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Keywords:

  • dopamine;
  • hippocampus;
  • microdialysis;
  • pilocarpine;
  • seizures;
  • serotonin

Abstract

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  2. Abstract
  3. References

The present microdialysis study evaluated the anticonvulsant activity of extracellular hippocampal dopamine (DA) and serotonin (5-HT) with concomitant assessment of the possible mutual interactions between these monoamines. The anticonvulsant effects of intrahippocampally applied DA and 5-HT concentrations were evaluated against pilocarpine-induced seizures in conscious rats. DA or 5-HT perfusions protected the rats from limbic seizures as long as extracellular DA or 5-HT concentrations ranged, respectively, between 70–400% and 80–350% increases compared with the baseline levels. Co-perfusion with the selective D2 blocker remoxipride or the selective 5-HT1A blocker WAY-100635 clearly abolished all anticonvulsant effects. These anticonvulsant effects were mediated independently since no mutual 5-HT and DA interactions were observed as long as extracellular DA and 5-HT levels remained within these protective ranges. Simultaneous D2 and 5-HT1A receptor blockade significantly aggravated pilocarpine-induced seizures. High extracellular DA (> 1000% increases) or 5-HT (> 900% increases) concentrations also worsened seizure outcome. The latter proconvulsive effects were associated with significant increases in extracellular glutamate (Glu) and mutual increases in extracellular monoamines. Our results suggest that, within a certain concentration range, DA and 5-HT contribute independently to the prevention of hippocampal epileptogenesis via, respectively, D2 and 5-HT1A receptor activation.

Abbreviations used
DA

dopamine

EC

extracellular

EcoG

electrocorticography

Glu

glutamate

5-HT

serotonin

LC

Liquid Chromatography

SSRI

selective serotonin reuptake inhibitor

SSS

Seizure Severity Score

TSSS

Total Seizure Severity Score

Several anti-epileptic drugs increase extracellular (EC) levels of dopamine (DA) and/or serotonin (5-HT) in brain areas involved in epileptogenesis (Biggs et al. 1992; Yan et al. 1992; Kaneko et al. 1993; Meshkibaf et al. 1995; Dailey et al. 1997a; Smolders et al. 1997a; Southam et al. 1998; Murakami et al. 2001). It is not clear whether these monoamine increases have a direct anticonvulsant effect, contribute to the total anticonvulsant effect, or are just a drug side-effect.

Behavioural and electrocorticographic studies in rats have shown that DA controls hippocampal excitability via opposing actions at D1 and D2 receptors (Barone et al. 1990, 1991; Bo et al. 1995). Seizure enhancement is presumed to be a specific feature of D1 receptor stimulation, whereas D2 receptor stimulation is anticonvulsant (Alam and Starr 1992, 1993). Hippocampal 5-HT can also produce an inhibitory response mediated by 5-HT1A receptors (Colino and Halliwell 1987; Salgado and Alkadhi 1995). 5-HT1A agonists increase seizure threshold and limit the development and propagation of seizure activity in the hippocampus (Zeise et al. 1994; Zhang et al. 1994; Lu and Gean 1998; Schmitz et al. 1998; Hernandez et al. 2002; Kanner and Balabanov 2002).

In spite of abundant data in favour of the inhibitory effects of DA and 5-HT, data from several experimental and clinical investigations suggest seizure facilitation as a consequence of therapeutic and toxic doses of different DA agonists, selective serotonin reuptake inhibitors (SSRIs) and even anti-epileptic drugs (Favale et al. 1995; Starr 1996; Salzberg and Vajda 2001). The underlying mechanisms for the paradoxical proconvulsant ability of anti-epileptics are poorly understood. Furthermore, significant increases in EC hippocampal DA and 5-HT concentration associated with pilocarpine-induced convulsions have been reported (Khan et al. 2000). The relationship between elevated brain monoamine levels and anti or proconvulsant effects remains a matter of debate. In vivo data regarding seizure-modulating effects of increasing DA and 5-HT concentrations administered into the brain could be useful to explain these conflicting data.

The present microdialysis study investigated dose-dependent effects of hippocampal DA and 5-HT perfusions against the development of pilocarpine-induced limbic seizures in freely moving rats. The involvement of D2 and 5-HT1A receptor activation was studied in another group of rats by co-perfusion of 4 µm remoxipride, a selective D2 antagonist, or 100 nm WAY-100635, a selective 5-HT1A antagonist. Anticonvulsant potency was assessed by observation of behavioural changes and electrocorticographic monitoring. Additionally, neuroanatomical, electrophysiological and biochemical studies have suggested a functional relationship between DA and 5-HT neuronal systems in prefrontal cortex, striatum and hippocampus (Benloucif and Galloway 1991; Parsons and Justice 1993; Dewey et al. 1995; Matsumoto et al. 1996; Sakaue et al. 2000). Mutual DA/5-HT interactions were observed in substantia nigra (Thorréet al. 1998a,b). To our knowledge, such a mutual relationship at the level of the hippocampus has not been studied in vivo and was therefore concomitantly evaluated in the present study.

