Modifications in muscarinic, dopaminergic and serotonergic receptors concentrations in the hippocampus and striatum of epileptic rats

Life Sciences 78 (2005) 253 – 258 www.elsevier.com/locate/lifescie

Modifications in muscarinic, dopaminergic and serotonergic receptors concentrations in the hippocampus and striatum of epileptic rats Rivelilson Mendes de Freitas b,*, Lissiana M.V. Aguiar b, Silvaˆnia M.M. Vasconcelos b, Francisca C.F. Sousa b, Glauce S.B. Viana b, Marta M.F. Fonteles a,b a

Department of Pharmacy, Laboratory of Neuropharmacology, School of Medicine, Federal University of Ceara´, Rua Cel. Nunes de Melo 1127, Fortaleza 60431-970, CE, Brazil b Department of Physiology and Pharmacology, Laboratory of Neuropharmacology, School of Medicine, Federal University of Ceara´, Rua Cel. Nunes de Melo 1127, Fortaleza 60431-970, CE, Brazil Received 8 October 2004; accepted 19 April 2005

Abstract The present study was undertaken in order to investigate the muscarinic (M1), dopaminergic (D1 and D2) and serotonergic (5-HT2) receptors densities in hippocampus and striatum of Wistar rats after status epilepticus (SE) induced by pilocarpine. The control group was treated with 0.9% saline. An other group of rats received pilocarpine (400 mg/kg, s.c.) and both groups were sacrificed 1 h after treatment. The results have shown that pilocarpine administration and resulting SE produced a downregulation of M1 receptor in hippocampus (41%) and striatum (51%) and an increase in the dissociation constant (K d) values in striatum (42%) alone. In both areas the 5-HT2 receptor density remained unaltered, but a reduction (50%) and an increase (15%) in the K d values were detected in striatum and hippocampus, respectively. D1 and D2 receptor densities in hippocampus and striatum remained unaltered meanwhile K d values for D1 receptor declined significantly, 33% in hippocampus and 26% in striatum. Similarly, K d values for D2 decreased 55% in hippocampus and 52% in striatum. From the preceding results, it is clear that there is a possible relation between alterations in muscarinic receptor density and others systems studied as well as they suggest that changes in dissociation constant can be responsible for the establishment of pilocarpine-induced SE by altering the affinity of neurotransmitters such as acetylcholine, dopamine and serotonine. D 2005 Elsevier Inc. All rights reserved. Keywords: Muscarinic; Dopaminergic; Serotonergic; Status epilepticus; Pilocarpine

Introduction Apart from stroke, epilepsy is the most common neurological disorder with 0.5% of prevalence (Browne and Holmes, 2001). The pilocarpine treatment in rats is an experimental model largely used to study the pathophysiology of seizures and status epilepticus (SE) (Kulkarni and George, 1997; Freitas et al., 2003a). This seizure model can demonstrate the pro-convulsive and damaging effects of pilocarpine (Teskey et al., 2004). Pilocarpine model produces a sequence of behavioural changes such as peripheral cholinergic signs, tremors, staring * Corresponding author. Department of Physiology and Pharmacology, Federal University of Ceara´, Rua Frederico Severo, 201, Ap 103 Bl 07, Cep: 60830-310, Fortaleza, CE, Brazil. Tel./fax: +55 85 3274 6091. E-mail address: [email protected] (R.M. de Freitas). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.04.045

spells, facial automatisms and motor seizures, which develop progressively within 1 –2 h into a long-lasting SE (Cavalheiro et al., 1994; Freitas et al., 2003b) and shows electroencephalographic alterations that are similar to those from the human temporal lobe epilepsy (Turski et al., 1983; Marinho et al., 1998). This epilepsy model is characterized by an acute phase in which animals present a long-lasting SE (1– 2 days), by a seizure-free period (silent; 4 – 44 days, mean of 15 days) and by a chronic phase when spontaneous recurrent seizures (SRS) occur (Clifford et al., 1987; Cavalheiro et al., 1994). Several studies have been performed after the convulsive process (Erakovic et al., 2000; Costa-Lotufo et al., 2002; Freitas et al., 2003b) in order to clarify the biochemical changes that affect different neurotransmitters like adenosine, norepinephrine, dopamine, serotonin, glutamate, g-aminobutyric acid (GABA) (Khan et al., 2000; Hwang et al., 2004), as

