Pilocarpine-induced status epilepticus: Monoamine level, muscarinic and dopaminergic receptors alterations in striatum of young rats

Neuroscience Letters 383 (2005) 165–170

Pilocarpine-induced status epilepticus: Monoamine level, muscarinic and dopaminergic receptors alterations in striatum of young rats V.S. Nascimento, A.A. Oliveira, R.M. Freitas ∗ , F.C.F. Sousa, S.M.M. Vasconcelos, G.S.B. Viana, M.M.F. Fonteles Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceara, Rua Cel. Nunes de Melo 1127, Rua Frederico Severo 201, Ap 103, Bl 07, 60431-270 Fortaleza, CE, Brazil Received 11 February 2005; received in revised form 20 March 2005; accepted 2 April 2005

Abstract Behavioural changes, muscarinic and dopaminergic receptors density and levels of monoamines were measured in striatum of rats after pilocarpine-induced status epilepticus (SE). Wistar rats at the age of 21 days were treated with pilocarpine (400 mg/kg; subcutaneously) whilst the control group was treated with 0.9% saline (s.c.). Both groups were sacrificed 1 h following the treatment. SE induced a muscarinic receptor downregulation of 64% in pilocarpine group. This effect was also observed to be 57% in D1 and 32% in D2 . In the dissociation constant (Kd ) values in muscarinic and D1 receptor no alterations were verified. On the other hand, the Kd value for D2 was observed to increase 41%. High performance liquid chromatography determinations showed 63, 35, 77 and 64% decreases in dopamine, 3-methoxy-phenylacetic acid, serotonin and 5-hydroxyindoleacetic acid contents, respectively. The homovanilic acid level was verified to increase 119%. The noradrenaline content was unaltered. A direct evidence of monoamine levels alterations can be verified during seizure activity and receptor density changes appear to occur in an accentuated way in immature brain during the estabilishment of SE induced by pilocarpine. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Pilocarpine; Monoamines; Muscarinic receptor; Dopaminergic receptor; Striatum; Status epilepticus

Human temporal lobe epilepsy can be characterized by a permanent change in neurotransmitter systems and receptor densities. Rodent models of pilocarpine-induced temporal lobe epilepsy have provided information regarding behavioural and neurochemical characteristics associated with seizure activity [2,3]. The acute treatment with a high dose of pilocarpine, a muscarinic cholinergic agonist, results in behavioural changes, seizures and brain injury in young and adult rats [1–4]. In addition, the administration of pilocarpine in rodents results in status epilepticus (SE) and, 24 h following the treatment, mortality. [6]. Little is known about the relation between the effects of seizure and age. Therefore, we deemed it important to study the seizures and SE in order to elicit the different alterations in immature brain. Age-related susceptibility to pilocarpine treatment might occur due to differences in neural systems, ∗

Corresponding author. Tel.: +55 85 3274 6091; fax: +55 85 3274 6091. E-mail address: [email protected] (R.M. Freitas).

0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.04.006

which are essential for the development of seizure activity and which could explain the behavioural and neurochemical changes in young and adult animals [7]. Cerebral monoaminergic neurons found in adult rats can be detected during the embryonic period and these neurons could explain the seizure process development in young rats [8,9]. The seizures induced by pilocarpine can be blocked by prior atropine treatment, showing the involvement of the cholinergic system in seizures and SE [7]. It is not established how other neurotransmitters such as noradrenaline, dopamine, serotonin, glutamate and ␥-aminobutyric acid (GABA) play a role in the maintenance and/or propagation of seizures [3,10,11], SE and development of cerebral changes in young and adult animals [12–14]. Thus, it is important to study the monoamine alterations related to pilocarpineinduced seizure model in young animals. In order to detect the involvement of cholinergic and dopaminergic systems in pilocarpine model in 21-dayold rats, we have studied behavioural alterations, striatal

