Different times of withdrawal from cocaine administration cause changes in muscarinic and dopaminergic receptors in rat premotor cortex

Neuroscience Letters 312 (2001) 129–132 www.elsevier.com/locate/neulet

Different times of withdrawal from cocaine administration cause changes in muscarinic and dopaminergic receptors in rat premotor cortex D.S. Maceˆdo, F.C.F. Sousa, S.M.M. Vasconcelos, V.T.M. Lima, G.S.B. Viana* Department of Physiology and Pharmacology, Federal University of Ceara´, Rua Cel. Nunes de Melo 1127, Fortaleza 60431-970, CE, Brazil Received 1 May 2001; received in revised form 3 August 2001; accepted 7 August 2001

Abstract The present work studied neurochemical changes in rat premotor cortex 30 min, 1 and 5 days after withdrawal from cocaine repeated administration (20 and 30 mg/kg, intraperitoneally, daily for 7 days). Binding assays were performed in 10% homogenates, and ligands used were [ 3H]-N-methylscopolamine, [ 3H]-SCH 23390, and [ 3H]-spiroperidol for muscarinic, D1- and D2-like receptors, respectively. Levels of cyclic AMP (cAMP) and cyclic guanosine monophosphate (cGMP) were determined using a commercial kit. Scatchard analyses of muscarinic receptors showed an upregulation after 1 and 5 days withdrawal. While D2-like receptors were upregulated at all withdrawal periods, D1-like receptors were upregulated only at the 30 min withdrawal, and returned to normal levels after 1 day of the last injection. In relation to cAMP levels, the repeated cocaine administration, 1 day after the last injection produced a decrease (around 26%) with both doses, while a 67% increase was seen in cGMP levels with the 30 mg/kg dose. These findings indicate lasting neurochemical changes in premotor cortex caused by cocaine which remained after different withdrawal periods. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cocaine; Dopaminergic receptors; Muscarinic receptors; Cyclic AMP; Cyclic guanosine monophosphate; Premotor cortex

Cocaine studies [9,12,25] have focused mainly on the nucleus accumbens. This brain area receives projections from dopaminergic neurons present in the ventral tegmental area (VTA), and is thought to mediate both the locomotor activating and rewarding properties of cocaine. More recent studies have examined the effect of withdrawal from repeated cocaine administration on biochemical parameters associated with mesolimbic neurons. Results indicate that there are major and long-lasting changes in these neurons during the withdrawal period [11]. On the other hand, very little is known about the effects of cocaine on brain premotor cortex. Since cocaine is also a local anesthetic, this area is mainly studied for this purpose. Some work [26] using patch-clamp technique found that, 3 days after discontinuation of repeated cocaine administration, nucleus accumbens neurons were less responsive to depolarizing current injections. These effects were not mimicked by repeated injections of the local anesthetic lidocaine and not observed in neurons within the motor cortex. * Corresponding author. Tel.: 155-85-288-8337; fax: 155-85288-8333. E-mail address: [email protected] (G.S.B. Viana).

According to Saper et al., 2000 [19], the premotor cortex is a set of interconnected areas in the frontal lobe just rostral to the motor cortex. This brain area receives inputs mainly from three sources: (1) the motor nuclei in the ventroanterior and ventrolateral thalamus (which receive input from the basal ganglia and cerebellum); (2) the primary somatosensory cortex and parietal association cortex (which provide information about the ongoing motor response); and (3) the prefrontal association cortex. Since most of the works with cocaine were performed in the nucleus accumbens, and considering that cocaine stimulates cortically projecting cholinergic neurons, we decided to study muscarinic and dopaminergic receptors densities as well as cyclic nucleotide levels on rat premotor cortex after cocaine administration for 7 days and 30 min, 1 and 5 days withdrawal periods. Male Wistar rats (180–200 g), from the Animal House of the Federal University of Ceara´, were treated daily for 7 days with cocaine (20 and 30 mg/kg, intraperitoneally (i.p.)). Controls received the same volume of saline. Animals were decapitated 30 min, 1 and 5 days after the last injection, and immediately their brains were dissected