Materials and methods

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Chemicals and reagents

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DA.HCl, 5-HT.HCl, glutamate (Glu), pilocarpine.HCl and WAY-100635 were supplied by Sigma (St. Louis, MO, USA). Remoxipride was obtained from Tocris (Avonmouth, UK). All other chemicals were analytical reagent grade or better and were supplied by Merck (Darmstadt, Germany). Aqueous solutions were made with purified water (Seralpur pro 90 CN, Belgolabo, Overijse, Belgium) and filtered through a 0.2 µm membrane filter. The aqueous perfusion medium for the microdialysis experiments contained 147 mm NaCl, 2.3 mm CaCl2 and 4 mm KCl (modified Ringer's). During perfusion, DA and 5-HT were protected from oxidation by addition of 12.5 µm ascorbic acid to the perfusion fluid. An antioxidant solution containing 3.3 mm l-cystein, 0.27 mm Na2EDTA, 12.5 µm ascorbic acid and 100 mm acetic acid was added to all dialysate samples to prevent oxidation of the collected DA and 5-HT in the dialysates.

Microdialysis

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All protocols for the animal experiments described in this study were carried out according to the European Guidelines on Animal Experimentation and were approved by the Ethical Committee of the university. Male albino Wistar rats, weighing 260–320 g, were anaesthetized with an intraperitoneal (i.p.) injection of a mixture of ketamine HCl/diazepam (58 : 4.5 mg/kg). A cannula with a replaceable inner guide (CMA Microdialysis, Stockholm, Sweden) was implanted stereotaxically in the dorsal hippocampus 3 mm above the final probe membrane position (CA1–CA3 region). The co-ordinates were 4.6 mm lateral and 5.6 mm anterior to bregma, and 4.6 mm ventral starting from the dura (Paxinos and Watson 1986). Post-operative analgesia was assured by a single injection of ketoprofen (4 mg/kg i.p.). Immediately after surgery, the inner guide was replaced with a 3 mm CMA 12 microdialysis probe (CMA Microdialysis) and was continuously perfused with modified Ringer's solution at a constant flow rate of 2 µL/min (CMA 100 microdialysis pump, CMA Microdialysis). Dialysate sampling was started at a minimum of 24 h after surgery to allow animals time to recover sufficiently. The rats were housed in experimental cages with access to food and water ad libitum. During the experiment, the flow rate was kept constant at 2 µL/min and dialysates were collected every 20 min from the freely moving animals. All dialysate samples were analysed for DA and 5-HT content. Probe positioning was histologically verified (Fig. 1). In some experimental groups, samples were split for Glu determination. Pharmacological manipulations were carried out according to the respective protocols.

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Figure 1. Position of the probe in the dorsal hippocampus. The arrow indicates the most ventral point of the probe.

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Group 1 (n = 7): pilocarpine control group

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After six basal dialysate collections (samples 1–6) with modified Ringer's solution, the perfusion fluid was switched to modified Ringer's with ascorbic acid (samples 7–11). Convulsions were evoked by intrahippocampal administration of 10 mm pilocarpine for 40 min (samples 12–13). The microdialysis fluid was then switched back to the modified Ringer's with ascorbic acid solution for another five sampling intervals (samples 14–18).

Group 2 (n = 4) and 3 (n = 4): effect of remoxipride or WAY-100635 on pilocarpine-induced seizures

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The same protocol as described for group 1 was followed, but with addition of 4 µm remoxipride or 100 nm WAY-100635 to the perfusate for 240 min (samples 7–18).

Group 4 (n = 4): effect of combined remoxipride and WAY-100635 perfusion on pilocarpine-induced seizures

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The same protocol as described for group 2 was followed, but with perfusion of 4 µm remoxipride and 100 nm WAY-100635.

Group 5 (n = 27): effect of intrahippocampal perfusion of DA on pilocarpine-induced seizures

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After six basal dialysate collections (samples 1–6) with modified Ringer's, a specific DA concentration of 1 nm (n = 6), 2 nm (n = 6), 4 nm (n = 5), 8 nm (n = 5) or 10 nm (n = 5) was administered intrahippocampally via the probe for 100 min (samples 7–11), initiating a stable plateau dialysate concentration. The perfusion fluid was switched to a solution containing the appropriate DA concentration and 10 mm pilocarpine for two sampling intervals (samples 12–13). Finally, the perfusate was switched back to the initial DA solution (samples 14–18).