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well as the muscarinic and dopaminergic receptor densities in hippocampus (Marinho et al., 1998) and striatum (Freitas et al., 2003a). It is likely that these systems may play a role in the SE process, but when and how it happens has yet to be determined. By using this epilepsy model, we can observe a toxicity induced by an initial cholinergic phase, and a distinct noncholinergic phase occurs through of an excessive glutamate and aspartate releases induced by SE (Fritschy et al., 1999). It is currently hypothesized that some pathological processes such as SE can occur under an increased glutamate release which activates a higher number of glutamatergic receptors, leading to neuronal necrosis by elevating [Ca+ 2]i and activating potentially destructive Ca+ 2-dependent enzymes (Mc Donald et al., 1991; Fritschy et al., 1999). The increase of glutamatergic receptor densities has been observed during the acute phase of seizures as previously described by Freitas et al. (2004) suggesting that these receptors present an important role in the establishment and propagation of SE. The role of cholinergic and dopaminergic receptors in the process of seizures is not thoroughly clear and several authors have suggested that the activation of cholinergic receptors is necessary in the seizures initiation (Turski et al., 1989), and that other systems (noradrenergic, serotonergic, GABAergic and glutamatergic) may be related to the seizures and SE propagation (Turski et al., 1987; Teskey et al., 2004). Therefore, it is important to investigate different receptor densities after SE induced by pilocarpine. Considering that receptor density alterations can be involved in SE mechanisms, this work was performed in order to measure the (M1) muscarinic, (D1 and D2) dopaminergic and (5-HT2) serotonergic receptors concentrations in hippocampus and striatum of adult rats after SE induced by pilocarpine.

Experimental protocol Control animals received 0.9% saline subcutaneously (s.c.) (Control group; n = 32) and in the experimental group the animals were treated with a single dose of pilocarpine hydrochloride (400 mg/kg, s.c., n = 32). Behavioural changes were observed for 1 h. The parameters assessed were: number of peripheral cholinergic signs, tremors, stereotyped movements, seizures, SE. The SE was defined as continuous seizures for a period longer than 30 min. SE was induced by method of Turski et al., 1983. The pilocarpine group (n = 16) was constituted by those rats that presented seizures, SE for a period longer than 30 min and that did not died after 1 h of observation from the treatment. Then, both pilocarpine and control group were sacrificed by decapitation 1 h after the treatment and their brains were dissected on ice to remove the hippocampus and striatum for muscarinic, dopaminergic and serotonergic receptor densities determinations. M1 muscarinic receptor densities determinations

Male Wistar rats (250 – 280 g; 2-month-old) were used. Animals were housed in cages with free access to food and water and were kept with standard light – dark cycle (lights on at 07:00 hours a.m.). The experiments were performed according to the Guide for the care and use of laboratory of the US Department of Health and Human Services, Washington, DC (1985).

Receptor density values were measured by binding assays with 10% homogenates prepared (w\v) in 150 mM sodium phosphate buffer, pH 7.4, using [3H]-NMS as the ligand (Dombrowski et al., 1983). This non-selective ligand binding to all subtypes of muscarinic receptors was carried out. The M1-like receptors assay was performed with [3H]-NMS in the presence of 100 AM carbachol for blocking M2 sites. Total homogenates (0.05 –0.10 mg protein) were incubated with 2.38 nM of [3H]-NMS. Non-specific binding was determined in the presence of atropine (12.5 mM). The mixture was incubated for 30 min at 37 -C and the final volume was 0.2 ml. The incubation time was 30 min at 37 -C, and the final volume was 0.2 ml. After all incubation period the samples were filtered through Whatman GF/B filters. Filters were washed three times with 4 ml of ice cold saline, dried and added to 3 ml of a toluene-based scintillation cocktail. Radioactivity was measured with a Beckman scintillation counter model LS 6500 and results expressed as fentomoles per milligram of protein (fmol/ mg protein) (B max) and nanomolar (nM) (K d). Protein was determined using bovine serum albumin as the standard (Lowry et al., 1951). B max and K d were calculated by the Instat Program for PC computers.

