Effects of carbamazepine on hippocampal serotonergic system

Epilepsy Research 31 (1998) 187 – 198

Effects of carbamazepine on hippocampal serotonergic system Motohiro Okada a,*, Takayuki Hirano b, Kazuhisa Mizuno a, Yuko Kawata a, Kazumaru Wada a, Takuya Murakami a, Hiroichi Tasaki a, Sunano Kaneko a b

a Department of Neuropsychiatry, School of Medicine, Hirosaki Uni6ersity, Hirosaki 036, Japan Department of Biochemistry Research, Mianami-Hanamaki National Hospital, Hanamaki 025, Japan

Received 17 October 1997; received in revised form 17 March 1998; accepted 28 March 1998

Abstract To establish the mechanism of action of the antiepileptic and psychotropic effects of carbamazepine (CBZ), its effects on serotonin (5-HT) transmission, metabolism and re-uptake activity in the rat hippocampus were studied. After acute and chronic administrations of 25 mg/kg CBZ, the plasma concentration of CBZ was found to be within the therapeutic range, whereas both acute and chronic administrations of 50 and 100 mg/kg CBZ resulted in a supratherapeutic plasma concentration. Acute administration of the therapeutic dose of CBZ resulted in an increase in the hippocampal extracellular and total level of 5-HT, its metabolite, 5-hydroxydoleacetic acid (5-HIAA) and its precursor, 5-hydroxytryptophan (5-HTP). The acute administration of 50 mg/kg CBZ resulted in an increase in the hippocampal levels of extracellular 5-HT and 5-HIAA as well as in the total levels of 5-HTP, whereas hippocampal levels of extracellular 5-HTP, total 5-HT and 5-HIAA remained unaffected. CBZ at a dose of 100 mg/kg decreased the levels of all of these substances. After chronic administration, 25 mg/kg/day CBZ increased hippocampal total levels of 5-HT, 5-HTP and 5-HIAA, whereas 100 mg/kg/day CBZ decreased all of these total levels. CBZ at a dose of 50 mg/kg/day decreased total levels of 5-HT, however neither total levels of 5-HIAA nor 5-HTP were affected. Both therapeutic and supratherapeutic plasma concentrations of CBZ inhibited 5-HTP accumulation, and did not affect 5-HT re-uptake activity in vitro. These results suggest that a therapeutic concentration of CBZ enhances 5-HT turnover and transmission, whereas a supratherapeutic concentration of CBZ inhibits 5-HT turnover and transmission without affecting 5-HT re-uptake activity. These effects of CBZ on serotonergic systems may be, at least partially, involved in the mechanisms of action of CBZ. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Carbamazepine; Epilepsy; Microdialysis; Serotonin release; Serotonin metabolism

1. Introduction * Corresponding author. Tel.: + 81 172 395066; fax: + 81 172 395067; e-mail: [email protected]

The antiepileptic drug carbamazepine (CBZ), 5-carbamoyl-5H-dibenz[b,f ]azepine was synthe-

0920-1211/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0920-1211(98)00025-4

188

M. Okada et al. / Epilepsy Research 31 (1998) 187–198

sized about 40 years ago by Schinder (1960). The agent has been shown to be effective in the treatment of simple partial, complex partial and generalized tonic-tonic seizures, but is ineffective against generalized absence seizures (Loiseau and Duche, 1995). CBZ and phenytoin (PHT) are the drugs of first choice in the treatment of these epileptic disorders, however CBZ has recently been demonstrated to be more effective than PHT in the treatment of bipolar disorders, trigeminal neuralgia (Fromm et al., 1984), schizophrenia (Neppe, 1982), acute and chronic bipolar disorder (Okuma et al., 1990), anxiety disorders (panic disorder, posttraumatic stress disorder), alcohol and sedative hypnotic withdrawal states, and behavioural dyscontrol syndromes (Keck et al., 1992). Numerous investigators have already studied the effects of CBZ on monoaminergic systems (Kaneko et al., 1980; Pratt et al., 1985; Elphic et al., 1990; Kaneko et al., 1993; Mizuno et al., 1994), because central monoaminergic function has been related to the etiology of several psychiatric and neurological disorders. We have already demonstrated the biphasic effects of CBZ on dopaminergic transmission and metabolism, in which therapeutic concentrations of CBZ enhance both striatal and hippocampal dopaminergic transmission and metabolism, whereas supratherapeutic concentrations reduce them (Okada et al., 1996a, 1997b). In spite of these studies, it remains unclear whether or not CBZ yields biphasic effects on serotonergic function which is also involved in the pathogenesis of epilepsy and other disorders (for example, bipolar disorder, trigeminal neuralgia, schizophrenia, anxiety disorder). It has been reported that CBZ inhibits several types of Na + and Ca2 + channel activities (Schwarz and Grigat, 1989; Kito et al., 1994; Macdonald, 1995; Yoshimura et al., 1995), and the inhibition of these channels should reduce serotonin (5-HT) release (Sharp et al., 1990). Contrary to this hypothesis, we have already demonstrated that therapeutic concentrations of CBZ increased extracellular and total 5-HT levels in the rat hippocampus (Kaneko et al., 1993; Mizuno et al., 1994). Therefore, the present study was undertaken to establish the concentration-dependent effects of CBZ on hippocampal serotonergic metabolism and transmission.