Group 6 (n = 19): effect of intrahippocampal perfusion of 5-HT on pilocarpine-induced seizures

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The same protocol as described for group 5 was followed, but with 5-HT perfusions of 2 nm (n = 6), 4 nm (n = 5), 8 nm (n = 3) or 10 nm (n = 5).

Group 7 (n = 10): effect of the D2 antagonist remoxipride on the anticonvulsant effects of DA

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The same protocol as described for group 5 was repeated with the use of the anticonvulsant DA perfusate concentration (1 and 2 nm, as determined in group 5 experiments) and addition of 4 µm remoxipride to the perfusion fluid during collections 7 to 18.

Group 8 (n = 5): effect of the 5-HT1A antagonist WAY-100635 on the anticonvulsant effects of 5-HT

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The same protocol as described for group 6 was followed, but with administration of the protective 5-HT concentration (2 nm, as determined in group 6 experiments) and co-administration of 100 nm WAY-100635.

Group 9 (n = 31): effects of anti and proconvulsant DA and 5-HT concentrations on hippocampal Glu levels

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The effect of DA (2 and 10 nm) and 5-HT (2 and 10 nm) perfusion on the hippocampal EC Glu overflow was investigated. After six basal collections (samples 1–6), DA or 5-HT was added to the perfusion fluid for another five collection periods (samples 7–11). The microdialysates obtained were analysed for hippocampal Glu content.

Electrocorticography (EcoG)

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Combined microdialysis–ECoG was performed in at least two animals of each experimental group. During the surgical procedure performed to implant the microdialysis probe, two additional screws were positioned at both sides of the sutura sagittalis to fix the electrodes. Both screws were placed upon the surface of the dura mater and functioned as a reference and a working electrode. A ground electrode was positioned subcutaneously.

Seizure Severity Score

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For each collection period, seizure severity assessment was based on the observation of behavioural manifestations. We adapted the Seizure Severity Score (SSS) from Racine's scale (Racine 1972) to take into account the typical behavioural changes associated with pilocarpine-induced motor seizures. This scale consists of six stages which correspond to the successive developmental stages of motor seizures: (0) normal non-epileptic activity; (1) mouth and facial movements, hyperactivity, grooming, sniffing, scratching, wet dog shakes; (2) head nodding, staring, tremor; (3) forelimb clonus, forelimb extension; (4) rearing, salivating; (5) falling, status epilepticus. Seizure severity was then determined by summation of the SSSs of each collection period, resulting in a Total Seizure Severity Score (TSSS) for each individual animal.

Chromatographic assays

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For the analysis of DA and 5-HT, an off-line microbore liquid chromatography (LC) assay (C8, 5 µm; 100 × 1 mm; Unijet, Bioanalytical Systems, Wess Lafayette, IN, USA) was used with automatic injection (10 µL) of the samples, as described previously in detail (Sarre et al. 1997). In summary, the assay was based on ion-pair reversed phase chromatography, coupled to single-channel amperometric detection (Decade, Antec, Leiden, the Netherlands).

Chromatographic conditions and pre-column derivatization procedures for Glu analysis have been described previously (Smolders et al. 1995, 1996). Glu determination was performed by gradient, reversed-phase, microbore LC with fluorescence detection after pre-column derivatization with o-phthalaldehyde/β-mercaptoethanol.

Statistical analysis

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All results presented in the figures are expressed as the mean EC DA and 5-HT concentration in nm, or Glu concentration in µm, with SEM. These data were corrected for in vivo recovery across the dialysis membrane, estimated by the well established Lönnroth point-of-no-net-flux method (Lönnroth et al. 1987). Basal values represent the mean transmitter concentrations as obtained under basal conditions, i.e. during the first six collections of each experiment. For experiments in which DA and 5-HT were perfused into the hippocampus, the EC concentrations for the transmitter in casu were derived by subtracting the dialysate concentrations from the perfusate concentration (Figs 3a and 4a). This concentration, representing the amount of compound that effectively reaches the EC medium, was added to the basal levels. The mean plateau concentration, established by two collections after the start of the monoamine perfusion (samples 8–11), is depicted with Roman symbols (mean ± SEM). The acquired TSSSs are represented as such. Statistical analysis of the alterations of neurotransmitter concentrations within one group was performed by one-way anova for repeated measurements, followed by a Fisher's protected least significant difference (PLSD) post hoc test (α = 0.05). The Mann–Whitney test (α = 0.05) was used for comparison of mean neurotransmitter concentrations and mean TSSSs between groups at a certain time point.