Drugs

5-HT2 serotonergic receptor densities determinations

Pilocarpine hydrochloride was purchased from ICN (CA, USA) and Atropine sulfate from Sigma (MO, USA). Radioligands, [3H] N-methylscopolamine methyl chloride ([3H] NMS, 85 Ci/mmol) and [3H]-spiperone (114 Ci/mmol) were provided by Amersham Pharmacia Biotech (NJ, USA) and New England Nuclear (USA), respectively. The radioligands [3H]-SCH-23390 (109 Ci/mmol, Amersham, Uppsala, Sweden) and [3H]-spiroperidol ([3H]-spiro, 114 Ci/mmol; and New England Nuclear, USA) were provided. All other drugs were from analytical grade.

Tissues were homogenized in 2 ml of 0.05 M Tris – HCl pH 7.4. The homogenate 10% (w/v) prepared was centrifuged for 15 min at 20,000 g at 4 -C. The supernatant was discarded and the pellet was washed three times with the same volume of 0.05 M Tris – HCl. The final pellet was resuspended in 0.3 ml of the same buffer for subsequent determination of [3H]spiperone binding. As described in 2.6, in D2-like receptors determination, [3H]-spiperone was used to determine 5-HT2 receptors, according to the method described by Peroutka and Snyder (1979).

Material and methods Animals

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Table 1 Scatchard analyses of serotonergic and muscarinic binding in hippocampus of adults rats after pilocarpine-induced SE Treatment

Hippocampus 5-HT2-like B max (fmol mg

Control group Pilocarpine group

M1-like 1

of protein)

158.21 T19.94 (8) 168.18 T 5.38 (4)

1

K d (nM)

B max (fmol mg

1.17 T 0.01 (8) 1.34 T 0.01 (4)a

115.76 T 7.46 (8) 68.00 T 2.45 (4)a

of protein)

K d (nM) 1.15 T 0.02 (8) 1.15 T 0.03 (4)

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

The assay consisted of the membrane (0.3 –0.5 mg protein) incubated in 50 mM Tris buffer (pH 7.4) consisting of 0.1% ascorbic acid, 120 mM of NaCl, 5 mM of KCl, 2 mM of CaCl2, in the presence of 4.72 nM [3H]-spiperone and dopamine 100 AM to block D2-like receptors. Nonspecific binding was defined by the addition of cyproheptadine 100 AM. The incubation time was 30 min at 37 -C, and the final volume was 0.2 ml. The procedure with the samples was performed as described to muscarinic receptor in 2.4. Dopaminergic receptor densities determinations Dopaminergic D1- and D2-like receptors were assayed. The methods of Meltzer et al. (1989) for D1-like receptor and of Kessler et al. (1991) for D2-like receptor were used. Control and pilocarpine groups were decapitated 1 h after treatment, and the hippocampus and striatum were dissected on ice for the preparation of 10% homogenates (w/v). The homogenates containing 60– 120 Ag of protein were incubated in 50 AM Tris –HCl buffer, pH 7.4, in the presence of 10 AM mianserin (Organon, Sa˜o Paulo, Brazil), to block serotonergic receptors, in the case of D2-like receptor binding assay. Several ligand concentrations of [3H]-SCH-23390 from 0.135 to 6.75 nM, or [3H]-spiroperidol from 0.09 to 4.76 nM, were used for D1- and D2-like receptors, respectively, in a final volume of 0.2 ml. Reaction media were incubated at 37 -C for 60 min, and the reaction was terminated by filtration through Whatman GF/B filter paper on a cell harvester apparatus from Brandel (Gaitjersburg, MD, USA). Filters were washed 5 times with cold saline, dried in the oven for 2 h at 60 -C, and placed in vials containing 3 ml of a toluene (Vetec) based scintillation cocktail. Radioactivity was measured with a Beckman LS 100 counter with 61.6% efficiency. Specific binding was calculated as the total minus nonspecific binding in the presence of 5 AM

dopamine (Sigma, St. Louis, MO, USA) containing 1 mg/ml ascorbic acid in order to prevent dopamine degradation. The procedure with the samples was performed as described to muscarinic receptor in 2.4. Statistic analyses Student –Newman – Keuls test was used for multiple comparison of means of two groups of data whose differences were considered statistically significant at p < 0.05. Differences in experimental groups were determined by analyses of variance (ANOVA) two-tailed. Results Behavioural alterations of adult rats after treatment with pilocarpine Immediately after pilocarpine administration, animals persistently presented behavioural changes, including initial akinesia, ataxic lurching, peripheral cholinergic signs (miosis, piloerection, chromodacriorrhea, diarrhea and masticatory automatisms), stereotyped movements (continuous sniffing, paw licking, rearing and wet dog shakes that persisted for 10– 15 min), clonic movements of forelimbs, head bobbing and tremors. These behavioural changes progressed to motor limbic seizures as previously described by Turski et al. (1983). Limbic seizures lasted for 30 – 50 min evolving to SE in rats. No case of fatality was observed in any of the experiments. Serotonergic and muscarinic receptor densities in hippocampus and striatum of adult rats pilocarpine-induced SE The total number of [3H]-NMS and [3H]-spiperone binding sites were obtained from the hippocampus and striatum