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(M1 + M2 ) muscarinic and (D1 and D2 ) dopaminergic receptor densities and levels of neurotransmitters after SE induced by pilocarpine. Male Wistar rats (21 days old; 40–50 g) were used. Animals were housed in cages with free access to food and water before being treated with 0.9% saline (s.c.; control group) or pilocarpine (400 mg/kg; s.c.). All of the animals were kept with standard light–dark cycle (lights on at 07.00 a.m.). The experiments were performed according to the Guide for the care and use of laboratory of the US of Department of Health and Human Services, Washington, DC, 1985. Pilocarpine hydrochloride was purchased from ICN (CA, USA). Atropine sulfate was purchased from Sigma (MO, USA). Radioligand: [3 H]-N-methylscopolamine methyl chloride ([3 H]-NMS, 85Ci/mmol) was provided by Amersham Pharmacia Biotech (NJ, USA). The radioligands: [3 H]SCH 23390 ([3 H]-spiro, 87 Ci/mmol) and [3 H]-spiroperidol ([3 H]-spiro, 114 Ci/mmol) were provided by Amersham Pharmacia Biotech (NJ, USA) and New England Nuclear (NJ, USA), respectively. All other drugs were of analytical grade. Control animals received 0.9% saline subcutaneously (s.c.) (control group; n = 37) and in the experimental group, the animals received a subcutaneous injection of pilocarpine hydrochloride (400 mg/kg, s.c., n = 60). Behavioural changes were observed for 1 h. The parameters observed were: number of peripheral cholinergic signs, tremors, stereotyped movements, seizures, SE and mortality. The SE was defined as continuous seizures for a period longer than 30 min. SE was induced by method of Turski et al. [36]. Mortality was recorded for 1 h after pilocarpine-induced SE. The pilocarpine group (n = 19) was constituted by those rats that presented seizures, SE for a period longer than 30 min and that did not die for 1 h of observation. The pilocarpine and control groups were sacrificed by decapitation 1 h after the treatment and their brains were dissected on ice to remove the striatum for determination of monoamine levels and muscarinic (M1 + M2 ) and dopaminergic (D1 - and D2 like) receptor-binding assays. In both binding assays, receptor density (Bmax ) and dissociation constant (Kd ) were determined and expressed as fentomoles per milligram of protein (fmoles) and nM, respectively. Animals were decapitated 1 h after the administration of pilocarpine (pilocarpine group, n = 4) or 0.9% saline (control group, n = 8), and immediately their brains were dissected. The striatum was used to prepare 10% homogenates (10%, w/v). Muscarinic receptors were measured using [3 H]-N-methylscopolamine ([3 H]-NMS), according to Dombrowski et al. [16]. Total homogenates corresponding to 50–100 ␮g protein were prepared in a 150 mM sodium phosphate buffer, pH 7.4, containing 0.119–5.95 nM of [3 H]-NMS in a final volume of 0.2 mL. After incubation at 37 ◦ C 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 an oven at 60 ◦ C, 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 nonspecific binding performed in the presence of atropine (12.5 ␮M), and results are reported as fentomoles per milligram of protein. Protein was determined by Lowry et al. [25] using bovine serum albumin as standard. Densities of D1 - and D2 -like receptors were determined according to the methods described by Meltzer et al. [29] and Kessler et al. [23]. In the case of D1 receptors, the specific ligand [3 H]-SCH 23390 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 [3 H]-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, [3 H]-spiroperidol was utilized. Total homogenates were incubated in a 50 mM Tris–HCl buffer, pH 7.4, containing 5 ␮M mianserin for blocking serotonergic receptors and 0.102–7.14 nM [3 H]-spiroperidol in a final volume of 0.2 mL. In both cases (D1 and D2 receptor assays), specific binding was defined as total minus nonspecifc binding carried out in the presence of 10 ␮M butaclamol. After incubation at 37 ◦ C for 60 min, experiments proceeded as described above for the muscarinic binding. The striatum of control animal (n = 13) and pilocarpine group (n = 7) were used for the preparation of a 10% homogenate (10%, w/v). Brain tissue samples were sonicated in 0.5–1 mL of 0.1 M perchloric acid (HClO4 , Qeel, SP, Brazil) for 30 s and centrifuged for 15 min at 26.000 × g, at 4 ◦ C. Then, a 20 ␮l supernatant aliquot was injected directly into the high performance liquid chromatograph (HPLC) column. For the monoamines analyses, a CLC-ODS(M) Shimadzu column was used. The mobile phase was 0.163 M citric acid (Vetec, RJ, Brazil), pH 3.0, containing 0.02 mM of ethylenediaminetetraacetic acid (EDTA, Vetec, RJ, Brazil), with 0.69 mM sodium octanesulfonic acid (SOS-Sigma, St. Louis, MO, USA), as ion pairing reagent, 4% (v/v) acetonitrile (Carlo Erba Reagenti, MI, Italy) and 1.7% (v/v) tetrahydrofurane (Sigma). Noradrenaline (NE), dopamine (DA), 4-hydroxy-3-methoxy-phenylacetic acid (DOPAC), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and homovanilic acid (HVA) were electrochemically detected using an amperometric detector (Model L-ECD-6A; Shimadzu Corp., Japan) by oxidation on a glassy carbon electrode at 0.85 V relative to a Ag–AgCl reference electrode. Amounts of neurotransmitters and metabolites in the supernatants were calculated by comparing their peak heights with that of standards determined at the same day. Results were expressed in ng/g wet tissue (Fig. 1). Differences in experimental groups were determined by analysis of variance (ANOVA). The Student–Newman–Keuls test was used for multiple comparison of means of two groups