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Figure 3. Effects of intrahippocampal perfusion of different DA concentrations on EC DA, EC 5-HT levels (in nm) (mean ± SEM) and seizure severity. (a) For the basal collections (1–6), the EC DA concentration is expressed as the mean of the first six stable EC concentrations. Each of the remaining bars represents a 20 min collection period. According to the initial different DA concentrations perfused, different EC DA plateau levels were established (8–11). The Roman symbols (I-V) represent the mean DA plateau concentrations (mean ± SEM) obtained after, respectively, modified Ringer's with ascorbic acid, 1–2 nm, 4 nm, 8 nm and 10 nm DA perfusion. The asterisks denote only the first values significantly different from the basal value (p < 0.05) (Statistics: anova). (b) Each bar represents the mean corresponding hippocampal EC 5-HT plateau level (mean ± SEM) that was obtained during the corresponding stable DA plateau concentration (8–11). The asterisk denotes values significantly different from the control value (I) (p < 0.05) (Statistics: Mann–Whitney test). (c) For each individual animal the relationship between the mean DA plateau concentration and the TSSS, with (□, ◊) and without (▪, ◆) 4 µm remoxipride co-perfusion, is depicted. Diamonds and squares, respectively, represent the TSSSs obtained in the pilocarpine control animals and in animals receiving pilocarpine and different DA concentrations. The arrow indicates the shift in TSSSs occurring after remoxipride co-perfusion. The results can be divided into five clusters corresponding to the previously defined mean DA plateau concentrations (I-V).

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Figure 4. Effects of intrahippocampal perfusion of different 5-HT concentrations on EC 5-HT, EC DA levels (in nm) (mean ± SEM) and seizure severity. (a) For the basal collections (1–6), the EC 5-HT concentration is expressed as the mean of the first six stable EC concentrations. Each of the remaining bars represents a 20 min collection period. According to the initial different 5-HT concentrations perfused, different EC 5-HT plateau levels were established (8–11). The Roman symbols (I-V) represent the mean 5-HT plateau concentrations (mean ± SEM) obtained after, respectively, modified Ringer's with ascorbic acid, 2 nm, 4 nm, 8 nm and 10 nm 5-HT perfusion. The asterisks denote only the first values significantly different from the basal value (p < 0.05) (Statistics: anova). (b) Each bar represents the mean hippocampal EC DA plateau level (mean ± SEM) that was obtained during the corresponding stable 5-HT plateau concentration (8–11). The asterisk denotes values significantly different from the control value (I′) (p < 0.05) (Statistics: Mann–Whitney test). (c) For each individual animal the relationship between the mean 5-HT plateau concentration and the TSSS, with (○,▵) and without (•,▴) 100 nm WAY-100635 co-perfusion, is depicted. Triangles and circles, respectively, represent the TSSSs obtained after control 10 mm pilocarpine perfusion and after co-perfusion of pilocarpine with the different 5-HT concentrations. The arrow indicates the shift in TSSSs occurring after WAY-100635 co-perfusion. The results can be divided into five clusters corresponding to the previously defined mean 5-HT plateau concentrations (I-V).

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Probe recovery

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The mean in vivo recovery (mean ± SEM) obtained from the slope of the Lönnroth plot was 35.61 ± 3.80% (n = 8), 40.71 ± 4.09% (n = 8) and 36.35 ± 3.69% (n = 6) for, respectively, DA, 5-HT and Glu.

Basal DA and 5-HT concentrations in hippocampus of conscious rats

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Basal hippocampal concentrations (mean ± SEM) corrected for probe recovery were 0.376 ± 0.018 nm (n = 47) for DA, 0.543 ± 0.027 nm (n = 44) for 5-HT and 1.469 ± 0.314 µm for Glu (n = 16).

Group 1: pilocarpine control group

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During the perfusion of pilocarpine (collections 12–13) the rats mainly exhibited hyperactivity, wet dog shakes and tremor. This preconvulsive behaviour then evolved to full blown seizures (from collection 14–15 on) characterized by salivating, rearing and falling. The mean TSSS was 14.7 ± 0.5 (mean ± SEM) (Figs 3c and 4c). ECoG recordings revealed that the focally-evoked seizures immediately became secondary generalized, indicated by cortical seizure activity at the contralateral side of the focus. Intrahippocampal administration of pilocarpine (Fig. 2) significantly increased basal hippocampal neurotransmitter overflow. Both DA and 5-HT levels were significantly elevated from collection 12 on, with maximal increases of 280% (p = 0 < 0.001) and 230% (p < 0.001), respectively (collection 13). Cessation of pilocarpine administration resulted in restoration of the basal DA and 5-HT levels, while recurrent periods of seizure activity were sustained during the subsequent collection periods. The basal levels were not influenced by the addition of ascorbic acid to the perfusate.

