Serotonin inhibits epileptiform discharge by activation of 5-HT1A receptors in CA1 pyramidal neurons

Neurophamncology, Vol. 36, No. 1102, pp. 1705-1712, 1997 0 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0028.3908/98 $19.00 + 0.00

Pergamon PII: SOO2&3908(9i’)O0134-2

Serotonin. Inhibits Epileptiform Discharge by Activation of 54IT1~ Receptors in CA1 Pyramidal Neurons DELANTHI SALGADO-COMMISSARIAT Department of Pharmacological

and KARIM A. ALKADHI*

and Pharmaceutical Sciences University of Houston, Houston, TX 772045515, US.A. (Accepted 25 June 1997)

Summary-The imti-epileptiform effect of serotonin was characterized in cellular models of epilepsy using electrophysiological recording techniques. In the bicuculline model, both serotonin (20 PM) and its ~-HTIA agonist, 8hydrox:y-2-(di-n-propylamino)tetralin (8-OH-DPAT, 10 PM) completely blocked the epileptiform discharge and caused membrane hyperpolarization and reduction in input resistance. These effects were completely antagclnized by the ~-HT~A receptor antagonist N-t-butyl-3(4-[2-methoxyphenyl]piperazin-l-y1)-2phenyl-propanamide(WAY 100135) (10 PM). Epileptiform discharge induced by positive current injection was also blocked by serotonin. The presence of WAY 100135 renders serotonin ineffective in the same model. In the bicuculline model, epileptiform discharge blocked by serotonin reappeared and was also intensified when BaClz was added to the medium. To rule out the possibility of serotonin-induced hyperpolarization strengthening the inhibitory effect of endogenous Mg2+ on glutamate N-methyl-D-aspartic acid (NMDA) receptor we studied the antiepileptic effect of serotonin in the 0 Mg2+ model. Spontaneous activity and evoked bursts seen with the 0 Mg2+ model were completely blocked by serotonin. WAY 100135 completely antagonized serotonin effects in this model as well. This study provides evidence suggesting that in rat CA1 pyramidal neurorrs, serotonin can inhibit epileptiform activity in a variety of accepted epilepsy cellular models and that inhibition of epileptiform bursts by serotonin may be mediated by activation of the 5-HTiA receptor subtype. @ 1998 Elsevier Science Ltd. All rights reserved.

Keywords-Hippocampus,

Epilepsy model, 8-OH-DPAT, Bicuculline,

That serotonin

may be involved in limiting epileptiform activity was first proposed by Bonnycastle et al. (1957) who suggested that certain anticonvulsant agents increased brain serotonin levels. Since then, a number of studies have been conducted on various animal models of epilepsy (Loscher and Czuczwar, 1985; Sparks and Buckholtz, 1985; Daile:y et al., 1992a,b; Pasini et al., 1992, 1996; Wada et al., 1992,1995; Yan et al., 1994a,b).

Most of these studies were done on whole animals and hence did not target any specific region of the brain. The involvement of endogenous serotonin in the control of excessive activity in the brain has been suggested by studies involving the use of the serotonin transport inhibitor fluoxetine. In one model, the genetically epilepsy-prone rats (GEPRs) where marked deficit of brain serotonin may be the cause of their seizure susceptibility (Dailey et al., 1992a), it was demonstrated that the serotonin transport inhibitor produced anticonvulsant effects (Dailey et al., 1992b; Yan et al., 1995). In another study, systemic as well as focal *To whom correspondence should be addressed. Tel: (713) 743-1212; Fax:(713) 743-1229; E-mail: [email protected].

Fluoxetine.

injections of fluoxetine in the substantia nigra protected rats from limbic seizures (Pasini et al., 1992, 1996). However, although chronic intraperitonial injection of fluoxetine increased seizure threshold, acute single injection was ineffective on hippocampal seizures in rats (Wada et al., 1995). Earlier, we reported an inhibitory effects of serotonin on hippocampal CA1 pyramidal neurons of the SpragueDawley rat (Salgado and Alkadhi, 1995). Similar results were obtained in CA1 neurons of the GEPR (SalgadoCommissariat and Alkadhi, 1996) whose brain is reported to be deficient in serotonin (Dailey et al., 1992a). Since serotonin mediates multiple actions through activation of various receptor subtypes (Zifa and Fillion, 1992; Hoyer et al., 1994), it is important to identify the receptor subtype by which it effects the inhibition of epileptiform activity. One of these subtypes, the 5-HTi* receptor, exists at a high density in the CA1 area of the hippocampus (Pazos and Palacios, 1985). On neurons in this brain area, serotonin produces a profound inhibitory response associated with membrane hyperpolarization that is believed to be mediated by activation of ~-HT~A receptor subtype (Andrade and Nicoll, 1987;