2. Materials and methods

2.1. Experimental animals Male Wistar rats (Clea, Japan), weighing 250– 300 g and housed at a constant temperature (259 2°C) with a 12-h light-dark cycle were used for the following experiments. The experimental protocols used in this study were approved by the ethical committee of Hirosaki University. Blood samples were drawn from the jugular vein, and CBZ levels were determined by high-performance liquid chromatography (HPLC) (Juergens, 1987).

2.2. Determination of the effects of CBZ on 5 -HT metabolism 2.2.1. Chronic administration Rats were group-housed (four rats per cage) and given free access to food and water. Their body weight was measured at 2-day intervals for the duration of the experiment. Their diet contained either 0, 25, 50 or 100 mg/kg body weight/ day CBZ (n= 6). After consuming the CBZ-treated food for 21 days, the rats were irradiated by microwave at 7.0 kW× 1.0 s (TMW6402a, Toshiba, Japan) (Okada et al., 1997b). 2.2.2. Acute administration Rats were administered with either 0, 25, 50 or 100 mg/kg body weight CBZ dissolved in saline/ dimethylsulfoxide (DMSO; 50/50 v/v), by intraperitoneal (i.p.) injection (1 ml/kg body weight; n= 6) (Okada et al., 1995, 1997b,c). 2.2.3. Acute administration with 3 -hydroxybenzylhydrazine (NSD1015) As an index of tryptophan hydroxylase activity (5-HT turnover) in the rat hippocampus in vivo, the accumulation of 5-hydroxytryptophan (5HTP) was measured. Graded doses of CBZ were administered (i.p.) 1 h before the administration of a central amino acid decarboxylase (AADC) inhibitor, NSD1015 (100 mg/kg, i.p.) (Elphic et al., 1990; Okada et al., 1995, 1997b). The rats were irradiated by microwave at 7.0 kW× 1.0 s (n= 6) 1 h later.

M. Okada et al. / Epilepsy Research 31 (1998) 187–198

2.2.4. Sample preparation Rat brains were dissected according to the method of Glowinski and Iversen (1966). Tissues were weighed before being frozen at − 80°C. The frozen tissues were placed into 1.5-ml microtubes and homogenized with the aid of an ultrasonic cell disrupter (HOM-100, Iwaki, Japan) in 500 ml of chilled 0.2 mM perchloric acid containing 100 ng isoproternol as an internal standard. After centrifugation at 6000×g for 20 min at 4°C, the aqueous layer was mixed with 150 ml sodium acetate (1 mM) to adjust the pH to 3.0, and then filtered through a 0.45-mm filter (HLCDISK 3, Kanto Kagaku, Japan). Aliquots (10 ml) of these filtrates were injected into the HPLC apparatus, which was equipped with an electrochemical detector (ECD-HPLC) system (Okada et al., 1997c). 2.3. Microdialysis 2.3.1. Determination of extracellular 5 -HT and its metabolite The rats were anesthetized with diethylether and placed in a stereotaxic frame. A dialysis probe (0.22 mm diameter, 3 mm exposed membrane; Eicom, Japan) was implanted into the hippocampus (A= −5.8 mm, L =4.8 mm, V = −4.0 mm, relative to the bregma) (Okada et al., 1992, 1997d). The perfusion experiments were started 24–36 h after probe insertion. The effects of CBZ on the extracellular levels of hippocampal 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were studied using a modified Ringer’s solution (MRS) containing (in mM): 147 Na + , 2.7 K + , 1.2 Ca2 + , 1.0 Mg2 + , 153.1 Cl − and 0.2 ascorbate (Okada et al., 1996a). This solution was buffered to a pH of 7.4 using 2 mM phosphate and 1.1 mM Tris buffer (Okada et al., 1996b). MRS was perfused at a rate of 1 ml/min, and injections of the dialysate into the ECD-HPLC system were repeated automatically every 20 min using an autoinjector (AS-10, Eicom, Japan). When the coefficients of variation (CV) of the data reached less than 5% for 60 min (stabilization), either 0, 20, 50 or 100 mg/kg body weight CBZ was administered (i.p.). Further data were recorded for 120 min after CBZ administration (Okada et al., 1997b).

189

2.3.2. Determination of extracellular le6els of 5 -HT precursor In order to study the effects of CBZ on hippocampal extracellular levels of the 5-HT precursor, 5-HTP, perfusion of the dialysis probe with MRS containing 10 mM of NSD1015 (NMRS) was commenced (Hashiguti et al., 1993). NMRS was perfused at a rate of 1 ml/min, and injections of the dialysate into the ECD-HPLC system were repeated automatically every 20 min using an autoinjector (AS-10, Eicom, Japan). When the coefficients of variation (CV) of the data reached less than 5% for 60 min (stabilization), either 0, 20, 50 or 100 mg/kg body weight CBZ was administered (i.p.). Further data were recorded for 120 min after CBZ administration. 2.4. ECD-HPLC system conditions An HPLC system (EP-10, Eicom, Japan) equipped with an ECD (ECD-100, Eicom, Japan) and a graphite carbon electrode set at + 750 mV (versus an Ag/AgCl reference electrode) was used. The analytical column (Prospher RP-18, 70×4 mm internal diameter, particle size 5 mm) was purchased from Kanto Kagaku (Japan). The mobile phase contained 0.2 M citrate/0.02 M sodium acetate buffer, containing 3% (v/v) methanol, 130 mg/l octansulfonic sodium and 0.1 mM ethylenediaminetetraacetic acid-2Na. The final pH was 2.5, the column temperature was maintained at 25°C, and the flow was rate set at 1.0 ml/min (Okada et al., 1997d).

