvement of opioid systems could not be established. 1997 Elsevier Science Ireland Ltd. Keywords: Cannabinoids; Electrophysiology; Ventral tegmental area
Marijuana is the most widely consumed illicit drug, a fact which could be used to argue that its chemical constituents act within the central nervous system (CNS) to produce positive reinforcement. Although the psychoactive ingredient in marijuana, D9-tetrahydrocannabinol (THC), may affect a variety of neurotransmitter systems through interactions with specific cannabinoid receptors, there is a compelling body of data linking D9-THC-dopamine interactions to some of the more global behavioral effects seen in animals following cannabinoid administration [2,10,15–17]. In fact D9-THC is presumed to mediate the reinforcing actions of marijuana through stimulation of dopamine neurotransmission in limbic areas of the brain [7]. Psychoactive cannabinoids increase extracellular concentrations of dopamine in the nucleus accumbens, an effect which is tetrodotoxin sensitive, calcium dependent and enhanced by haloperidol [4]; concordant with an impulse-dependent mechanism of * Tel.: +1 520 6264359; fax: +1 520 6264182; e-mail: [email protected]
action. The D9-THC-induced efflux of dopamine was also reported to be attenuated by naloxone, thereby implicating endogenous opioids in the dopaminergic effects of marijuana. However, it has been found that other D9-THCevoked behavioral changes are not sensitive to naloxone [19]. Recently we examined the effects of systemically administered cannabinoids on the activity of midbrain dopamine neurons within the rat [6]. Both D9-THC and the synthetic cannabinoid compound WIN 55,212–2 produced increased firing of dopamine neurons within the VTA and substantia nigra pars compacta. Furthermore, the inactivity of the nonpsychoactive cannabidiol and of the inactive enantiomer WIN 55,212–3 strongly suggested that the stimulatory effects we observed were cannabinoid receptor mediated. However, in order to validate this presumption the present study was designed to assess the D9-THC-induced stimulation of dopamine cell firing in the presence of the specific CB1 receptor antagonist SR141716A [13]. Moreover, to address the possible involvement of endogenous opioid sys-
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tems in these dopaminergic stimulatory changes we also tested D9-THC in animals treated with the opioid receptor blocker naloxone. Adult male Sprague–Dawley rats (Harlan Sprague Dawley Inc.) weighing 250–350 g were used in all experiments. Chloral hydrate (350 mg/kg, i.p.) was used for the induction and maintenance of anesthesia throughout the recording period. This anesthetic had the advantage over ketamine which appears to attenuate the response of VTA neurons to the cannabinoids (unpublished observation). The preparation of the animals and methods for extracellular recordings from single dopamine neurons in the midbrain VTA have been detailed elsewhere [5]. Presumptive dopamine neurons were identified according to the following criteria: (1) biphasic (or triphasic) positive-negative waveform action potentials: (2) action potential duration .2 ms; (3) firing rates ,10 spikes/s; and (4) regular to bursting patterns of firing [3]. Once a neuron meeting these characteristics was found, the activity was monitored for approximately 5 min before injections were begun. Only one cell per animal was tested. Light microscopic verification of electrode placement was also made at the conclusion of the experiment [5]. All D9-THC injections were i.v. and separated by interinjection intervals of 2–4 min in a cumulative dosing paradigm of 0.125, 0.25, 0.5, 1, and 2 mg/kg resulting in a total cumulative dose of 3.875 mg/kg. SR141716A was administered i.p. as a bolus of either 0.5 or 2 mg/kg approximately 15 min prior to the first injection of D9-THC. Naloxone treatments of 1 or 10 mg/kg were also given i.p. 10 min before commencing the cannabinoid injections. D9-THC and SR141716A were prepared in the following vehicle (vol/vol): 10% Tween 80, 20% dimethyl sulfoxide (DMSO) and 70% distilled water. An aliquot of stock D9THC (100 mg/ml ethanol) was placed in a vial and evaporated to dryness under a stream of argon gas. DMSO was then added and vortexed, followed by Tween 80 and vortexed, and lastly water and vortexed. Naloxone HCl was dissolved in 165 mM NaCl. Solutions were prepared fresh for each experiment. SR141716A (N-piperidin-1-yl)5-(4-chlorophenyl)-1(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxiamide hydrochloride) was provided by Dr. Carmona-Rinaldi, Sanofi Research. D9-tetrahydrocannabinol and naloxone were purchased from Sigma Chemical Co. Cumulative dosing with i.v. D9-THC in vehicle-pretreated animals produced a dose-dependent increase in the firing rate of neurons identified as dopaminergic in nature (Fig. 1, top panel). Peak changes in rate averaged 55% at the maximum cumulative dose of 3.875 mg/kg (Fig. 2A). In contrast, pretreatment with SR141716A prevented D9THC-evoked excitation of VTA neurons (Fig. 1, bottom panel). Following 0.5 or 2 mg/kg SR141716A, the maximum change in firing rate in response to D9-THC was down to 30 and 6% over baseline, reductions of 45 and 90%, respectively (Fig. 2A). Basal firing rates of the three treatment groups (vehicle, 0.5 and 2 mg/kg SR141716A) were
not significantly different (3.1 ± 0.4, 3.6 ± 0.3 and 3.5 ± 0.6 spikes/s ± SEM). Following 2 mg/kg SR141716A alone, spontaneous firing rates declined by 9% which albeit a small change was significant when analyzed by the paired t-test. The DMSO/Tween/water vehicle alone was previously found to be ineffective [6]. The increase in firing rate elicited by D9-THC was also accompanied by increases in the percent of action potentials contained in bursts going from a basal level of 10–35% following 3.875 mg/kg D9-THC. SR141716A pretreatment also reduced the amount of burst firing to a maximum of 14% in the 0.5 mg/kg group and to 20% in the 2 mg/kg group. In contrast to the effects of SR141716A, naloxone pretreatment failed to blunt the response of VTA dopamine neurons to D9-THC. For example after 1 and 10 mg/kg naloxone, dopamine cell firing rates in response to D9THC increased to 58.5 and 51% above baseline, respectively (Fig. 2B); not significantly different from controls (two-way ANOVA). Basal firing rates were also not affected by naloxone. The results of the present study are the first to show that D9-THC, the psychoactive ingredient in marijuana, stimulates VTA dopamine neuronal activity through activation of
Fig. 1. Single dopamine neurons representative of responses to D9-THC following pretreatment with vehicle or the CB1 receptor antagonist SR141716A. Numbers on the ordinate refer to number of spikes/s converted from spikes collected in 10 s bins. Abscissa is time with the horizontal line below top panel denoting a period of 5 min. Open and closed circles show time of pretreatment with 1 ml/kg i.p. vehicle (V) or 2 mg/kg i.p. SR141716A. Control injections of i.v. saline (S) and D9-THC were given at the arrowheads. Doses of THC were as follows: 0.125, 0.25, 0.5, 1 and 2 mg/kg.