Table 2 Scatchard analyses of serotonergic and muscarinic binding in striatum of adults rats after pilocarpine-induced SE Groups

Striatum 5-HT2-like B max (fmol mg

Control group Pilocarpine group

M1-like 1

235.74 T 19.76 (8) 233.06 T 1.55 (4)

of protein)

1

K d (nM)

B max (fmol mg

3.54 T 0.37 (8) 1.77 T 0.13 (4)a

199.43 T 18.69 (8) 98.00 T 1.29 (4)a

of protein)

K d (nM) 1.29 T 0.09 (8) 1.83 T 0.05 (4)a

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

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Table 3 Scatchard analyses of dopaminergic binding in hippocampus of adults rats after pilocarpine-induced SE Treatment

Hippocampus D1-like B max (fmol mg

Control group Pilocarpine group

D2-like 1

of protein)

319.00 T 11.65 (8) 312.00 T 14.02 (4)

K d (nM)

B max (fmol mg

1.77 T 0.02 (8) 1.19 T 0.01 (4)a

364.04 T 7.77 (8) 386.75 T 3.07 (4)

1

of protein)

K d (nM) 3.19 T 0.48 (8) 1.52 T 0.03 (4)a

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

membranes of 2-month-old rats after pilocarpine-induced SE (Tables 1 and 2), respectively. The hippocampal M1 receptor density had a 41% decreased [T(10) = 4.408; p < 0.0013], but 5-HT2 density was not altered [T(10) = 0.3448; p = N.S.] (Table 1). However, a significant 15% increase in 5-HT2 K d values [T(10) = 6.962, p < 0.0001] was detected. M1 K d values [T(10) = 0.1195, p = N.S.] in this area remained the same (Table 1). Striatal scatchard analyses (Table 2) revealed that 5-HT2-like receptor density (B max) [T(10) = 0.07543; p = N.S.] remained unaltered, but the K d values decreased 50% [T(10) = 3.302, p < 0.0080]. On the other hand, M1-like receptor density values showed decrease of 51% [T(10) = 11.859; p < 0.0001], and a 42% increase in the K d values [T(10) = 4.462, p < 0.0012] was noticed as compared to control group. Dopaminergic receptor densities in hippocampus and striatum of adult rats pilocarpine-induced SE The total number of [3H]-SCH 23390 and [3H]-spiroperidol binding sites were obtained from the hippocampus and striatum membranes of 2-month-old animals after SE (Tables 3 and 4), respectively. After SE, D1 dopaminergic receptor in the hippocampus [T(10) = 0.3524; p = N.S.] and striatum [T(10) = 0.06667; p = N.S.] remained unaltered. Nevertheless, the K d values measurements remarkably declined 26% and 33% in the hippocampus [T(10) = 19.463, p < 0.0001] and striatum [T(10) = 3.607, p < 0.0048], respectively. In addition, D2 remained unaltered in the hippocampus [T(10) = 1.982; p = N.S.] and striatum [T(14) = 1.404; p = N.S.], but the K d values showed reductions of 55% and 52% in the striatum [T (8) = 2.920, p < 0.0193] and hippocampus [T(10) = 14.484, p < 0.0001], respectively.