V.S. Nascimento et al. / Neuroscience Letters 383 (2005) 165–170

Fig. 1. Determination of monoamines and metabolites levels in striatum after pilocarpine-induced status epilepticus. Male rats (250–280 g, 2 months old) were treated with a single dose of pilocarpine (400 mg/kg, s.c.; n = 7) and the control group with 0.9% saline (n = 13). Animals were submitted to a 1 h observation and afterwards sacrificed. Each bar represents mean ± S.E.M. of the number of animals shown in parentheses. a p < 0.05 as compared to control animals (ANOVA) and the differences in experimental groups were determined by Student–Newman–Keuls test. Dopamine (DA) and its metabolites 4-hydroxy-3-methoxy-phenylacetic acid (DOPAC) and homovanilic acid (HVA). Noradrenaline (NE), serotonin (5-HT) and its metabolite 5hydroxyindoleacetic acid (5-HIAA). Table 1 Behavioural alterations in 21-day-old rats after treatment with pilocarpine Groups

Behavioural alterations (%) PCS

21-day-old rats Control group 00 Pilocarpine group 100

Tremors SM

Seizures SE Mortality

00 71

00 86

00 100

00 86

00 68

Male rats (250–280 g, 2-month-old) were treated with a single dose of pilocarpine (400 mg/kg, s.c.; n = 60) and the control group with 0.9% saline (n = 60). Animals were submitted to a 1 h observation and afterwards sacrificed. In the control group no alterations were noticed in the observed parameters. The values represent behavioural alterations in percentages. PCS: peripheral cholinergic signs; SM: stereotyped movements; SE: status epilepticus.

of data whose differences were considered statistically significant at p < 0.05. As shown in Table 1, within 5–10 min after pilocarpine administration (400 mg/kg), all of the 21-day-old animals presented peripheral cholinergic signs (miosis, piloerection,