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on ice. The premotor cortex was used to prepare 10% homogenates. Muscarinic receptors were measured using [ 3H]-Nmethylscopolamine, ([ 3H]-NMS, 85 Ci/mmol, New England Nuclear, Boston, MA), according to Dombrowski et al. [5] with some modifications. Total homogenates corresponding to 50–100 mg protein were prepared in a 150 mM sodium phosphate buffer, pH 7.4, containing 0.119 to 5.95 nM [ 3H]-NMS in a final volume of 0.2 ml. After incubation at 378C for 30 min, time to reach equilibrium, the reaction was terminated by filtering the incubation mixture through Whatman GF/B filters in a cell harvester apparatus from Brandel, USA. The filters were then washed five times with 4 ml of ice-cold saline, dried for at least 2 h in the oven at 608C, and placed in vials with 3 ml of toluene-based scintillation fluid. The radioactivity was measured with a Beckman scintillation counter, model 6500, USA, at a count efficiency of 67%. Specific binding was calculated as total minus non-specific binding performed in the presence of atropine (12.5 mM), and results are reported as femtomoles per milligram of protein. Protein was determined by Lowry et al. [15], using bovine serum albumin as standard. Densities of D1- and D2-like receptors were determined according to methods described by Kessler et al. and Meltzer et al. [8,16]. In the case of D1 receptors, the specific ligand [ 3H]-SCH 23390 (87 Ci/mmol from New England Nuclear, USA) was used. Total homogenates were incubated in 50 mM Tris–HCl buffer, (pH 7.4) with the following composition (mM): NaCl (120), CaCl2 (2), MgCl2 (1), NaEDTA (1), and ascorbic acid (1). Concentrations of [ 3H]SCH 23390 ranging from 0.115 to 9.2 nM in a final volume of 0.2 ml were used. For the determination of D2 receptors, the specific ligand, [ 3H]-spiroperidol (114 Ci/mmol, from New England Nuclear, USA) was utilized. Total homoge-

nates were incubated in a 50 mM Tris–HCl buffer, (pH 7.4) containing 5 mM mianserin for blocking serotonergic receptors and 0.102–7.14 nM [ 3H]-spiroperidol in a final volume of 0.2 ml. In both cases (D1 and D2 receptor assays), specific binding was defined as total minus non-specific binding carried out in the presence of 10 mM butaclamol. After incubation at 378C, 60 min, experiments proceeded as described above for the muscarinic binding. Cyclic AMP (cAMP) or cyclic guanosine monophosphate (cGMP) levels were determined with an assay kit from Amersham International, UK. The determination is based on the competition between unlabelled cAMP or cGMP and a fixed quantity of the tritium labeled compound for binding to a protein or an antiserum (in the case of cGMP) which has a high specificity and affinity for cAMP or cGMP. The results for cAMP or cGMP assays are expressed as pmol/ mg protein. The data for all experiments are expressed as means ^ SEM. ANOVA was used for the statistical analysis followed by the Student–Newman–Keuls test to identify differences between experimental groups. Values of P , 0:05 were considered significant. Table 1 shows an upregulation of muscarinic receptors density, after 1 day of cocaine withdrawal, (118.7% increase) only with the dose of 30 mg/kg, in relation to controls and to the dose of 20 mg/kg [Fð2; 15Þ ¼ 29:781 P , 0:01], with no alteration in dissociation constant values [Fð2; 15Þ ¼ 1:638, N. S.]. After 5 days withdrawal, there was an increase of 88.4 and 74.3% in muscarinic receptors density with cocaine doses of 20 mg/kg [Fð2; 9Þ ¼ 8:026, P , 0:05] and 30 mg/ kg [Fð2; 9Þ ¼ 8:026, P , 0:05], respectively with a decrease in Kd value observed only with the higher dose [Fð2; 9Þ ¼ 5:868, P , 0:05]. There was no change in receptor density after 30 min withdrawal [Fð2; 12Þ ¼ 0:1501, N.

Table 1 Effects of cocaine on muscarinic, D1 and D2 dopaminergic receptor densities (Bmax) and Kd values in rat premotor cortex a Cocaine (mg/kg)

30 min withdrawal 0 20 30 1 day withdrawal 0 20 30 5 days withdrawal 0 20 30 a

M1 1 M2

D1

Bmax (fmol/mg protein)

Kd (nM)

273.6 ^ 36.05 (7) 243.1 ^ 30.49 (3) 264.6 ^ 29.36 (3)

1.17 ^ 0.17 (7) 153.2 ^ 11.73 (13) 0.48 ^ 0.081 (3) 368.1 ^ 44.77 (3) b 0.68 ^ 0.308 (3) 257.6 ^ 30.35 (3) b

281.0 ^ 41.75 (6) 1.27 ^ 0.15 (6) 247.9 ^ 31.48 (5) 0.89 ^ 0.13 (5) 614.6 ^ 31.40 (5) b,c 1.29 ^ 0.22 (5) 278.2 ^ 15.51 (4) 524.0 ^ 27.95 (3) b 484.9 ^ 88.50 (3) b

Bmax (fmol/mg protein)

153.2 ^ 11.73 (13) 107.4 ^ 11.66 (5) 103.7 ^ 13.98 (5)

1.32 ^ 0.172 (4) 153.2 ^ 11.73 (13) 0.85 ^ 0.06 (3) 122.9 ^ 21.97 (3) 0.55 ^ 0.21 (3) b 182.8 ^ 43.11 (3)