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Fig. 1. (A) Inhibition of an epileptiform burst with the selective ~-HT~A receptor agonist, 8-OH-DPAT. The bursts were evoked by applying a single submaximal stimulus to presynaptic nerves (the Schaffer collateral pathway) in the presence of bicuculline (BIC). In the absence of bicuculline the same stimulus evoked only an EPSP. This figure shows results from one of three neurons tested (RMP and resistance: before 8-OH-DPAT: -73 mV, 65 MR; after: -77 mV, 45 MQ). (B) Complete inhibition of intracellularly-evoked discharge by serotonin (5HT). The burst was evoked by intracellular injection of a positive current (1.7 nA) for 100 msec (Rh4P and resistance: CONTROL: -78 mV, 65 MR; after 5-HT: -86 mV, 45 MQ). (C) Antagonism of the 5-HT-mediated inhibition of action potential bursts in the presence of the 5-I-ITt* receptor antagonist, WAY 100135 (10 PM). No discharge was seen when the slice was superfused with WAY 100135 alone (left) or with a mixture of WAY 100135 and 5-HT (right). The epileptiform discharge was evoked by direct stimulation with a 100 msec positive current. Lower tracings represent current injected. This experiment represents three other similarly treated neurons (RMP and resistance: WAY100125: -82 mV, 32,5 MQ: after 5-HT: -79 mV, 33.5 MO).

Serotonin inhibits epileptiform discharge

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Fig. 2. Pretreatment with the 54ITr~ receptor antagonist, WAY 100135 (10 PM) prevented the usual 5-HTinduced hyperpolarization and reduction in membrane input resistance. Membrane input resistance (A) and resting potential (B) were recorded from a group of four neurons before treatment (control), after treatment with WAY 100135 and mixture of WAY 100135 and 5-HT. No significant difference (one-way analysis of variance, P > 0.05) was observed amon,ggroups. Also shown, (A) changes in membrane input resistance and (B) resting potential (B)

with 5-HT treatment in another series (five to eight neurons). *Significant difference from control is indicated (paired t-test, P < 0.05). Vertical lines are SEM. (C) The 5-HTt* receptor antagonist, WAY 100135 prevented the 5-HT-mediated inhibition of epileptiform bursts evoked in the presence of bicuculline. After treatment with bicuculline and development of epileptiform discharge in response to subthreshold presynaptic nerve stimulation, the neuron was exposed to WAY 100135 (10pM) alone, then to WAY 100135 and 5-HT (20pM) in the continuing presence of bicuculline. These results were seen in three Cdl pyramidal neurons, traces shown are

from one of these neurons.

Colino and Halliwell, 1987; Beck et al., 1992). The present study was conducted to investigate the effects of 5-HTi* receptor agonist and antagonist on cellular models of epilepsy in hippocampal CA1 pyramidal neurons, using conventional electrophysiological techniques. By virtue of the technique employed, we were able to target a specific region of the brain, namely the hippocampus, which is known to be involved in limbic seizures which are generally most resistant to current drug therapy. METHODS

All experiments were conducted on brain slices of male Sprague-Dawley rats (15&300 g). The methods

used in this study are similar to those described earlier (Salgado and Alkadhi, 1995). Briefly, the rat was decapitated and the brain quickly removed from the skull, and placed in ice cold oxygenated (95% 02, 5% CO2) artificial cerebrospinal fluid [ACSF (n&I): NaCl, 127; CaC12, 2.5; KCl, 4.7; MgC12, 1.2; NaHCOs, 22 and NaHzPOs, 1.2; Glucose, 11.0: pH 7.4.). Brain slices (cu 500 pm thick) were obtained from both hemispheres using a tissue slicer (Vibroslice, Campden Instruments Ltd, London, U.K.). After a recovery period, a single slice was placed in a recording chamber, immobilized between two nylon nets, and continuously superfused with oxygenated ACSF at 32°C. Conventional electrophysiological techniques were used for intracellular recording from pyramidal CA1 neurons with glass

