Address correspondence and reprint requests to Dr. F.E. Dudek at Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, CO 80523, U.S.A. E-mail: email@example.com
Summary: Purpose: Fluoxetine is a selective serotonin [5-hydroxytryptamine (5-HT)] reuptake inhibitor (SSRI) commonly used to treat depression. Some uncontrolled clinical studies have reported that SSRIs increase seizures, but animal experiments with evoked-seizure models have suggested that SSRIs at therapeutic doses decrease seizure susceptibility. We tested the hypothesis that fluoxetine and trifluoromethylphenylpiperazine (TFMPP, a nonselective 5-HT–receptor agonist) reduce the frequency of spontaneous motor seizures in pilocarpine-treated rats.
Methods: Fluoxetine (20 mg/kg) and TFMPP (5 mg/kg) were administered to rats with pilocarpine-induced epilepsy. Phenobarbital (PB; 10 mg/kg) was a positive control, and saline (i.e., 0.5 ml) controlled for the injection protocol. Each rat received each treatment (intraperitoneally) once per day for 5 consecutive days with 1 week between treatments. Rats were continuously video-monitored for the last 72 h of each treatment.
Results: When compared with saline over the entire 72-h observation period, PB and fluoxetine treatment, but not TFMPP, reduced the spontaneous-seizure rate. Plots of magnitude of the drug effect as a function of seizure frequency after saline treatment revealed larger drug effects for fluoxetine and PB in the rats with the highest control seizure rate. When the data from the five rats with the highest seizure frequency in saline were analyzed for the first 6 h after treatment, TFMPP also significantly reduced seizure frequency.
Conclusions: Animal models with spontaneous seizures can be used to screen potential antiepileptic drugs, and fluoxetine and TFMPP reduce spontaneous seizures in the pilocarpine model of temporal lobe epilepsy.
Individuals with epilepsy have a higher incidence of psychiatric disorders than do those without epilepsy (1,2). Depression is the most frequently reported psychiatric condition in epilepsy patients (3). Epidemiologic studies suggest the comorbidity of patients with both epilepsy and depression to be as high as 66%(4). The most widely used antidepressant drugs are the selective serotonin reuptake inhibitors (SSRIs). Several uncontrolled clinical case studies of patients with depression have reported that SSRI administration increased the seizure frequency (5–8). Many experimental studies have reported SSRI administration to decrease evoked seizures in animals (9–14). The controversy whether SSRIs increase or decrease epileptic activity remains to be resolved.
Neurons containing serotonin (5-HT) are localized mainly in the raphe nuclei of the brainstem, and provide diffuse innervation throughout the brain. Currently it is believed that 5-HT has as many as 13–16 different receptor subtypes in seven classes (15–17). SSRI treatment increases overall levels of 5-HT in the extracellular space, therefore affecting all of the different receptor subtypes. Previous studies have shown that the 1A and 2C receptor subtypes may have an antiepileptic effect (14,18–21), whereas the 5-HT 1B receptor subtype may regulate presynaptic release of neurotransmitters (15,22–24). Trifluoromethylphenylpiperazine (TFMPP) is generally considered to be a mixed agonist for 5-HT receptors (25).
Most evaluations of experimental antiepileptic drugs (AEDs) have used evoked seizure activity (e.g., 9–11), often in normal animals (e.g., 14,26). Animal models that develop a chronic epileptic state with spontaneous seizures, and that have behavioral, electrographic, and anatomic characteristics similar to those of human temporal lobe epilepsy, may be more appropriate for AED testing. Few animal studies, however, have reported AED testing in a spontaneous-seizure model (27). Pilocarpine, a muscarinic cholinergic receptor agonist, causes status epilepticus and spontaneous recurrent seizures that can be observed after a latent period of 2–4 weeks (28–30). The pilocarpine model of temporal lobe epilepsy shows histologic alterations in the hippocampus similar to those of mesial temporal sclerosis and may therefore be useful for testing the effects of experimental AEDs.
By using rats with pilocarpine-induced epilepsy, we tested the hypothesis that fluoxetine (an SSRI) and TFMPP (a mixed 5-HT–receptor agonist) would decrease the frequency of spontaneous motor seizures. Phenobarbital (PB) was administered as the positive control (27). We used a paired design (i.e., each animal served as its own control) to determine drug effects on the frequency of spontaneous motor seizures relative to treatment with saline. We found that PB, fluoxetine and TFMPP—each tested with a single dosage—significantly decreased the frequency of epileptic seizures, but the duration of effect was different (fluoxetine > PB > TFMPP). These experiments suggest that 5-HT receptors reduce the frequency of spontaneous recurrent seizures and thus have an antiepileptic action in the pilocarpine-treated rat.