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Fig. 2. Cumulative dose-response effects of D9-THC on VTA dopamine neurons in presence of SR141716A (A) or naloxone (B). Injections of THC were given as described in Fig. 1 legend. SR and naloxone pretreatments were i.p. and given approximately 15 and 10 min, respectively, prior to the commencement of cannabinoid administration. Percent changes in firing rate were calculated on the activity in 500 spikes prior to each drug injection over activity in 500 spikes in the post-saline period prior to the first D9-THC injection.
CB1 cannabinoid receptors. This conclusion is based upon the observation that D9-THC-induced excitation of dopamine neuronal firing is attenuated in animals pretreated with the CB1-receptor selective antagonist SR141716A. This finding further validates the results of a recent study where D9-THC and the active cannabimimetic WIN 55,212–2 elicited increases in VTA firing rates, while the non-psychoactive cannabidiol and the inactive enantiomer WIN 55,212–3 failed to do so [6]. Thus, it would appear that the activation of cannabinoid CB1 receptors mediates the excitatory effects of D9-THC on mesolimbic dopamine neurons, even though the site or ionic mechanism(s) that mediate this stimulatory effect remains unknown. The doses of SR141716A used here to antagonize the effects of D9-THC on VTA firing are similar to doses used in other studies. For example, in the rat the AD50 for blocking D9-THC impairment in radial arm maze performance
161
was 2.4 mg/kg [9], while that required for attenuating D9THC-induced antinociception was 2 mg/kg [12]. In a WIN 55,212–2 discrimination task the ED50 for SR141716A antagonism was 1.6 mg/kg [11]. Moreover, the ED50 for inhibiting [3H]CP55,940 in an ex vivo binding study was found to be 1.1 mg/kg i.p., and 0.39 mg/kg to inhibit the specific binding of [3H]SR141716A [14,15]. Thus, it would be reasonable to conclude that the 0.5 and 2 mg/kg doses used here sufficiently blocked central CB1 receptors. Furthermore, the partial blockade of D9-THC-induced increases in VTA firing with the smaller 0.5 mg/kg dose of SR141716A would support the competitive nature of this antagonism. The fact that SR141716A did produce a small but significant decrease in firing rate raises the possibility that endogenous cannabinoids may play a tonic role in regulating VTA baseline neuronal activity. The present findings also strongly suggest that D9-THC’s effects on dopamine neuronal activity are not mediated through central opioid receptors. Earlier behavioral studies showing cross tolerance between morphine and D9-THC [1], and naloxone blockade of D9-THC-induced antinociception had implicated a role for endogenous opioid systems in the central actions of cannabinoids [20]. These findings received additional support from the fact that naloxone administration produced opiate-like withdrawal signs in animals perinatally exposed to D9-THC [18]. Also, high concentrations of cannabinoids displaced radiolabeled dihydromorphine and naloxone binding from m receptors and DPDPE from d receptors [1,18]. However, more recent studies have shown that naloxone by various routes of administration and up to doses as high as 60 mg/kg was not able to block or reverse cannabinoid-induced antinociception [19]. Furthermore, D9-THC-induced inhibition of adenylate cyclase was insensitive to naloxone [19]. Nevertheless, the findings of Gardner et al. [8] that low dose naloxone attenuated the D9-THC augmentation of intracranial self-stimulation did suggest that cannabinoid modulation of reward circuits of the brain involved endogenous opioids. This supposition was also supported by the finding that mesolimbic dopamine release following D9-THC was sensitive to low doses of naloxone [4]. However, the present results clearly show that the ability of D9-THC to activate VTA dopamine neurons is not altered by opiate receptor blockade, even at doses considerably higher than those used elsewhere [7], or by an order of magnitude greater than that needed to block m, d and k opioid receptors. The low dose naloxone used here has been shown to effectively block morphine-induced stimulation of VTA dopamine neurons [5]. Thus, if the effects of D9-THC on ICSS and limbic dopamine release occur through activation of midbrain dopamine neurons, endogenous opioid systems do not appear to be involved in this stimulatory action of D9-THC. In conclusion, the present data show that increased mesolimbic dopamine neuronal activity following D9-THC occurs via activation of central CB1 cannabinoid receptors. Since VTA-mesolimbic pathways have been found to play a
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