Discussion Hippocampal and striatal M1 receptors can be directly involved in the convulsive process during and after SE in adult rats. However, the dissociation constant (K d) values were measured in the striatum indicating a reduction in ligand affinity (acetylcholine) in this area during SE and seizures. The M1 donwregulation observed in this areas is an immediate compensatory mechanism due to the increase in acetylcholine concentration produced by pilocarpine during the acute phase of seizures. The present results have shown that the 5-HT2 receptor density was not altered in any of the areas considered. Nevertheless, these findings have interestingly demonstrated a decrease and an increase in 5-HT2 receptor K d values in striatum and hippocampus after SE, respectively, suggesting that affinity of 5-HT for 5-HT2 receptor increase in striatum and decrease in hippocampus, under this conditions. Serotonergic receptor (5-HT2) can mediate the opposite function of the M1receptor in the regulation of epileptic activity. M1 receptor stimulation after administration of pilocarpine induces epileptic activity in adult and young rats. This effect is prevented by muscarinic receptor blockade (Marinho et al., 1997). Conceivably, M1 receptor can be located in some brain structures that control the spread of the convulsive activity, which might be facilitated by 5-HT2 receptor stimulation in other areas. Nevertheless, the role of 5-HT in the development of seizure activity remains an unresolved issue. The serotonin can exert convulsive and anticonvulsive effects mediated by 5-HT2and 5-HT1 receptors stimulation, respectively (O’Dell et al., 2000). The activation of serotonergic system by 5-HT2 receptor in hippocampus and striatum can facilitate the seizures induced by excessive muscarinic stimulation of the limbic system areas.

Table 4 Scatchard analyses of dopaminergic binding in striatum of adults rats after pilocarpine-induced SE Groups

Striatum D1-like B max (fmol mg

Control group Pilocarpine group

D2-like 1

346.19 T 21.40 (8) 348.61 T 28.43 (4)

of protein)

K d (nM)

B max (fmol mg

1.65 T 0.08 (8) 1.22 T 0.02 (4)a

378.4 T 23.4 (8) 316.9 T 25.59 (4)

1

of protein)

K d (nM) 3.19 T 0.48 (8) 1.45 T 0.08 (4)a

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

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It is evident that the effects of acetylcholine on other modulatory neurotransmitters such as glutamate, norepinephrine, dopamine and serotonin may be involved direct and/or indirectly in cholinergic seizures (Hirsch et al., 1992; Hwang et al., 2004; Teskey et al., 2004). Our data support the hypothesis that a decrease in M1 muscarinic receptor density as well as the unaltered 5-HT2 serotonergic receptor density may be involved in the changes of neuronal function followed by seizures and SE induced by pilocarpine. In addition, there were no alterations in D1 and D2 receptors, suggesting that an indirect involvement of dopaminergic system in the SE mechanisms may exist. Dopaminergic receptors mediate opposite functions in the regulation of seizures. Thus, D1, but not D2, receptor stimulation reduces the threshold for pilocarpine induced epileptic activity in rats. This effect is prevented by D1receptor blockade. Hippocampal D1 receptors control the installation of seizures, which might be inhibited by D2 receptor stimulation (Alam and Starr, 1993). Dopaminergic receptors K d values had significant decreases in both cerebral areas studied. Our results suggest that during SE an increase in the brain dopaminergic ligand affinity for D1 and D2 receptors occur and facilitate the propagation and maintenance of seizures. Different receptors, neuronal damage and neurochemical changes have been implicated in the mechanism of pilocarpineinduced seizures (Persinger et al., 1993; Raza et al., 2001; Riban et al., 2004). Activation of M1 muscarinic receptor consists in the first step for seizure activity and other systems appear to mediate seizure mechanisms (Marinho et al., 1998). Previous results have shown a downregulation of GABAergic receptors in the different cerebral areas of adult rats during pilocarpine-induced SE (Freitas et al., 2004). The GABA, the principal inhibitory neurotransmitter in the cortex cerebral, sustains the inhibitory tone that counterbalances neuronal excitation (Fritschy et al., 1999). When this balance is perturbed, seizures may occur (Treiman, 2001). Glutamatergic receptor densities were augmented in the hippocampus, striatum and frontal cortex, suggesting that this effect in these receptors can be required during SE (Raza et al., 2001; Freitas et al., 2004). It is known that GABA and glutamate can exert anti- and pro-convulsive effects in the seizures and SE induced by pilocarpine, respectively (Costa-Lotufo et al., 2002). Different alterations in the receptor densities in hippocampus and striatum after subsequent pilocarpine-induced SE were observed, suggesting that a possible relation among the receptors is essential for the establishment of epileptic focus. However, the relation between SE and receptor density alterations has always been easier to propose rather than to demonstrate and more studies including area others, neurotransmitters and different receptors should be carried out in order to identify the essential mechanisms for generation and spread pilocarpine-induced SE. In conclusion, our findings show that acute compensatory physiological alterations in density and dissociation constant values of different receptors studied are important and occur during the epileptic phenomenon. The consequences of these changes during the acute phase of limbic seizures in mature

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