D2 Kd (nM)

Bmax (fmol/mg protein)

3.85 ^ 0.347 (13) 283.2 ^ 13.6 (18) 4.61 ^ 0.87 (3) 532.8 ^ 17.61 (3) b 3.57 ^ 0.599(3) 541.9 ^ 32.4 (3) b

Kd (nM)

3.14 ^ 0.38 (17) 5.08 ^ 1.59(3) 4.54 ^ 0.86 (3)

3.85 ^ 0.347 (13) 283.2 ^ 13.6 (18) 3.14 ^ 0.38 (17) 3.67 ^ 0.57 (5) 327.9 ^ 29.77 (5) 6.56 ^ 1.22 (5) b 1.92 ^ 0.10 (5) b,c 438.5 ^ 33.40 (4) b,c 6.28 ^ 0.57 (4) b 3.85 ^ 0.347 (13) 283.2 ^ 13.6 (18) 1.81 ^ 0.38 (3) 497.6 ^ 83.3 (3) b 6.12 ^ 2.48 (3) 458.3 ^ 36.6 (3) b

3.14 ^ 0.38 (17) 4.06 ^ 1.23 (3) 3.19 ^ 0.82 (3)

Animals were treated daily for 1 week with cocaine 20 and 30 mg/kg, i.p., and measurements were done 30 min, 1 and 5 days after the last injection. Data are reported as means ^ SEM for the number of experiments shown in parentheses. For statistical analyses, ANOVA and the Student–Newman–Keuls test as a post-hoc test were used. b,cP , 0:05 as compared to controls and cocaine 20 mg/kg, respectively.

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S.], or Kd value [Fð2; 12Þ ¼ 3:144, N. S.] with either doses. An upregulation of D1-receptors of 140.3 and 68.1% with cocaine 20 mg/kg [Fð2; 18Þ ¼ 25:219, P , 0:001] and 30 mg/kg [Fð2; 18Þ ¼ 25:219, P , 0:05], respectively occurred only after a 30 min withdrawal. No significant changes were seen in Kd value [Fð2; 18Þ ¼ 0:5848, N. S.]. After 1 day withdrawal there was a significant difference only in kd value with the higher dose, no difference was seen in Bmax values (Bmax, [Fð2; 22Þ ¼ 4:575, N. S.]; kd, 20 mg/kg [Fð2; 22Þ ¼ 5:452, N. S.], 30 mg/kg [Fð2; 22Þ ¼ 5:452, P , 0:05]) nor after 5 day withdrawal (Bmax, [Fð2; 18Þ ¼ 1:212, N. S.]; kd, [Fð2; 18Þ ¼ 3:949, N. S.]). D2-like receptors presented a significant increase of 88 and 91.3% after 30 min withdrawal with the dose of 20 and 30 mg/kg [Fð2; 23Þ ¼ 47:211, P , 0:001], respectively. After 1 day withdrawal there was an upregulation of 54.8% with the higher dose in relation to control and to cocaine 20 mg/kg [Fð2; 26Þ ¼ 10:959, P , 0:001), while Kd increased with both doses studied [Fð2; 25Þ ¼ 313:77, P , 0:001]. After 5 days withdrawal, an increase of 75.7 and 61.8% was seen with cocaine 20 and 30 mg/kg [Fð2; 23Þ ¼ 17:095, P , 0:001], respectively. On the other hand, no difference was seen in dissociation constant values of D2 receptors after 30 min and 5 days withdrawal period (30 min, [Fð2; 22Þ ¼ 2:195, N. S.]; 5 days, [Fð2; 22Þ ¼ 0:4166, N. S.]). Table 2 presents a significant decrease (around 27%) with both doses of cocaine [Fð2; 29Þ ¼ 4:838, P , 0:05] in cAMP levels. In the case of cGMP levels, there was an increase of 66.7% only with the higher dose in relation to controls and to cocaine 20 mg/kg [Fð2; 30Þ ¼ 5:994, P , 0:01]. The present work showed that cocaine upregulated D1receptors density only after 30 min withdrawal, and this receptor density returned to normal levels after 1 day withdrawal. This dopaminergic receptor subtype is believed to have a role in the mediation of reinforcing effects of cocaine. According to Staley & Mash, [22], during chronic administration of cocaine, D1 receptor numbers in the olfactory tubercle, nucleus accumbens, ventral pallidum and substantia nigra were elevated, but within a couple of Table 2 Effects of cocaine repeated treatment and early withdrawal on cyclic nucleotide levels a Groups

cAMP (pmol/mg protein)

cGMP (pmol/mg protein)

Control Cocaine 20 mg/kg Cocaine 30 mg/kg

7.71 ^ 0.55 (12) 5.80 ^ 0.51 (12) b 5.49 ^ 0.26 (6) b

0.246 ^ 0.027 (16) 0.260 ^ 0.033 (9) 0.410 ^ 0.029 (6) b,c

a Animals were treated daily for 1 week with cocaine 20 and 30 mg/kg, i.p., and measurements were taken 1 day after the last injection. Data are reported as means ^ SEM for the number of experiments shown in parentheses. For statistical analyses, ANOVA and the Student–Newman–Keuls test as a post-hoc test were used. b,cP , 0:05 as compared to controls and cocaine 20 mg/kg, respectively.