2.5. Determination of 5 -HT re-uptake acti6ity The effects of CBZ on 5-HT re-uptake were determined in the hippocampal slices of ten male Wistar rats (body weight 250–300 g). The levels of CBZ in the incubation medium, including 1% methanol (v/v), ranged from 1 to 300 mM. The same amount of methanol was added to the controls. The determination of 5-HT re-uptake activity was performed according to the method of Hirano et al. (1990). Radioactive material, [3H]-5HT, whose specific activity was 500.35 mCi/mmol, was obtained from NEN Research Products (Japan).

190

M. Okada et al. / Epilepsy Research 31 (1998) 187–198

2.6. Statistics The differences between the mean hippocampal extracellular levels of 5-HT, 5-HIAA and 5-HTP for control and drug treated experiments were analyzed using one-way analyses of variance (ANOVA) with a randomized blocked design; Dunnett’s multiple comparison test was also applied. Completely randomized two-way ANOVA and Tukey’s multiple comparison test were applied to the analysis of the differences between the effects of 25-, 50- and 100-mg/kg doses of CBZ on extracellular levels of 5-HT, 5-HIAA and 5-HTP in the hippocampus. Completely randomized oneway ANOVA and Dunnett’s multiple comparison test were applied to the analysis of the effects of vehicle (saline/DMSO, 50/50, v/v) and CBZ on the total levels of 5-HTP, 5-HIAA and 5-HTP in the hippocampus. Differences of P B0.05 were considered to be significant.

3. Results

3.1. Plasma concentration of CBZ The mean plasma CBZ concentrations 2 h after CBZ administration (25, 50 and 100 mg/kg, i.p.) were 33.69 5.7, 139.5930.5 and 226.09 13.9 mM, respectively. The mean plasma CBZ concentrations 3 weeks after CBZ oral administration (25, 50 and 100 mg/kg/day, p.o.) were 23.19 2.3, 84.8 9 32.6 and 107.2 92.15 mM, respectively. However, 2 h after administrations of either 25, 50 or 100 mg/kg CBZ (i.p.) followed 1 h later by the central AADC inhibitor NSD1015 (100 mg/ kg, i.p.), the mean plasma CBZ levels were 70.09 6.1, 173.195.2 and 277.5926.5 mM, respectively. Thus, although both acute and chronic administrations of 25 mg/kg CBZ (i.p.) were within therapeutic range, co-administration of 25 mg/kg CBZ (i.p.) with NSD1015 (100 mg/ kg, i.p.) resulted in supratherapeutic plasma levels of the former (Masuda et al., 1979). Both acute and chronic administrations of 50 and 100 mg/kg CBZ (i.p.) resulted in supratherapeutic plasma concentrations of CBZ, irrespective of whether or not it was administered together with NSD1015.

3.2. Effects of CBZ on extracellular le6els of 5 -HT, 5 -HIAA and 5 -HTP DMSO, used as a solvent for CBZ, showed no significant effect on the levels of total or extracellular 5-HT, 5-HIAA and 5-HTP (data not shown) (Okada et al., 1992, 1997b,c).

3.2.1. Effects of CBZ on extracellular 5 -HT and 5 -HIAA le6els Previous in vitro experiments (Okada et al., 1997a) have demonstrated that a recovery rate of probes for 5-HT (external to internal probes) was 16.5393.42% (data not shown), indicating that the basal hippocampal extracellular 5-HT level in this study was 7.239 0.33 fmol/sample/20 min. Thus, the estimated basal hippocampal extracellular 5-HT level was 43.75 9 2.01 fmol/20 ml (2.1990.10 nM). The effects of CBZ extracellular levels of hippocampal 5-HT and 5-HIAA are shown in Fig. 1a and b, respectively. Both 25 and 50 mg/kg CBZ (i.p.) significantly increased extracellular levels of hippocampal 5-HT and 5-HIAA (PB 0.01). The elevation of hippocampal extracellular 5-HT and 5-HIAA levels induced by 25 mg/kg CBZ (i.p.) was higher than that induced by 50 mg/kg CBZ (i.p.). Conversely, 100 mg/kg CBZ (i.p.) significantly decreased extracellular levels of hippocampal 5-HT and 5-HIAA (PB0.01), in a dose-dependent manner. 3.2.2. Effects of CBZ on extracellular le6els of 5 -HTP Although the hippocampal extracellular 5-HTP level in the perfusate was undetectable under the condition of perfusion with MRS, the hippocampal extracellular levels of 5-HTP were 0.429 0.06 pmol/sample for 20 min under the condition of perfusion with NMRS. In vitro experiments (Okada et al., 1997a) have demonstrated that a recovery rate of probes for 5-HTP (external to internal probes) was 17.4495.34% (data not shown). Thus, the estimated mean level of basal hippocampal extracellular 5-HTP in that study was 2.419 0.32 pmol/20 ml (120.049 16.16 nM), under the condition of perfusion with NMRS.