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Figure 2. Effects of intrahippocampal perfusion of pilocarpine on EC hippocampal DA and 5-HT levels (in nm) (mean ± SEM) with and without co-perfusion of remoxipride and/or WAY-100635. For the basal collections (1–6), the concentration is expressed as the mean of the first six dialysate concentrations. EC hippocampal concentrations of DA (a) and 5-HT (b) in animals receiving either modified Ringer's with ascorbic acid, 4 µm remoxipride alone, 100 nm WAY-100635 alone or combined 4 µm remoxipride and 100 nm WAY-100635 intrahippocampally (7–18) are shown before (7–11), during (12–13) and after (14–18) intrahippocampal administration of 10 mm pilocarpine. Each bar represents a 20 min collection period. Asterisks denote only the first values significantly different from the corresponding baseline value (p < 0.05) (Statistics: anova).

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Group 2: effect of remoxipride on pilocarpine-induced seizures

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Remoxipride perfusion had no effect on basal DA and 5-HT overflow, and on the pilocarpine-induced monoamine and seizure profile (Fig. 2). Intrahippocampal co-administration of pilocarpine and remoxipride resulted in comparable significant increases in EC DA and 5-HT from collection 12 on (p = 0.001). All rats developed limbic seizures with typical ECoG patterns. Selective blockade of D2 receptors alone did not influence seizure severity since the mean TSSS of 14.5 ± 1.0 (Fig. 3c) was not significantly different from the group 1 control conditions.

Group 3: effect of WAY-100635 on pilocarpine-induced seizures

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WAY-100635 had no effect on basal hippocampal DA or 5-HT levels, or on the pilocarpine-induced neurotransmitter profile (Fig. 2). All rats developed seizures comparable with the group 1 control animals, resulting in a mean TSSS of 15.3 ± 0.6 (Fig. 4c).

Group 4: effect of combined remoxipride and WAY-100635 perfusion on pilocarpine-induced seizures

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Simultaneous administration of 4 µm remoxipride and 100 nm WAY-100635 did not affect basal EC hippocampal DA and 5-HT concentrations, or the typical pilocarpine-induced monoaminergic profile (Fig. 2). All rats developed seizures that were significantly more severe when compared with the other control group animals. This clearly resulted in an increased mean TSSS of 21.0 ± 0.8 (p < 0.05).

Group 5: effect of intrahippocampal perfusion of DA on pilocarpine-induced seizures

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(a) Effect on baseline DA and 5-HT release.  Stable EC DA plateau levels were established 40 min after initiation of the DA perfusion (Fig. 3a). Exogenously-perfused DA dose-dependently increased EC plateau levels (I–VI). An EC DA concentration range of 0.686 ± 0.016 (II) to 3.433 ± 0.116 nm (V) had no effect on EC 5-HT levels when compared with basal levels (Fig. 3b). At the highest DA plateau level (VI) (4.869 ± 0.187 nm), a significant increase in the EC hippocampal 5-HT concentration was observed (26%, p < 0.05) (Fig. 3b).

(b) Effect on pilocarpine-induced seizures.  Rats perfused with 1 and 2 nm DA, initiating EC plateau levels II (0.686 ± 0.016 nm) and III (1. 1782 ± 0.105 nm), were protected against pilocarpine-induced seizures. ECoG recordings showed no signs of seizure activity. Wet dog shakes and hyperactivity were only seen occasionally. This was reflected in a significant decrease in mean TSSS (II: 1.0 ± 0.4, III: 0.5 ± 0.3) when compared with control conditions (p < 0.05) (Fig. 3c). Animals in which plateau level IV (2.051 ± 0.131 nm) was initiated were free from full-blown seizures but not protected from partial seizure activity. This partial protection was associated with significant decreases in TSSSs (7.6 ± 0.7; p < 0.05) compared with the control group. Rats with levels V (3.433 ± 0.116 nm) and VI (4.869 ± 0.187 nm) developed limbic seizures as described for the pilocarpine control group. The latter group (VI) additionally developed status epilepticus, a condition that was rarely seen in control group animals. This proconvulsive behaviour was reflected in significant increases in TSSSs (20.6 ± 0.8; p < 0.05) (Fig. 3c).

Group 6: effect of intrahippocampal perfusion of 5-HT on pilocarpine-induced seizures

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(a) Effect on baseline DA and 5-HT release.  Comparable data as for group 5 experiments were obtained. Forty minutes after initiation of the local 5-HT perfusion, stable EC 5-HT plateau levels were measured (Fig. 4a). The dose-dependently increasing 5-HT plateau levels II′ (1.332 ± 0.156 nm), III′ (2.276 ± 0.058 nm) and IV′ (3.486 ± 0.115 nm) did not significantly affect the EC DA overflow when compared with basal levels (Fig. 4b). However, significant increases in EC DA were seen when the highest EC 5-HT plateau level (V′ = 5.565 ± 0.337 nm) (55%; p < 0.05) was reached (Fig. 4b).