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chromodacriorrhea, diarrhea) and stereotyped movements (continuous sniffing, paw licking and rearing). The rate of 71% of young (43/60) animals presented tremors. Eighty-six percent of the animals presented seizures which progressed to SE (52 out of 60 animals). The mortality rate was 68% during the behavioural study. In the control group, no behavioural alterations were noticed (Table 1). Data from (3 H)-NMS, (3 H)-SCH 23390 and (3 H)spiroperidol bindings in striatum of 21-day-old rats are presented in Table 2. The total number of (3 H)-NMS, (3 H)-SCH 23390 and (3 H)-spiroperidol-binding sites was obtained from the striatum membranes. The rats showed a (M1 + M2 ) muscarinic receptor downregulation of 65% in the striatum after administration of pilocarpine [T(9) = 8.967; p < 0.0001] as compared to control. In the pilocarpine group it was noticed a significant decrease of 57% in D1 dopaminergic receptor [T(10) = 5.021; p < 0.0005] and a significant decrease of 32% in D2 receptor density as compared to control group [T(10)=3.838; p < 0.0033]. In the Kd values of muscarinic receptor no alterations were detected [T(14) = 1.494; p = n.s.]. The D1 dopaminergic receptor remained unaltered [T(14) = 1.594; p = n.s.]. On the other hand, it was observed a significant increase of 41% [T(8) = 5.917; p < 0.0004] in Kd values from D2 (Table 2). The treatment with a single dose of pilocarpine produced significant decreases of 63, 35, 77 and 64% in DA [T(18) = 3.647; p < 0.0018]; DOPAC [T(22) = 2.120; p < 0.0455]; 5-HT [T(17) = 2.720; p < 0.0146] and 5-HIAA [T(15) = 6.248, p < 0.0001], respectively. In contrast, the HVA concentration was increased of 119% [T(9) = 8.211; p < 0.0001]. In the NE content no alterations were detected [T(18) = 0.2174; p = n.s.] (Fig. 1). Several neurotransmitter systems are modified during the brain development and may be implicated in the mechanism of pilocarpine-induced seizures and SE. In immature brain, it was observed a decrease in muscarinic receptor density in the striatum and a higher sensitivity of the M1 cholinergic receptor to pilocarpine [18]. Muscarinic receptors reach adult levels between 21 and 40 days of age. However, other studies detected muscarinic receptor numbers close to adult levels at the age of 12–14 days. The presence of those receptors in 21-day-old rats could contribute to the installation seizure activity induced by pilocarpine [20–22].

Table 2 Muscarinic and dopaminergic receptors density in striatum of young rat after pilocarpine-induced status epilepticus Groups

21-day-old rats Control group Pilocarpine group

M1 + M2

D1 -Like

D2 -like

Bmax (fmol/mg protein)

Kd (nM)

Bmax (fmol/mg protein)

Kd (nM)

Bmax (fmol/mg protein)

Kd (nM)

271.7 ± 15.68 (8) 93.75 ± 11.01 (4)a

1.33 ± 0.08 (8) 1.26 ± 0.09 (4)

311.9 ± 23.5 (8) 134.1 ± 14.4 (4)a

1.35 ± 0.08 (8) 1.37 ± 0.21 (4)

373.4 ± 19.9 (8) 253.0 ± 18.3 (4)a

1.67 ± 0.08 (8) 2.35 ± 0.07 (4)a

Male rats (250–280 g, 2-month-old) were treated with a single dose of pilocarpine (400 mg/kg, s.c.; n = 4) and the control group with 0.9% saline (n = 8). Animals were submitted to a 1 h observation and afterwards sacrificed. Each bar represents mean ± S.E.M., of the number of animals shown in parentheses. a p < 0.05 as compared to control animals (ANOVA) and the differences in experimental groups were determined by Student–Newman–Keuls test.