M. Okada et al. / Epilepsy Research 31 (1998) 187–198

The effects of CBZ on extracellular levels of 5-HTP in the rat hippocampus, under the condition of perfusion with NMRS, are shown in Fig. 2. Administration of 25 mg/kg CBZ (i.p.) significantly increased hippocampal levels of extracellular 5-HTP (P B0.01). On the other hand, 100 mg/kg CBZ (i.p.) significantly decreased hippocampal 5-HTP levels (P B0.01). Administration of 50 mg/kg CBZ (i.p.) had no effect on hippocampal 5-HTP levels.

3.3. Effects of acute and chronic CBZ administration on total le6els of 5 -HT, 5 -HIAA and 5 -HTP in the hippocampus The hippocampal total levels of 5-HT, 5-HIAA and 5-HTP were 3.77 9 0.32, 1.97 9 0.16 and 0.0299 0.004 nmol/g wet weight brain tissue (nmol/ g wwbt), respectively.

3.3.1. Effects of acute administration of CBZ on total le6els of 5 -HT, 5 -HIAA and 5 -HTP in the hippocampus The total levels of 5-HT, 5-HIAA and 5-HTP

191

in the hippocampus 2 h after CBZ administration (25, 50 or 100 mg/kg, i.p.) are shown in Fig. 3a, b and c, respectively. By 2 h after the 25 mg/kg CBZ injection (i.p.), the hippocampal total levels of 5-HT, 5-HIAA and 5-HTP increased significantly (PB0.05), while at the same time interval following the 100 mg/kg CBZ injection (i.p.) these levels were significantly decreased (PB 0.05). The total levels of hippocampal 5-HTP levels were significantly increased (PB 0.01) by 50 mg/kg of CBZ (i.p.), while the same dose of CBZ had no effect on total levels of either hippocampal 5-HT or 5-HIAA.

3.3.2. Effects of chronic administration of CBZ on total le6els of 5 -HT, 5 -HIAA and 5 -HTP in the hippocampus Total levels of 5-HT, 5-HIAA and 5-HTP after chronic CBZ administration for 3 weeks are shown in Fig. 4a, b and c, respectively. The total 5-HT, 5-HIAA and 5-HTP levels were increased significantly (PB 0.05) by a daily dose of 25 mg/

Fig. 1. Effects of CBZ on extracelluar levels of 5-HT and 5-HIAA. Extracelluar levels of hippocampal 5-HT (a) and 5-HIAA (b) were measured in perfusates for 60 min during the pre-drug period (control), and for 120 min after administrations of 25 (closed circles), 50 (opened circles) or 100 (opened squares) mg/kg CBZ (i.p.). The ordinate represents the extracelluar levels of 5-HIAA (% control) and the abscissa shows the time in minutes. The data are expressed as percentage (mean 9S.D., n =6) of control values. Comparisons were made between mean values obtained before and after CBZ administration (*PB0.05, **PB0.01) by one-way ANOVA with randomized blocked design and Dunnett’s multiple comparison test.

192

M. Okada et al. / Epilepsy Research 31 (1998) 187–198

not affect hippocampal 5-HT re-uptake activity (data not shown).

3.5. Effects of CBZ on hippocampal 5 -HTP accumulation The effects of CBZ on hippocampal 5-HTP accumulation are shown in Fig. 5. The hippocampal accumulation level (control) of 5-HTP was 596.569 57.44 pmol/g wwbt. All doses of CBZ significantly inhibited hippocampal 5-HTP accumulation in a dose-dependent manner (PB 0.05).

4. Discussion

Fig. 2. Effects of CBZ on extracelluar levels of 5-HTP. Extracelluar levels of DOPAC and HVA were measured in hippocampal perfusates which included 10 mM of NSD1015 for 60 min during the pre-drug period (control) and for 120 min after administration of 25 (closed circles), 50 (opened circles) or 100 (opened squares) mg/kg CBZ. The ordinate represents the extracelluar levels of DOPAC or HCA (% control) and the abscissa shows the time in minutes. We expressed the data as percentage (mean 9 S.D., n= 6) of control values. Comparisons were made between mean values obtained before and after CBZ administration (*PB 0.05, **PB0.01) by one-way ANOVA with randomized blocked design and Dunnett’s multiple comparison test.

kg CBZ, whereas these levels were significantly decreased by a daily dose of 100 mg/kg CBZ (PB 0.05). A daily dose of 50 mg/kg CBZ significantly decreased the total levels of hippocampal 5-HT (P B 0.05), but the total levels of hippocampal 5-HTP and 5-HIAA remained unchanged.