(b) Effect on pilocarpine-induced seizures.  Perfused with 2 nm 5-HT, introducing EC 5-HT level II′, protected rats from pilocarpine-induced seizures (TSSS: 0.8 ± 0.4). Animals with EC 5-HT levels III′, IV′ and V′, respectively, experienced partial seizure protection (TSSS: 8.6 ± 0.5), no protection (TSSS: 15.0 ± 1.0) and proconvulsive behaviour (mean TSSS: 21.0 ± 0.3) (Fig. 4c). When compared with pilocarpine control conditions (I′), respectively, a significant decrease (II′ and III′: p < 0.05), no difference (IV′) and a significant increase (V′: p < 0.05) in mean TSSSs was seen.

Group 7: effect of the D2 antagonist remoxipride on the anticonvulsant effects of DA

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For this group of experiments the DA concentrations shown to be anticonvulsant during group 5 experiments were selected. The protective effect of 1 and 2 nm DA was blocked by co-perfusion with 4 µm remoxipride. ECoG recordings clearly showed epileptic seizure patterns. This was reflected in the TSSSs shifting towards control values (1 nm DA: 14.3 ± 0.6; 2 nm DA: 14.2 ± 0.9) (Fig. 4c). EC hippocampal monoamine levels were not significantly different from the transmitter changes observed in the corresponding experiments without D2 blockade (data not shown).

Group 8: effect of the 5-HT1A antagonist WAY-100635 on the anticonvulsant effects of 5-HT

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The protective effect of 2 nm 5-HT perfusion was blocked by co-perfusion with 100 nm WAY-100635. Again, TSSSs shifted towards control group values (14.2 ± 0.6) (Fig. 4c). EC neurotransmitter changes were similar to those observed in group 5 (data not shown).

Group 9: effects of anti and proconvulsant DA and 5-HT concentrations on hippocampal Glu levels

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No significant changes in extracellular Glu levels were observed during perfusion of anticonvulsant DA and 5-HT concentrations. Proconvulsive concentrations of DA and 5-HT (10 nm) significantly increased hippocampal Glu levels by, respectively, 41 and 74% (p < 0.05) (Fig. 5).

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Figure 5. Effects of intrahippocampal perfusion of DA and 5-HT on EC hippocampal Glu concentrations (in µm) (mean ± SEM). For the basal collections (1–6), the concentration is expressed as the mean of the first six stable Glu dialysate concentrations. Each following bar represents a 20 min collection period. (a) EC hippocampal Glu concentrations in animals receiving either 2 or 10 nm DA via the microdialysis probe (7–11) are depicted. (b) Changes in EC hippocampal Glu concentrations during 2 and 10 nm 5-HT perfusion (7–11) are shown. Asterisks denote only the first values significantly different from the corresponding baseline value (p < 0.05) (Statistics: anova).

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Anticonvulsant effects of hippocampal DA and 5-HT: role of D2 and 5-HT1A receptors

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Since monoamines can exert variable seizure-modulating responses, depending on animal species, seizure model and monoaminergic drug dose administered (Weinshenker and Szot 2002), the present study was designed to elucidate the contribution of the hippocampal DA-ergic and 5-HT-ergic system in seizure generation and prevention. Intrahippocampal administration of DA (1 and 2 nm) or 5-HT (2 nm), prior to pilocarpine, led to the determination of so-called anticonvulsant ranges. For DA, this anticonvulsant range corresponds to a 70–430% increase in basal levels while for 5-HT, an 80–350% increase was proven to be protective. Therapeutic doses of sodium valproate, carbamazepine, antiepilepsirine, lamotrigine and zonisamide were reported to induce monoamine increases corresponding to our determined anticonvulsant ranges (Biggs et al. 1992; Yan et al. 1992; Baf et al. 1994a,b; Dailey et al. 1996, 1997a, 1997b; Smolders et al. 1997b; Graumlich et al. 1999; Murakami et al. 2001). Interestingly, these ranges also seem applicable to compounds that mediate direct or indirect DA or 5-HT facilitating and anticonvulsant effects, such as fluoxetine, MK-801, amphetamine and dex-fenfluramine (Yan et al. 1994a,b; Puig de Parada et al. 1995; Smolders et al. 1997b; Hanson et al. 1999; Rocher and Gardier 2001; Hernandez et al. 2002). Moreover, these ranges seem to be independent of animal species, seizure model or route of drug administration. The current findings confirm electrophysiological in vitro inhibitory responses obtained after direct application of DA (10–100 µm) and 5-HT (20 µm) to CA1 hippocampal neurones (Suppes et al. 1985; Salgado and Alkadhi 1995). To our knowledge, this is the first in vivo study with exogenous DA or 5-HT administration proving that the anticonvulsant effects exerted by hippocampal DA and 5-HT levels are mediated by, respectively, the D2 and 5-HT1A receptors. Indeed, even with DA or 5-HT concentrations within the determined anticonvulsant ranges, seizures can be provoked after selective blockade of these receptor subtypes.