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Other researches have shown that in young and adult rats, cholinergic activation is essential to initiate the convulsive process because atropine can inhibit seizures and brain injury induced by pilocarpine [15,37]. An earlier study suggested that the M1 muscarinic receptor play a role in mediating the initiation of pilocarpine-induced seizures. Those data also suggest that, in this condition, M2 receptor may be acting in the attempt to attenuate the neuronal activity mediated by the M1 receptor during seizures and SE [24,26,27]. Ontogenic studies have shown an age-dependent differences in the neurochemical changes in animals treated with pilocarpine that could be justified by the existence of complex alterations in mature and immature brains regarding epilepsy and also due to the presence of different neurotransmitter systems [10,18,20]. In the present work, a striatal muscarinic receptor downregulation in pilocarpine group was verified, which is similar to what happens in adult animals [18]. Nevertheless, after seizures and SE no changes in the receptor sensitivity were detected. This area may be considered, therefore, important and essential for development of seizures and SE, as previously reported by Viana et al. [39] and Freitas et al. [19]. The activation of muscarinic receptor is the first step for seizure activity and other systems (noradrenergic, serotonergic, dopaminergic, GABAergic and glutamatergic) appear to mediate propagation and/or maintenance of seizures [17,24,28,30]. The relation between muscarinic and dopaminergic receptors during the seizures and SE induced by pilocarpine is not clear. However, previous studies have demonstrated that receptor stimulation of D1 , but not D2 , reduces the threshold for pilocarpine-induced epileptic activity in rats [5,30–32]. It is known that dopamine exert pro- and anticonvulsive effects by D1 and D2 receptor stimulation, respectively [20,23,33]. In our study, pilocarpine produced a decrease of D1 and D2 dopaminergic receptors densities. None alteration for Kd value was detected in D1 receptor, suggesting that the ligand affinity for this receptor does not change in this area during the first hour of the acute phase of seizures. On the other hand, in D2 , Kd value presented a 41% increase, indicating a lower ligand affinity for D2 receptor. Freitas et al. [19] demonstrated a decrease in Kd value and absence of alterations in receptor D2 in striatum of adult rats after seizures and SE induced by pilocarpine. Our results suggest that dopaminergic receptors are directly or indirectly modified after cholinergic system activation in different ages. There is evidence that the effects of acetylcholine on other modulatory neurotransmitters such as noradrenaline, dopamine, GABA, glutamate and serotonin, may be involved in cholinergic seizures and SE [34,35]. It has been observed that during the subsequent pilocarpine-induced limbic seizures, extracellular glutamate, GABA [10] and DA [6] levels in hippocampus were increased, suggesting neuronal vesicular release. However, more studies including other brain areas should be carried out to identify the importance of monoamines in the seizure process [36].

Significant differences in monoamine contents were evident in striatum during development of seizures and SE induced by pilocarpine [18,20] and in human epilepsy [37]. Concomitantly, the decrease of DA level, associated with an increase in the metabolization rate, was noticed in young rats [18]. Our work showed that the levels of DA and its metabolite DOPAC and of 5-HT, decreased in striatum of young rat, suggesting a function for these monoamines in seizures induced by pilocarpine. Earlier studies with adult animals have demonstrated similar effects in levels of DA, DOPAC and 5-HT but it is important to enhance that the reduction in DA and 5-HT contents are greater in young animals [18]. Simultaneously to the dopamine reduction, a downregulation in D1 and D2 was observed, but this alteration was greater in D1 than in D2 . These findings suggest that a possible physiological response may occur to reduce of dopamine affinity for D2 receptor measured by the increase of its Kd . There is evidence of an inhibitory role of dopamine mediated by D2 receptor in depressing the hyperexcitability of hippocampal and striatal neurons involved in the mechanism of SE and in development of neurochemical changes [16,37,38]. It seems that in the pilocarpine model of seizures, D2 agonists exert a powerful anticonvulsive effect which is mediated by this receptor in the striatum, but not in the substantia nigra [33,34,39]. The NE content may not be important during the seizure process in immature striatum once no alterations in this monoamine were noticed. The simultaneous decreases in 5HT level and its metabolization rate are consitent with the hypothesis that a decrease in the synthesis and release rates of 5-HT in young rats can occur during SE. With regard to 5-HIAA content, it appears to be modified in function of age since it did not present any changes in mature brain [18] while in immature one it was observed a reduction. In the HVA level, dopamine metabolite, an increase was verified in striatum of adult rats [18] whilst our results demonstrated an important increase of this metabolite. It was observed that during the subsequent pilocarpine-induced SE, monoamine levels, muscarinic and dopaminergic receptors content in striatum were altered, suggesting possible neuronal changes after the administration of pilocarpine. In conclusion, our findings show that acute compensatory physiological alterations in monoamine concentration, density and dissociation constant values of different receptors studied are important and occur during the epileptic phenomenon. The consequences of this changes during the acute phase of limbic seizures in immature brain contribute for the establishment of SE in this epilepsy model.

Acknowledgments This work had financial support from the Brazilian National Research Council (CNPq). R.M.F. and V.S.N. are fellows from CNPq. A.A.O. is fellow from CAPES.

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The technical assistance of Maria Vilani Rodrigues Bastos and Stˆenio Gardel Maia are gratefully acknowledged.

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