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days returned to normal levels. However, in the striatum, the D1 receptor density decreases, and this effect remains up to 2 weeks after cocaine withdrawal. There are no data in the literature concerning possible alterations in the premotor cortex under the influence of cocaine. Leshner, [14] reported that D2 receptors may be involved in motor behaviors observed in cocaine addiction, since D2 agonists induced drug-seeking behaviors. Koob et al. [10], suggest that this observed effect of cocaine could be due to the increased amount of D2 receptors in the corpus striatum involved in motor behavior. Our results indicated that D2 receptors are increased in the premotor cortex after all periods of withdrawal studied, also suggesting a role of D2 receptors in this cerebral area in the reinforcing effects of cocaine. Sharkey et al. [20], observed that cocaine inhibits muscarinic receptors in rat heart and brain. Our work found that cocaine in premotor cortex produced an upregulation in muscarinic receptors with the 30 mg/kg dose in 1 day withdrawal, and both doses (20 and 30 mg/kg) in 5 days withdrawal, suggesting a kind of compensatory mechanism involving this receptor with cocaine withdrawal, since we saw no alterations in 30 min withdrawal. Sousa et al. [21], using striatum homogenates found that cocaine 5 and 10 mg/ kg produced a dose-related increase in muscarinic receptor density. Consolo et al. [3], using microdialysis to study acetylcholine transmission in nucleus accumbens, found that D1 receptor stimulates ACh release in the shell and core on rat nucleus accumbens. It is also known [13,24] that in dorsal striatum exists an inhibitory control on cholinergic interneurons by dopamine acting at the D2 receptors. In this work, we found that when D1 receptors density normalize (1 day withdrawal), muscarinic receptors starts to upregulate, while D2 receptors are upregulated in all withdrawal periods. Probably, there is also a kind of regulation of ACh release mediated by D1 and D2 receptors in this brain area. The concurrent influence of dopaminergic, opioid, and muscarinic neurotransmitter systems has been shown [18] in mediating some of the toxic effects of cocaine, including cocaine-induced lethality. Other data [18] indicated that, although 5-HT transporter and 5-HT2 receptor sites appear to be primary sites related to cocaine-induced convulsions, M1 and sigma receptors binding sites also play important modulatory roles in this response. It has been demonstrated [4] that cortical ACh release is greatly increased by cocaine, suggesting that cocaine stimulates cortically projecting cholinergic neurons. In addiction, earlier experiments [2] showed significant increases in cortical ChAT activity after cocaine administration. According to Miserendino and Nestler, [17], much of the current work on cocaine action on neurotransmitter receptors has focused on functional changes in dopamine release and dopamine receptors, however mechanisms beyond the receptor level might also be critically involved in cocaine effects, mainly related to the development and expression of behavioral sensitization. In this work, we observed that cAMP levels were decreased with both doses of cocaine 1

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day after the last injection. This can be related, at least in part, to the upregulation of D2 receptors, since the molecular mechanism of action of this receptor involves inhibition of adenilyl cyclase and decrease in cAMP levels. On the other hand, there was an increase in cGMP levels only with the higher cocaine dose. Kalivas & Duffy, [7], reported that systemic cocaine administration increases extracellular glutamate by indirect stimulation of D1 receptors in the VTA which indicates a potential role for the mechanism of sensitization-based psychopathologies. Moreover, behavioral sensitization to repeated psychostimulant administration in rats is prevented by injections of either D1 [1,23] or NMDA receptors antagonist in the VTA [6]. In the premotor cortex, it seems that D2-receptors play an important role on the regulation of glutamate release, because in this brain area there was an increase in D2 receptors only with the higher dose studied (1 day withdrawal), the same occurring with cGMP levels. Since glutamate interaction with NMDA receptor is the principal pathway for production of cGMP, we can speculate that D2-receptors might regulate glutamate release in the premotor cortex area. Most of the works in the literature have focused on changes remaining in the nucleus accumbens after withdrawal from cocaine administration. This work demonstrated that some changes after cocaine administration are also found in premotor cortex after cocaine discontinuation, regarding to dopaminergic D1, D2 and muscarinic receptors as well as cyclic nucleotides.

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