Proconvulsant effects of hippocampal DA and 5-HT

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Perfusion of rat hippocampus with 4, 8 and 10 nm DA or 5-HT gives rise to non-protective EC concentrations. In particular, 10 nm perfusions are proconvulsant, since seizures were more severe compared with control conditions. Okada et al. (1997) also showed that toxic phenytoin doses trigger generalized tonic-clonic seizures accompanied by approximately 600% increases in hippocampal and striatal monoamine levels, which were considered partially responsible for this paradoxical intoxication. Carbamazepine and lamotrigine have also been associated with similar toxic effects and interferences with monoaminergic function. Apparently, as long as pharmacologically-induced hippocampal DA and 5-HT increases remain within the anticonvulsant range, seizures remain absent, whereas seizures as a side-effect of monoaminergic drugs are to be expected after inducing high monoamine levels.

Which neurobiochemical processes can be held responsible for these paradoxical effects?

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Dex-fenfluramine, amphetamine analogues and apomorphine are anticonvulsant under most circumstances (Weinshenker and Szot 2002; Zagnoni and Albano 2002) but possess in vivo epileptogenic effects at high doses (Hanson et al. 1999). On the basis of similar neurobiochemical effects (monoamine increases in hippocampus, striatum and nucleus accumbens), some authors have suggested that these substances may share a common neurotoxic mechanism of action (Ricaurte et al. 1991; Sabol et al. 1992; Stephans and Yamamoto 1994) in which Glu might play a crucial role (Sonsalla et al. 1991; Colado et al. 1993; Okada et al. 1995; Ohmori et al. 1996; Bouron and Reuter 1999). Elevated Glu levels are presumed to elicit morphological neuronal changes, are involved in NMDA receptor-mediated excitotoxicity and play a critical role in epileptogenesis (Isokawa and Mello 1991; Meldrum 1994; Smolders et al. 1997a, 2002). Toxic doses of dex-fenfluramine (10 mg/kg) and methamphetamine (5 mg/kg), respectively, increased 5-HT (> 1000%) and DA (> 2000%) overflow in different brain regions, thereby abundantly exceeding the currently determined anticonvulsant ranges and thus reaching proconvulsant concentrations (Sabol et al. 1992; Puig de Parada et al. 1995; Rocher and Gardier 2001). In most cases, these monoamine increases were accompanied by concomitant increases in Glu, similar to the Glu increases observed after 10 nm DA or 5-HT perfusion in this study. Our results clearly show facilitatory effects on hippocampal Glu, with aggravation of the pilocarpine-induced seizure symptoms, during 10 nm DA and 5-HT co-perfusion. We also demonstrated that such facilitation does not occur at lower perfusion concentrations.

Alteration of the excitation-inhibition balance

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Excitatory and inhibitory actions of DA have been reported in hippocampus (Barone et al. 1991; Starr 1996). High DA concentrations enhance Glu release via D1/D5 receptor stimulation, while low concentrations reduce excitatory responses via D2 receptors (Hsu 1996; Dailey et al. 1997b; Bouron and Reuter 1999). Both excitatory and inhibitory hippocampal transmission can be reduced via 5-HT3 receptor activation (Zeise et al. 1994; Zhang et al. 1994; Schmitz et al. 1998; Pralong et al. 2002). Additionally, hippocampal 5-HT reduces Glu release by acting on presynaptic 5-HT1A receptors (Schmitz et al. 1995b; Mauler et al. 2001). During selective 5-HT1A blockade, 5-HT produces fast excitation probably mediated by 5-HT2C receptors (Beck 1992). Moreover, 5-HT inhibits GABA-ergic hippocampal interneurones via presynaptic 5-HT1A receptors (Schmitz et al. 1995a). The inhibitory effect of 5-HT on Glu-ergic transmission may therefore be partially counterbalanced by a 5-HT-mediated disinhibition of the principal hippocampal output cells (Schmitz et al. 1998). Under normal circumstances, the D2 and 5-HT1A influences on seizure activity are expected to be larger than those mediated by D1/D5, 5-HT2C and 5-HT1A receptors on GABA-ergic interneurones, since the brain is not spontaneously epileptic. This is the first study providing evidence that the balance can dose-dependently tip the other way and increase the risk of seizures.