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microelectrodes (1.0 mm, Kwik-fil, WPI, Sarasota, Fl, U.S.A.), filled with 4M potassium acetate (tip resistance: 80-16OMR). Action potentials were evoked either directly (intracellular positive current injection) or indirectly by stimulation of the presynaptic nerves at the Schaffer collateral path using a bipolar electrode. Electrical signals were amplified by an Axoclamp 2A amplifier and displayed on an oscilloscope (Kikusui, DSS 5040, Wakasaki, Japan). Signals were stored on video cassettes using a PCM Data recorder (Vetter Model 200, Rebersburg, PA, U.S.A.), and digitized tracings were obtained by capturing signals on a digital oscilloscope (Lecroy 9310, Chestnut Ridge, NY, U.S.A.) and printing output via a laser printer (LaserJet III, Hewlett Packard, Greeley, CO, U.S.A.). Statistical methods Unpaired Student’s t-test was used to compare the means of the two groups. A difference was considered significant when P c 0.05. RESULTS Bicuculline model Epileptiform discharge was induced by a single stimulus to the presynaptic nerves of CA1 pyramidal neurons pretreated with bicuculline (10 PM). The bicuculline-induced epileptiform model is an established experimental paradigm for epilepsy (Schwartzkroin, 1986). Serotonin Earlier work from this laboratory (Salgado and Alkadhi, 1995) showed that serotonin blocked the bicuculline-induced epileptiform activity [see also Fig. 3(A)] as well as bursts evoked by direct intracellular stimulation [Fig. l(B)]. The effects of serotonin were accompanied by a membrane hyperpolarization and decrease in membrane input resistance [Fig. 2(A and B)]. 8-OH-DPAT In order to ascertain the serotonin receptor subtype involved in the inhibition of epileptiform activity, we tested the selective 5-HTl* receptor agonist, 8-hydroxy2-(di-n-propylamino)tetralin (8-OH-DPAT, 1 PM) on the bicuculline (10 PM)-treated slices. This series of experiments indicates that 8-OH-DPAT completely blocks the bursts of action potentials evoked with single stimuli in the presence of bicuculline [Fig. l(A)]. The epileptiform bursts returned when the superfusate was switched to ACSF that contained bicuculline only (not shown). Hyperpolarization and reduction in membrane input resistance accompanied the inhibition of the epileptiform bursts. WAY 100135. To provide additional evidence for the involvement of the 5-HTlA receptor in the serotoninmediated inhibition of epileptiform activity we used the selective ~-HT~A receptor antagonist N-t-butyl-3(4-[2-

methoxyphenyllpiperazin - 1 - yl) - 2 - phenyl - propanamide (WAY 100135). In one series of experiments, untreated slices were superfused with WAY 100135 (10 PM) for ca 10 rnin, following this, serotonin (20 PM) was included in the superfusate. Figure l(C) shows that, in the presence of WAY 100135, serotonin is unable to inhibit the action potential bursts evoked by intracellular positive current injection. The serotonin-mediated hyperpolarization and reduction of membrane input resistance were not observed in the presence of WAY 100135 [Fig. 2(A and B)]. By itself, WAY 100135 did not cause any significant change in membrane potential or membrane resistance. In another series of experiments we tested the 5-HT1* receptor antagonist on bicuculline-treated brain slices. Earlier experiments (Salgado and Alkadhi, 1995) showed that serotonin was effective in blocking the bicucullineinduced epileptiform activity [see also Fig. 3(A)]. In the present series, once epileptiform bursts were obtained in the presence of bicuculline, WAY 100135 was included in the superfusate. Figure 2(C) shows that in the presence of WAY 100135, serotonin (20 PM) did not suppress the synaptically-evoked bursts. Antagonism BaC12