3.4. Effects of CBZ on hippocampal 5 -HT re-uptake acti6ity The maximal inhibition of hippocampal 5-HT re-uptake activity was less than 10% and this was induced by the concentration of CBZ (300 mM). The IC50 values of CBZ for 5-HT re-uptake activity in hippocampal slices were more than 1 mM. Thus, concentrations of 1 – 300 mM of CBZ did

The minimum plasma concentration of CBZ that is effective against maximal electroshock seizures (MES) in rats has been shown to be 3.7 mg/ml (about 17 mM), while the neurotoxic plasma concentration is higher than 9.3 mg/ml (about 42 mM) (Masuda et al., 1979). In the present study, plasma CBZ levels after both acute and chronic administrations of 25 mg/kg CBZ remained within this therapeutic range, whereas doses of 50 and 100 mg/kg resulted in supratherapeutic levels. Thus, 25 mg/kg CBZ is a therapeutic dose, whereas 50 and 100 mg/kg are supratherapeutic. However, co-administration of a therapeutic dose of CBZ with NSD1015 (100 mg/kg) resulted in supratherapeutic plasma CBZ levels. In the present study, a therapeutic concentration of CBZ (acute administration of 25 mg/kg CBZ, i.p.) increased hippocampal extracellular and total levels of 5-HT, as well as those of its metabolite (5-HIAA) and precursor (5-HTP), indicating that a therapeutic concentration of CBZ enhances hippocampal serotonergic tone. On the other hand, acute administration of 100 mg/kg CBZ (i.p.) reduced hippocampal serotonergic tone, since a corresponding dose of CBZ decreased hippocampal extracellular and total levels of 5-HT, 5-HIAA and 5-HTP. The effects of acute administration of 50 mg/kg CBZ (i.p.) were not consistent, since a corresponding dose of CBZ increased the hippocampal extracellular levels of 5-HT and 5-HIAA, but did not affect the hippocampal extracellular levels of 5-HTP or the

M. Okada et al. / Epilepsy Research 31 (1998) 187–198

total levels of both 5-HT and 5-HIAA. In addition, the hippocampal total levels of 5-HTP were increased by acute administration of 50 mg/kg

193

CBZ (i.p.). These results suggest that a therapeutic concentration of CBZ enhances hippocampal serotonergic function, while a supratherapeutic

Fig. 3. Hippocampal total levels of 5-HT, its precursor and metabolite, 2 h after CBZ administrations. The levels of 5-HT (a), 5-HTP (b) and 5-HIAA (c) were measured in hippocampal brain tissues 2 h after administration of CBZ (25, 50 or 100 mg/kg, i.p.) dissolved in vehicle (saline/DMSO, 50/50 v/v). The abscissa represents the 5-HT, 5-HTP or 5-HIAA levels (mean 9 SD, n = 6) in nmol/g wet weight brain tissue (nmol/g wwbt), and the ordinate shows the CBZ doses (mg/kg body weight). Comparisons were made between mean values for control (vehicle) and CBZ administration (*PB0.05, **PB0.01) by one-way ANOVA with completely randomized design and Tukey’s multiple comparison test.

194

M. Okada et al. / Epilepsy Research 31 (1998) 187–198

Fig. 4. Hippocampal total levels of 5-HT, its precursor and metabolite after administration of CBZ for 3 weeks. The levels of 5-HT (a), 5-HTP (b) and 5-HIAA (c) were measured in hippocampal brain tissues after treatment with CBZ for 3 weeks (0, 25, 50 or 100 mg/kg, p.o.). The abscissa represents the 5-HT, 5-HTP and 5-HIAA levels (mean 9S.D., n = 6) in nmol/g wet weight brain tissue (nmol/g wwbt), and the ordinate shows the CBZ doses (mg/kg body weight). Comparisons were made between mean values for control (0 mg/kg/day CBZ) and CBZ administration (*PB 0.05, **PB 0.01) by one-way ANOVA with completely randomized design and Tukey’s multiple comparison test.

concentration of CBZ reduces it. The inconsistent results of acute administration of 50 mg/kg CBZ (i.p.) on the hippocampal serotonergic system

might be the result of an intermediate phenomenon between administration of 25 and 100 mg/kg CBZ, although the plasma concentration

M. Okada et al. / Epilepsy Research 31 (1998) 187–198

of CBZ 2 h after the administration of 50 mg/kg CBZ was within the supratherapeutic range. Furthermore, the effects of chronic administration of daily doses of CBZ from 25 to 100 mg/kg on hippocampal 5-HT metabolism were almost identical to the effects of acute CBZ administration. Thus, these results suggest that therapeutic concentrations of CBZ enhance hippocampal serotonergic transmission and turnover continuously, without affecting 5-HT re-uptake and monoamine oxidase (MAO) activity (Okada et al., 1997b). Our previous studies have also demonstrated that acute administration of 25 mg/kg CBZ (i.p.) increases hippocampal extracellular and total levels of 5-HT (Kaneko et al., 1993; Mizuno et al., 1994), however the results of this study are inconsistent with those of Pratt et al. (1985) and Elphic et al. (1990). In the report of Pratt et al. (1985), acute administration of both 50 and 100 mg/kg CBZ (i.p.) increased total 5-HT levels in whole brain tissue of Swiss S- or P-strain mice, without an elevation in the rate of incorporation of [3H]-5HT (Pratt et al., 1985). There is no ready explanation for this discrepancy, since these authors did

Fig. 5. Effects of CBZ on hippocampal 5-HTP accumulations. 5-HTP accumulation was measured in hippocampal brain tissues. Graded doses of CBZ (0, 25, 50 or 100 mg/kg, i.p.) were administered 1 h before NSD1015 (100 mg/kg, i.p.) and rats were irradiated by microwave at 7.0 kW × 1.0 s, 1 h later. The abscissa represents 5-HTP levels (mean9 S.D., n= 6) in pmol/g wet weight brain tissue (pmol/g wwbt) and the ordinate shows CBZ doses (mg/kg body weight/day). Comparisons were made between mean values for control and CBZ administration (*PB0.05, **PB0.01) by one-way ANOVA with completely randomized design and Tukey’s multiple comparison test.