Importance of DA and 5-HT increases in pilocarpine-induced seizures

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In the pilocarpine control experiments, significant hippocampal DA and 5-HT increases occur as a result of muscarinic receptor stimulation, thereby reaching the described anticonvulsant ranges (Turski et al. 1989; Smolders et al. 1997b). During this period only preconvulsive behavioural symptoms and partial seizure activity were most frequently observed. After cessation of the pilocarpine administration, hippocampal DA and 5-HT levels progressively returned to baseline levels, with concomitant aggravation of the seizure outcome. In this stage, full-blown seizures were observed in all animals. This suggests that the initial increased monoamine levels might mediate an inhibitory response, curtailing seizure activity via D2 and 5-HT1A receptor stimulation. Indeed, simultaneous selective blockade of both these receptor subtypes worsened seizure outcome, as evident from the significant increase in TSSS compared with the other control groups. Our previous studies showed that pilocarpine-induced seizures were accompanied by significant seizure-related increases in hippocampal Glu and GABA. Effective anticonvulsant treatment always abolished these Glu increases (Smolders et al. 1997a,b, 2002). We hypothesize that, in vivo, the Glu-ergic excitatory response not only exceeds the well established inhibitory GABA-ergic response but apparently, also, the currently reported monoaminergic inhibitory response. Since the Glu increases observed during 10 nm DA and 5-HT perfusions are in the same order of magnitude as those reported during pilocarpine control experiments, proconvulsant effects would not be expected. However, Glu increases during 10 nm monoamine and 10 mm pilocarpine co-perfusions are probably triggered by two distinct mechanisms disturbing Glu homeostasis. We therefore suggest that both mechanisms might give rise to additionally longer-lasting Glu increases and concomitant proconvulsant responses.

Mutual DA and 5-HT effects?

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In contrast to the well documented monoaminergic interactions in the basal ganglia, our data do not support the existence of such interactions in hippocampus (Benloucif and Galloway 1991; Parsons and Justice 1993; Dewey et al. 1995; Thorréet al. 1998a,b; Porras et al. 2003). Indeed, perfusate concentrations of up to 8 nm for both monoamines did not facilitate mutual release. Only at proconvulsant doses did mutual facilitatory effects occur. These in vivo data are in disagreement with previous in vitro studies demonstrating a dose-dependent functional stimulation of hippocampal 5-HT release mediated via DA receptors (Balfour and Iyaniwura 1985). Several 5-HT receptor subtypes seem implicated in the presynaptic facilitation of DA release in several brain areas: 5-HT4 in substantia nigra, 5-HT1 in striatum, 5-HT2 and 5-HT3 in nucleus accumbens and striatum (Benloucif and Galloway 1991; Parsons and Justice 1993; Dewey et al. 1995; Thorréet al. 1998a,b; Porras et al. 2003). To our knowledge, no such data are available for the hippocampus. We suggest that the mutual hippocampal monoamine increases only occur as a result of a non-selective, toxic-based mechanism. These mutual effects might be carrier-mediated. Indeed, when adding high DA concentrations to nigral slices, 5-HT was released by reversal of the carrier (Reubi and Emson 1978) and inhibition of 5-HT reuptake (Kelly et al. 1985).

Conclusion

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The present microdialysis study provides direct in vivo evidence concerning the importance of both DA-ergic and 5-HT-ergic hippocampal transmission in modulating limbic seizures. Intrahippocampal perfusion of ‘therapeutic’ DA and 5-HT concentrations independently prevent the development of pilocarpine-induced seizures via, respectively, D2 and 5-HT1A receptor stimulation. Paradoxically, establishment of high hippocampal DA and 5-HT concentrations results in a proconvulsant response, mediated by hippocampal Glu increases and mutual 5-HT and DA facilitation. Finally, this study opens perspectives for using direct and indirect DA and 5-HT agonists as future anti-epileptic drugs. The determination of anticonvulsant ranges will facilitate the screening of such compounds in animal models for epilepsy, since drug doses can be adjusted according to their pharmacological effects on hippocampal DA, 5-HT and Glu release.

Acknowledgements

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The authors acknowledge the excellent assistance of S. Gheuens, R. Berckmans, G. De Smet, C. De Rijck and R. M. Geens. We thank the Fund for Scientific Research Flanders (FWO-Vlaanderen-Belgium) and the Vrije Universiteit Brussel for financial support. RC and IS are, respectively, research and postdoctoral fellows of the FWO-Vlaanderen.

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