of serotonin

anti-epileptifonn

action by

It has been suggested that activation of the 5-HTl* receptor may lead to opening of a potassium channel which can be blocked by BaC12 (Colino and Halliwell, 1987; Andrade and Nicoll, 1987). To determine if the serotonin-mediated inhibition of epileptiform bursts evoked in the presence of bicuculline can be antagonized by blocking the potassium channel, we used BaC12. Addition of BaC12 (2 mM) not only reversed the inhibitory effect of serotonin on evoked epileptiform discharge; but also intensified bursting [Fig. 3(A)]. Additionally, when BaC12 was left in the superfusate containing serotonin and bicuculline, spontaneous bursting developed [Fig. 3(B and C)]. Both the spontaneous bursting and antagonism of antiepileptiform effects of serotonin were reversed when BaC12 was removed from the superfusate. The magnesium-free ACSF model It is possible that the excitatory effects of glutamate Nmethyl D-aspartic acid (NMDA) receptor activation is indirectly suppressed by the serotonin-induced membrane hyperpolarization which would strengthen the inhibitory effects of Mg2+ on this receptor (Bekkers and Stevens, 1993). To test this possibility, the normal superfusate was switched to Mg2+-free ACSF. In 4060 min, spontaneous epileptiform activity consisted of rhythmic bursting developed. Additionally, single stimuli to the Schaffer collateral pathway evoked bursts of action potentials. When serotonin (20 PM) was included in the superfusate, the spontaneous epileptiform activity as well as the burst evoked by subthreshold synaptic stimulation

Serotonin inhibits epileptiform discharge

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Fig. 4. Inhibition by 5HT of a synaptically evoked action potential burst in a CA1 pyramidal neuron in magnesium-free (0 Mg) ACSF (A). The figure represents consecutive tracings from a single neuron representing three other CA1 pyramidal neurons. Changes in (B) the membrane input resistance and (C) resting potential in the magnesium-free (0 Mg) epilepsy model before and after treatments with 5-HT (20 mM). Each bar is the mean of three neurons. *Significant difference (one-way analysis of variance, P c 0.05) in input resistance was observed between this group and the 0 Mg group only. **Significant difference in resting membrane potential was seen among these groups. Vertical lines represent SEM.

were eliminated [Fig. 4(A)]. As in other models, the inhibition of epileptiform activity by serotonin was characteristically accompanied by hyperpolarization and reduction of membrane input resistance [Fig. 4(B and C)]. The anti-epileptiform effects of serotonin in Mgzf-free ACSF was completely antagonized by the selective 5-HTrA antagonist, WAY 100135 (not shown).

Effect of jluoxetine It has been demonstrated that the serotonin transport inhibitor fluoxetine has anticonvulsant action in a variety of animal models of epilepsy (Dailey et al., 1992b; Pasini et al., 1992, 1996; Wada et al., 1995). We tested fluoxetine on untreated CA1 neurons (eight neurons) of Sprague-Dawley rats. In seven of these experiments, only a small transient hyperpolarization (2-3 mV; lasting ca 5 min) was observed in the presence of fluoxetine (lo N).

DISCUSSION The present experiments demonstrate that the selective 5-HTr* agonist, 8-OH-DPAT (Gozlan et al., 1983), mimics serotonin by causing inhibition of the epileptiform bursts produced in the presence of bicuculline. In a previous study we ruled out the involvement of the 5-HT2 and 5-HTs receptor subtypes in the anti-epileptiform action of serotonin (Salgado and Alkadhi, 1995). We thus assume that the antiepileptic activity of serotonin is due to activation of its 5-HTi* receptor subtype. However, a role for 5-HT2 receptor has been suggested in the development of seizures in hippocampus-kindled cats (Wada et al., 1992). When the 5-HTr* receptor agonist 8-OH-DPAT was tested in pentylenetetrazol-treated mice and rats, it either produced no effect, or in some animals, it decreased the seizure threshold (Loscher and Czuczwar, 1985). In the same study, the anticonvulsant effect of the serotonin precursor 5-hydroxytryptophan (5-HTP) was attenuated