195

not determine the plasma CBZ concentration (Pratt et al., 1985). A possible explanation for this, however, lies in the fact that they used whole mouse brain, because the present data suggest the existence of a regional selectivity in the effects of CBZ on 5-HT metabolism, and because our previous study also failed to observe changes in total levels of 5-HT in whole brain tissue of ddY mice following doses of both 50 and 100 mg/kg CBZ (i.p.) (Kaneko et al., 1980). In a 5-HTP accumulation study, Elphic et al. (1990) reported that acute administration of 50 mg/kg CBZ (i.p.) together with NSD1015 reduced hippocampal 5-HT synthesis in male C57B1/6/01a mice (Elphic et al., 1990). However, our present and previous results indicate that CBZ inhibits the monoamine precursor accumulation normally induced by NSD1015 (Okada et al., 1997b). The facilitation of 5-HT neurotransmission by 5-HTP (the endogenous 5-HT precursor), fenfluramine (the 5-HT releaser), fluoxetetine (5HT re-uptake inhibitor) and 5-methoxydimethyltryptamine (a non-selective 5-HT receptor agonist) has been shown to attenuate MES (Browning, 1987). Administration of high doses of 5-HTP or co-administration of low doses of 5-HTP with 5-HT re-uptake inhibitor also shows anticonvulsive activity (Buus Lassen, 1997). In addition, the selective 5-HT re-uptake inhibitor produces a dose-related decrease in the ED50 values for the various conventional antiepileptic drugs to protect against MES-induced tonic-extension seizures (Leander, 1992). In addition, the stimulation of 5-HT receptor function inhibits seizure activities via hyperpolarization of the neurons (Colino and Halliwell, 1987; Wada et al., 1993). Therefore, these results suggest the possibility that these stimulatory effects of therapeutic concentration of CBZ on hippocampal 5-HT function, at least partially, are involved in the mechanisms of antiepileptic action of CBZ. Recently, augmentation therapy has been used increasingly in the treatment of refractory depression. The efficacy of CBZ has been established in this strategy (De la Fuente and Mendlewicz, 1992). On the other hand, the co-administration of therapeutic doses of fluoxetine with a small dose of CBZ has been shown to produce toxic

196

M. Okada et al. / Epilepsy Research 31 (1998) 187–198

5-HT syndrome (Dursun et al., 1993). The repeated administration of selective 5-HT re-uptake inhibitors, which increase extracellular 5-HT levels dramatically (Okada et al., 1997b), produced desensitization of the 5-HT1A receptor, whereas some of these types of agents did not produce a down-regulation of the 5-HT2 receptor (Nelson et al., 1989; Chaupt et al., 1991). Therefore, these results suggest that the elevation of extracellular 5-HT acts most strongly on the 5-HT1A receptor subtype, without a concomitant induction of down-regulation of hippocampal 5-HT function, since, in the present study, both acute and chronic administrations of therapeutic doses of CBZ continuously enhanced hippocampal 5-HT turnover. This clinical and experimental evidence is in line with our present results showing that low doses of CBZ stimulate 5-HT synthesis and transmission without affecting either 5-HT re-uptake or MAO activity (Okada et al., 1997b). At the same time, the present results also give some idea of the mechanisms of the antipsychotic effects of CBZ. One of the major mechanisms of the antiepileptic action of CBZ was considered to be its inhibitory effects on voltageand frequency-dependent Na + channel activity (Schwarz and Grigat, 1989; Macdonald, 1995). CBZ also reduces Ca2 + influx, but the effects of CBZ on Ca2 + have not yet been clarified. Kito et al. (1994) demonstrated, using a patch-clamp technique, the lack of effect off CBZ on various subtypes of Ca2 + channels in the human neuroblastoma NB1 cell (Kito et al., 1994). On the other hand, Yoshimura et al. (1995) demonstrated that CBZ inhibited N-type Ca2 + channel function in cultured bovine adrenal medullary cells (Yoshimura et al., 1995). The inhibition of Na + and N-type Ca2 + channel activity has been shown to reduce basal striatal extracellular 5-HT levels (Sharp et al., 1990). However, in the present study, therapeutic concentration of CBZ increased hippocampal extracellular 5-HT levels. Therefore, the inhibition of Na + and Ca2 + channel activity does not explain the increased extracellular 5-HT levels induced by therapeutic concentrations of CBZ, but may explain the reduction of 5-HT levels induced by supratherapeutic concentrations of CBZ. In addition, we have already reported

that the enhancement of striatal adenosine A1 and A2 receptor functions stimulates hippocampal 5HT release (Okada et al., 1997d) and, simultaneously, suggested that CBZ might be an adenosine A1 receptor antagonist as well as an adenosine A2 receptor agonist (Okada et al., 1997a). These results therefore suggest that the inhibitory effects of CBZ on Na + and Ca2 + mobilization might be masked by other effects of CBZ (i.e. those on adenosine receptor and 5-HT turnover). Therefore, the effects of CBZ on 5-HT release via an effect on Na + channels can be either only excepted at supratherapeutic concentrations or during high frequency activation of neuronal circuits (Okada et al., 1997b,c). Further study is required to confirm this. In conclusion, the present data indicate that CBZ affects serotonergic functions biphasically, like its effects on dopaminergic functions (Okada et al., 1997b), depending upon the dose used; a therapeutic dose (25 mg/kg) enhances serotonergic function, whereas supratherapeutic doses (50 and 100 mg/kg) inhibit serotonergic function. These biphasic effects of CBZ on serotonergic function may support the anticonvulsive effect and may at least in part account for the psychotropic effects of therapeutic doses as well as the side-effects of supratherapeutic doses of this compound.