MATERIALS AND METHODS
Adult male Sprague–Dawley rats (n = 30; 150–200 g; Harlan) were treated with pilocarpine, kept in a standard light/dark cycle (i.e., lights on from 6 a.m. to 6 p.m.), and fed ad libitum. Rats were first injected with methyl-scopolamine (1 mg/kg, i.p.), a muscarinic-receptor antagonist, to prevent the peripheral effects of pilocarpine. The pilocarpine (320 mg/kg, i.p.) was administered 30–45 min after methyl-scopolamine. Approximately 30 min after pilocarpine treatment, rats began to have motor seizures. Seizures were scored according to a modification of the scale of Racine (31), and only motor seizures were considered (i.e., class I and II seizures were not scored, because they involve mouth and facial movements and head nodding, which were not clearly discernible under our monitoring conditions). Seizures were graded as follows: class III, rats displayed forelimb clonus with a lordotic posture; class IV, rats reared with a concomitant forelimb clonus; and class V, rats had a class IV seizure and fell. After 90 min of convulsive seizure activity (status epilepticus), sodium pentobarbital (PTB) was administered (17.5 mg/kg, i.p.) to stop the seizures. Once the seizures were successfully halted, all rats received lactated Ringer's (∼3–6 ml, s.c.). After treatment, the rats were allowed to recover and were housed in the vivarium for 5–6 months for direct observation of spontaneous seizures.
Spontaneous seizures and baseline analysis
All pilocarpine-treated rats (n = 30) were viewed directly during intervals of 1–2 h for 6 h/week. These observational periods occurred during the 12-h period when the lights were on. Behavioral observations of seizure activity were recorded with the same modified Racine (31) scale used during pilocarpine treatment. Behavioral monitoring of seizure activity was initiated 1–5 days after pilocarpine treatment. Only rats demonstrating spontaneous seizures were selected for further baseline analysis (n = 20), which consisted of three consecutive 24-h periods (12/12-h light cycle) of video monitoring and required two sessions (10 rats/session). The 10 animals that were observed to have the highest spontaneous-seizure rates were selected for further study.
Experimental drug treatment
Animals were randomly assigned to four different treatments (i.e., PB, fluoxetine, TFMPP, or saline) throughout the first week of drug administration. PB (Elkins-Sinn, Inc.) was administered at 10 mg/kg (see 32), and this dose was determined to be effective for reducing seizures in a pilot study. Saline was given as the control at an equal volume (i.e., 0.5 ml). Fluoxetine (gift from Eli Lilly & Co.) was administered at 20 mg/kg, and TFMPP (ICN Biomedicals Inc.), at 5 mg/kg (23). All treatments were intraperitoneal injections once per day (i.e., at 1:00 p.m.). Each rat received each treatment for 5 consecutive days with a 1-week recovery period between treatments (e.g., rat 1 received 5 days of TFMPP, 1 week of recovery, 5 days of saline, another week of recovery, 5 days of PB). Some animals were given two saline treatments, and consequently the protocol for these animals was extended to allow for the extra week. Figure 1 illustrates the drug-treatment protocol.
Rats were continuously video monitored for the last 72 h of each treatment (i.e., Wednesday, Thursday, and Friday). The behavior and seizures were recorded on 8-h videotapes from an MTI 65 Silicon Intensified Target camera. One trained technician, blind to all experimental conditions, viewed all videotapes. Seizure activity was rated with the same modified Racine (31) scale used after pilocarpine injections. Seizures were assessed by viewing behavioral postures (i.e., lordosis, straight tail, jumping/running, forelimb clonus, and/or rearing) during fast-forward observation of the videotapes. Once a behavioral posture was observed, the videotape was rewound to the beginning of the behavior and examined at real-time speed. Seizure frequencies for the different treatments were then compared by using repeated-measures, one-way analysis of variance (ANOVA) and a multiple-comparisons Student–Newman–Keuls (SNK) test.
Overall effects of drug treatments
The data were separated into 4-h periods over 72 h to determine if the drugs affected seizure frequency. No effect was apparent after saline treatment (Fig. 2A). PB and fluoxetine caused a marked and persistent decrease in seizure rate (Fig. 2B and C), whereas TFMPP appeared to cause a sharp but transient decrease in seizure rate (note the first 4-h period after drug administration in Fig. 2D). The difference between mean spontaneous-seizure rates after drug treatment versus after saline treatment, over the entire 72 h, was determined for each drug (Fig. 3A). This analysis (i.e., when assessed over the full 3-day observation period) indicated that administration of PB and fluoxetine, but not TFMPP, significantly reduced spontaneous-seizure frequency.