Serotonin inhibits epileptiform discharge by the 5-HT2 antagonist ketanserin suggesting that the 5HT2 receptor subtype may be involved in the serotoninmediated decrease in seizure susceptibility of some parts of the brain. However, since ketanserin has a high affinity for al -adrenoceptors, histamine and dopamine receptors (Janssen, 1983), it is possible that the effect of ketanserin is due to the involvement of these other receptors. Whole animal experiments suggest that the effect of serotonin on seizures may vary according to the model of epilepsy and/or species of animal. However, our study indicates that serotonin can inhibit epileptiform activity in several models of epilepsy in Sprague-Dawley rat brain slices. It is now known that 8-OH-DPAT acts as a full agonist at presynaptic 5-HTi* receptor, however, at postsynaptic 5HTIA receptor sites it is a partial agonist (Andrade and Nicoll, 1987; Blier and DeMontigny, 1990). Activation of the presynaptic 5-HTiA receptors of the dorsal raphe serotonergic neurons, also known as the somatodendritic autoreceptors, leads to a reduction in synthesis, and release of serotonin (De Montigny and Blier, 1992). Antagonism of the serotonin-mediated inhibition of epileptiform activity bly the novel selective 5-HTiA receptor antagonist, WAY 100135 (Fletcher et al., 1993) provided more convincing evidence that the 5HTi* receptor was involved. Interestingly, a serotoninmediated slow excitatory response was observed in hippocampal CA1 pyramidal neurons (Andrade and Chaput, 1991). This was attributed to 5-HT4 receptor activation which was unmasked when the 5-HTiA receptor was antagonized. However, in our study we did not see depolarization with WAY 100135 which may indicate that these oppclsing actions of serotonin did not play a significant role:, perhaps because the 5-HTi* receptor activation predominated. The serotonin-mediated inhibition of epileptiform activity was accompanied by membrane hyperpolarization which would strengthen the magnesium block of the NMDA glutamate receptors-ion channel complex (Herron et al., 1985; Mody et al., 1987; Bekkers and Stevens, 1993). Thus, part of the anticonvulsant action of serotonin could conceivably result from the indirect action of suppressing excitatory amino acid receptor activation. In the present study, serotonin inhibited epileptiform activity in the magnesium-free model as well, indicating that indirect action on NMDA receptors is not a major contributing factor to the anticonvulsant action of serotonin. However, a possible direct action of serotonin on this receptor can not be excluded by the present results. Conversely, in a study on purkinje cells, it was shown that the inhibitory action of serotonin is diminished in a low magnesium medium (Darrow et al., 1991). It was suggested1 that in low magnesium medium, the affinity of a ligand to the 5-HTi* receptor site was decreased. However, se.rotonin also produced a profound excitatory action in purkinje cells. This implies the presence of several subltypes of the serotonin receptor in this region of the brain. In the present study, we examined the CA1 hippocampal region of the brain which is known

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to have a high density of 5-HTi* receptor sites (Pazos and Palacios, 1985). This may be the reason why we did not detect a suppression of serotonin-mediated inhibition in the presence of magnesium. It has been suggested that ~-HT~A receptor activation leads to the opening of a potassium channel that shows inward rectification. It was possible to reverse the 5HTiA-mediated effects by treatment with BaC12 which may have blocked the 5-HTiA-activated potassium channel (Andrade and Nicoll, 1987; Okuhara and Beck, 1994). However, it is well known that Ba*+ is a potent blocker of most potassium channels and has both pre- and post-synaptic actions (Krnjevic et al., 1977). Additionally, Ba*+ can substitute for Ca*+ in permeating Ca2+ channels, although in the presence of Ca*+, Ba*+ severely curtails conductance. In our experiments, the serotonin-mediated inhibition of epileptiform activity was reversed in the presence of BaC12. However, due to possible multiple effects of Ba*+ no clear conclusion can be drawn as to the mechanism that underlies the effect of activation of the ~-HT~A receptor. It is likely that serotonin could have caused the release of y-aminobutyric acid (GABA) which in turn could have led to the changes in membrane properties we observed in the CA1 pyramidal neurons. This study rules out this possibility inasmuch as serotonin produces an inhibitory effect on epileptiform activity even when GABAA receptors are blocked with bicuculline. The failure of fluoxetine to modify activities in CA1 pyramidal neurons is rather surprising, but not unexpected in that our results are in accord with those reported by previous investigators (Andrade and Nicoll, 1987). This does not mean that there is no serotonin in this region inasmuch as the involvement of endogenous serotonin in the control of excessive activity in the brain has been suggested by whole animal studies involving the use of fluoxetine (Pasini et al., 1992, 1996; Wada et al., 1995, Dailey et al., 1995). However, in the hippocampus, although acute single injection was ineffective on hippocampal seizures in rats, a chronic intraperitonial injection of fluoxetine increased the seizure threshold (Wada et al., 1995). This could have been due to the inaccessibility of sites to the drug in the short term. In the brain slices, fluoxetine failed to have an effect because there might not be much serotonin left to be released due to the disruption of the normal connections in this preparation. In summary, the present results provide evidence suggesting that in Sprague-Dawley rat CA1 pyramidal neurons, serotonin inhibits epileptiform discharge in various models of epilepsy by activation of ~-HT~A receptors. REFERENCES Andrade R. and Chaput Y. (1991) 5-Hydroxytryptamine 4-like receptors mediate the slow excitatory response to serotonin in the rat hippocampus. Journal of Pharmucology and Experimental Therapeutics 257: 930-937.

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