Acknowledgements This study was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture (05454309 and 09770727), a Grant from the Hirosaki Research Institute for Neurosciences, a Grant from the Japan Epilepsy Research Foundation and a Grant from the Pharmacopsychiatry Research Foundation.

References Browning, R.A., 1987. The role of neurotransmitters in electricshock seizure models. In: Jobe, P.C., Laird, H.E. II (Eds.), Neurotransmitter and Epilepsy. Humana Press, Clifton, NJ, pp. 277 – 320.

M. Okada et al. / Epilepsy Research 31 (1998) 187–198 Buus Lassen, L., 1997. Potent and long-lasting potentiation of two 5-hydroxytryptophan-induced effects in mice by three selective 5-HT uptake inhibitors. Eur. J. Pharmacol. 47, 351 – 358. Chaupt, Y., de Montigny, C., Blier, P., 1991. Presynaptic and postsynaptic modifications of the serotonin system by longterm administration of antidepressant treatment. An in vivo electrophysiologic study in the rat. Neuropharmacology 5, 219 – 229. Colino, A., Halliwell, J.V., 1987. Differential modulation of the three separate K-conductances in hippocampal CA1 neurons by serotonin. Nature 328, 73–77. De la Fuente, J.N., Mendlewicz, J., 1992. Carbamazepine addition in tricyclic antidepressant-resistant unipolar depression. Biol. Psychiatry 32, 369–374. Dursun, S.M., Matthew, V.M., Reveley, M., 1993. Toxic serotonin syndrome after fluxetine plus carbamazepine. Lancet 342, 442 – 443. Elphic, M., Anderson, S.M.P., Hallis, K.F., Granhame-Smith, D.G., 1990. Effects of carbamazepine on 5-hydroxytryptamine function in rodents. Psychopharmacology 100, 49– 53. Fromm, G.H., Terrence, C.F., Maroon, J.C., 1984. Trigeminal neuralgia. Current concepts regarding etiology and pathogenesis. Arch. Neurol. 41, 1204–1207. Glowinski, J., Iversen, L., 1966. Regional studies of catecholamines in the rat brain. J. Neurochem. 13, 665–669. Hashiguti, H., Nakahara, D., Maruyama, W., Naoi, M., Ikeda, T., 1993. Simultaneous determination of in vivo hydroxylation of tyrosine and tryptophan in rat striatum by microdialysis-HPLC: relationship between dopamine and serotonin biosynthesis. J. Neural Transm. 93, 213– 223. Hirano, T., Kaneko, S., Otani, K., Kondo, T., Sasa, H., Fukushima, Y., 1990. Mechanisms of antimanic effects of zotepine. Neuroscience 16, 511–516. Juergens, U., 1987. Simultaneous determination of zonisamide and nine other anti-epileptic drugs and metabolites in serum. J. Chromatogr. 385, 233–240. Kaneko, S., Fukushima, Y., Sato, T., Hiramatsu, M., Mori, A., 1980. Effects of carbamazepine on catecholamine level in mouse brain. IRCS Med. Sci. 9, 80–81. Kaneko, S., Okada, M., Hirano, T., Otani, K., Fukushima, Y., 1993. Carbamazepine and zonisamide increase extracellular dopamine and serotonin levels in vitro, and carbamazepine does not antagonize adenosine effect in vitro: mechanisms of blockade of seizure spread. Jpn. J. Psychiatry Neurol. 47, 371 –373. Keck, P.E.J., McElroy, S.L., Friedman, L.M., 1992. Valproate and carbamazepine in the treatment of panic and posttraumatic stress disorders, withdrawal states, and behavioural dyscontrol syndromes. J. Clin. Psychopharmacol. 12, 36S– 41S. Kito, M., Maehara, M., Watanabe, K., 1994. Antiepileptic drugs — calcium current interaction in cultured human neuroblastoma cells. Seizure 3, 141–149.