Duration of drug effects
To evaluate the duration of the drug effect on seizure frequency, the data from Fig. 2 were divided into the three 24-h periods, and corresponding 4-h increments were averaged. After saline administration, the average seizure frequency fluctuated ∼0.30 seizures per hour, with no apparent effect of the injection on seizure rate (Fig. 4A). The PB-induced decrease in seizure frequency persisted until just before the next injection (Fig. 4B), whereas the fluoxetine-induced reduction in seizure frequency did not appear to show any recovery during the entire 24-h period between injections (Fig. 4C). TFMPP induced a decrease in seizure frequency immediately after the injection, which then appeared to return quickly to baseline (Fig. 4D). These results suggest that, when analyzed across the 24-h period, saline had little or no effect on seizure frequency; PB decreased seizure frequency for ∼20 h; fluoxetine decreased seizure frequency for ≥24 h; and TFMPP appeared to decrease seizure frequency for only a short period after drug administration. None of the drug treatments had a significant preferential effect on a particular seizure type (i.e., class III vs. IV vs. V; p > 0.05, SNK test).
Control seizure frequency and the magnitude of drug effects
The differences in seizure frequency between saline and drug for each animal were plotted to determine whether the magnitude of the differences across animals was related to the control seizure frequency. Figure 5 shows, for each treatment, the difference in seizure frequency after drug administration and saline as a function of the saline seizure frequency. Linear regression analyses were performed for PB (Fig. 5A), fluoxetine (Fig. 5B), and TFMPP (Fig. 5C). These results suggest that our ability to detect a decrease in seizure frequency during PB and fluoxetine treatments was greater in the pilocarpine-treated rats that had the highest seizure frequency under control conditions. The results from Fig. 5 suggested that a reanalysis of the data from Figs. 3A and 4 should be performed to include only those animals that experienced the highest seizure frequencies during saline treatment (n = 5).
Reanalysis of drug effect on rats with the highest seizure frequency in saline
The data from the five rats with the highest seizure frequencies after saline treatment (see Fig. 4) were analyzed to reassess alterations in seizure frequency for the 24-h period after drug administration. Under these conditions, saline injections may have induced a decrease in seizure frequency (Fig. 6A) lasting a few hours. Similar results to those in Fig. 3A were obtained for fluoxetine and PB (see Fig. 3B), except that fluoxetine was significantly more effective than PB (p < 0.05). Furthermore, TFMPP significantly reduced seizure frequency for several hours after treatment (see Figs. 3C and 6D). Therefore all of the experimental drugs decreased seizure frequency at these doses, but the duration of effect was fluoxetine > PB > TFMPP.
These experiments indicate that a single daily injection of PB, fluoxetine, or TFMPP reduces the frequency of spontaneous motor seizures in the pilocarpine model of temporal lobe epilepsy. Fluoxetine appeared to have a greater and longer effect than PB, which had a longer effect than TFMPP. Animals with a higher seizure frequency after saline treatment showed a decrease in seizure frequency that was larger in magnitude during PB and fluoxetine treatment than did rats with a lower seizure frequency under control conditions. The mixed 5-HT–receptor agonist, TFMPP, also decreased seizure frequency, further suggesting that 5-HT–receptor subtypes may be useful targets for future development of new AEDs.
This study used a paired design (i.e., each animal was its own control) to decrease the effect of interanimal variability. A few disadvantages of this experimental design, however, also became apparent during the study. Because each rat received each treatment, and each treatment was separated by 1 week, the duration of the study was >7 weeks. The long duration of the study combined with multiple intraperitoneal injections may have compromised the health of the rats. These problems, which are more likely to affect the animals with high baseline seizure rates, can be addressed in future studies by altering the experimental design.
The order of drug administration was varied across the different animals to reduce the possibility that one drug caused spurious effects on the testing of a subsequent drug. A 1-week period between tests also was used to avoid interactions between drug administrations. Although our experiments did not directly address this potential problem, the duration of effect of these drugs is short (i.e., hours to days) compared with the recovery period (>1 week). In future studies, additional video monitoring could provide better data on the time course of antiepileptic effects of drugs with a longer-lasting effect (e.g., fluoxetine).