197

Leander, J.D., 1992. Fluoxetine, a selective serotonin-uptake inhibitor, enhances the anticonvulsant effect of phenytoin, carbamazepine, and ameltolide (LY20116). Epilepsia 33, 573 – 576. Loiseau, P., Duche, B., 1995. Carbamazepine, clinical use. In: Levy, R.H., Mattson, R.H., Meldrum, B.S. (Eds.), Antiepileptic Drugs, 4th ed. Raven Press, New York, pp. 555 – 566. Macdonald, R.L., 1995. Carbamazepine, mechanisms of action. In: Levy, R.H., Mattson, R.H., Meldrum, B.S. (Eds.), Antiepileptic Drugs, 4th ed. Raven Press, New York, pp. 491 – 498. Masuda, Y., Utsui, Y., Shiraishi, Y., Karasawa, T., Yoshida, K., Shimizu, M., 1979. Relationships between plasma concentrations of diphenylhydantoin, phenobarbital, carbamazepine, and 3-sulfamoylmethyl-1,2-benzisoxazole (AD810), a new anticonvulsant agent, and their anticonvulsant or neurotoxic effects in experimental animals. Epilepsia 20, 623 – 633. Mizuno, K., Okada, M., Kaneko, S., Hirano, T., Tokinaga, N., Chiba, T., Otani, K., Fukushima, Y., 1994. Effects of zonisamide, carbamazepine and valproate on monoamine metabolism in rat striatum and hippocampus. Jpn. J. Psychiatry Neurol. 48, 406 – 408. Nelson, D.R., Thomas, D.R., Johnson, A.M., 1989. Pharmacological effects of paroxetine after repeated administration to animals. Acta Psychiatr. Scand. 80, 21 – 23. Neppe, V.M., 1982. Carbamazepine in the psychiatric patient. Lancet 2, 334. Okada, M., Kaneko, S., Hirano, T., Ishida, M., Kondo, T., Otani, K., Fukushima, Y., 1992. Effects of zonisamide on extracellular levels of monoamine and its metabolites, and on Ca2 + dependent dopamine release. Epilepsy Res. 13, 113 – 119. Okada, M., Kaneko, S., Hirano, T., Mizuno, K., Kondo, T., Otani, K., Fukushima, Y., 1995. Effects of zonisamide on dopaminergic system. Epilepsy Res. 22, 193 – 205. Okada, M., Mizuno, K., Kaneko, S., 1996a. Magnesium ion augmentation of inhibitory effects of adenosine on dopamine release in the rat striatum. Psychiatr. Clin. Neurosci. 50, 147 – 156. Okada, M., Mizuno, K., Kaneko, S., 1996b. Adenosine A1 and A2 receptors modulate extracellular dopamine levels in rat striatum. Neurosci. Lett. 212, 53 – 56. Okada, M., Kiryu, K., Kawata, Y., Mizuno, K., Wada, K., Tasaki, H., Kaneko, S., 1997a. Determination of the effects of caffeine and carbamazepine on striatal dopamine release by in vivo microdialysis. Eur. J. Pharmacol. 321, 181 – 188. Okada, M., Hirano, T., Mizuno, K., Chiba, T., Kawata, Y., Kiryu, K., Wada, K., Tasaki, H., Kaneko, S., 1997b. Biphasic effects of carbamazepine on the dopaminergic system in rat striatum and hippocampus. Epilepsy Res. 28, 143 – 153. Okada, M., Kawata, Y., Kiryu, K., Mizuno, K., Wada, K., Inomata, H., Tasaki, H., Kaneko, S., 1997c. Effects of non-toxic and toxic concentrations of phenytoin on monoamines levels in rat brain. Epilepsy Res. 28, 155 – 163.

198

M. Okada et al. / Epilepsy Research 31 (1998) 187–198 Na + currents in mammalian myelinated nerve fibers. Epilepsia 30, 286 – 294. Sharp, T., Bramwell, S.R., Grahame-Smith, D.G., 1990. Release of endogenous 5-hydroxy-tryptamine in rat ventral hippocampus evoked by electrical stimulation of the dorsal raphe nucleus as detected by microdialysis: sensitivity to tetrodotoxin, calcium and calcium antagonist. Neuroscience 39, 629 – 637. Yoshimura, R., Yanagihara, N., Terao, T., Minami, L., Abe, K., Izumi, F., 1995. Inhibition by carbamazepine of various ion channels — mediated catecholamine secretion in cultured bovine adrenal medullary cells. NaunynSchmiedeberg’s Arch. Pharmacol. 352, 297 – 303. Wada, Y., Nakamura, M., Hasegawa, H., Yamaguchi, N., 1993. Intra-hippocampal injection of 8-hydroxy-2(di-npropylamino) tetralin (8-OH-DPAT) inhibits partial and generalized seizure induced by kindling stimulation in cats. Neurosci. Lett. 159, 179 – 182.

Okada, M., Kawata, Y., Kiryu, K., Wada, K., Tasaki, H., Kaneko, S., 1997d. Effects of adenosine receptor subtypes on hippocampal extracellular serotonin level and serotonin reuptake activity. J. Neurochem. 69, 2581– 2588. Okuma, T., Yamashita, I., Takahashi, R., Itoh, H., Otuki, S., Watanabe, S., Hazama, H., Inagawa, K., 1990. Comparison of the antimanic efficacy of carbamazepine and lithium carbanate by double-blind controlled study. Pharmacopsychiatry 23, 143 – 150. Pratt, J.A., Jenner, P., Marsden, C.D., 1985. Comparison of the effects of benzodiazepines and other anticonvulsant drugs on synthesis and utilization of 5-HT in mouse brain. Neuropharmacology 24, 59–68. Schinder, W., 1960. New N-heterocyclic compounds, US Patent 2,948,718, August 9, 1960. Schwarz, J.R., Grigat, G., 1989. Phenytoin and carbamazepine: potential- and frequency-dependent block of

. .