Fluoxetine exerts its effect by blocking 5-HT reuptake at axon terminals, and causes an increase in the overall levels of 5-HT in the extracellular space (33). Yan et al. (34) found that an increase in brain 5-HT levels resulted in an anticonvulsant effect in genetically epilepsy-prone rats. Furthermore, recent studies have demonstrated that short-term administration of fluoxetine, in particular, causes a decrease in the evoked epileptiform activity observed in various rat models of seizures (10–12,35). The effects of fluoxetine on the rate of spontaneous seizures in rats with pilocarpine-induced epilepsy in this study are consistent with these findings.
One issue requiring further consideration is that the usual time required for the antidepressive effect of the SSRIs is 3–8 weeks (33), yet this study analyzed the effects of only 1 week (5 days) of administration. Some uncontrolled case reports involving human patients (5,7,8,36), as well as evoked-seizure models in animals (26), reported an increase in seizure activity. Whether prolonged administration of fluoxetine for several weeks in spontaneous-seizure models would lead to increases or decreases in seizure frequency is unknown, but this issue should be addressed with further experiments.
Another important issue is that this study examined only one dose of each drug, including fluoxetine (20 mg/kg). This dose is relatively high compared with those in other behavioral studies in animals (9,10,35,37), and therefore most likely produced the maximal effect of the drug. Some investigators reported lower doses to be more effective at decreasing seizure activity (35,36), whereas other studies reported similar high doses to be optimally effective (10,11). In contrast, some reports found that higher doses of fluoxetine increase seizure activity (36,38). The question of which doses of fluoxetine exert a therapeutic anticonvulsant effect versus a possible proconvulsant effect also will require further investigation.
Fluoxetine increases the overall levels of 5-HT, and TFMPP is a mixed agonist. Previous studies suggested TFMPP is an agonist for 1A, 1B, and 2C receptor subtypes (39–46). It has been suggested that the 5-HT 1A and 2C receptor subtypes may be directly responsible for the antiepileptic effects observed in previous studies (14,18–21). The 5-HT 1B receptor subtype may contribute to the decreased seizure frequency through its regulatory role in the presynaptic release of neurotransmitters such as glutamate (15,47,48). Although TFMPP did not significantly decrease seizure frequency when analyzed over the 24-h period after drug administration (Fig. 3A and B), a significant decrease in seizure frequency was clearly seen for several hours immediately after TFMPP administration (Figs. 3C and 6D). Although these data may suggest that 1A, 1B, and/or 2C receptor subtypes have an antiepileptic effect, the limitations of this agonist (including lack of dose–response data) indicate that additional experiments are needed before conclusions can be made regarding the potential antiepileptic effects of particular 5-HT–receptor subtypes.
Testing AEDs in an animal model with spontaneous seizures
Animal studies of the effect of fluoxetine on seizures have generally involved evoked seizures in normal or epilepsy-prone animals (e.g., 9–12,26). One possible caveat of evoked-seizure models in normal animals is that the mechanisms responsible for seizure generation may not be the same as those in epilepsy patients or animals. Pilocarpine-treated rats have served as a useful model of temporal lobe epilepsy, and they reproduce some of the key histopathologic features of human temporal lobe epilepsy. Nonetheless, it would be prudent to conduct similar studies in other models that have recurrent spontaneous seizures to reduce the possibility that the observed 5-HT drug effects result from some idiosyncrasy of the pilocarpine model. Furthermore, although rats with high seizure rates have obvious advantages, experimental data (presumably with a different design) are needed for rats with low seizure rates. Finally, our data relate only to motor seizures, and additional research on nonconvulsive electrographic seizures would be more relevant to the complex partial seizures characteristic of temporal lobe epilepsy.
PB, fluoxetine and TFMPP decreased the frequency of spontaneous motor seizures in the pilocarpine model of temporal lobe epilepsy. These results suggest that one or more subtypes of the 5-HT receptor (e.g., 1A, 1B, and/or 2C receptor subtypes) may be important targets for development of new AEDs.
This general type of experimental design in a spontaneous-seizure model appears to be useful for testing AEDs. Several methodologic considerations, such as baseline seizure frequency and duration of the study, are important factors for designing future experiments.
Acknowledgment: This research was supported by NS 16683 (F.E.D.). We thank P. Dou and D.J. Ferraro for technical assistance, and J. Hellier for help during preliminary studies. We also thank H. Shannon, J. Stables, and K. Staley for comments on a draft of the manuscript. We gratefully acknowledge the donation of fluoxetine from Eli Lilly & Company.