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Effects of serotonin on induced epileptiform activity in CA1 pyramidal neurons of genetically epilepsy prone rats
Brain Research 743 Ž1996. 212–216
Research report
Effects of serotonin on induced epileptiform activity in CA1 pyramidal neurons of genetically epilepsy prone rats Delanthi Salgado-Commissariat, Karim A. Alkadhi
)
Department of Pharmacological and Pharmaceutical Sciences UniÕersity of Houston, Houston, TX 77204-5515, USA Accepted 20 August 1996
Abstract The seizure susceptibility in genetically epilepsy prone rats ŽGEPRs. is reported to be caused by abnormalities in several neurotransmitter systems including the serotonergic system. Among the reported abnormalities is a decrease in brain serotonin content. Therefore, we examined the effects of exogenous serotonin on brain slices from the severe seizure strain of GEPRs ŽGEPR-9s.. We employed conventional electrophysiological techniques to record from CA1 pyramidal neurons of hippocampi of GEPR-9s. The membrane resting potential and input resistance of the GEPR-9 CA1 pyramidal neurons were not different from those of the Sprague–Dawley rats. Serotonin Ž20 mM. inhibited the directly and synaptically evoked action potentials in GEPR-9 CA1 neurons, as it did in the Sprague Dawley neurons, but only in some and not all of the neurons tested Žblocked the directly evoked potentials in 57% and synaptically evoked potentials in 33.3% of the total neurons.. This inhibition was also accompanied by hyperpolarization and reduction of membrane input resistance. In the bicuculline-treated brain slices of the GEPR-9, serotonin inhibited the epileptiform bursts causing concurrent hyperpolarization and reduction in membrane input resistance. The effects of the selective serotonin 5-HT1A receptor agonist, 8-OH-DPAT Ž20 mM. on GEPR-9 pyramidal CA1 neurons were similar to those of serotonin, except the magnitude of hyperpolarization and reduction of membrane input resistance were less than those produced by serotonin. We conclude that the apparent decrease in sensitivity of the GEPR-9 CA1 pyramidal neurons to serotonin may represent a deficiency of serotonin 5-HT1A receptor. Keywords: Genetically epilepsy-prone rat strain 9 ŽGEPR-9.; Epilepsy; Serotonin; Electrophysiology
1. Introduction The genetically epilepsy-prone rats ŽGEPRs., described by Dailey et al. w3x, were derived from Sprague–Dawley stock by a selective inbreeding process that resulted in a line of animals susceptible to audiogenic seizures. In this manner, two strains of GEPRs were developed: a moderate seizure strain ŽGEPR-3. and severe seizure strain ŽGEPR9.. They undergo seizures spontaneously and in response to sound, hyperthermia and many other seizure-provoking stimuli. Dailey et al. w4x measured serotonin levels in various regions of the GEPR brain and found a significant decrease in serotonin content in the hippocampus and five other discrete brain areas. They also reported that the GEPR-9 strain has significantly lower serotonin content in the striatum and cerebellum than GEPR-3 or control. Although this study suggests that the level of serotonin in the striatum may determine seizure severity, it does not indi) Corresponding author. Fax: q1 Ž713. 743-1229; E-mail: [email protected]
cate how this reduction in serotonin content relates to seizure susceptibility in the GEPRs. Furthermore, it is unclear in what specific areas in the brain the lack of serotonin content is most responsible for seizure susceptibility. The same group reported that, in the GEPR, serotonin transport inhibitors prevented audiogenic seizures in a dose-dependent manner. The antiepileptic actions of the transport inhibitors were accompanied by a corresponding increase in extracellular serotonin in the thalamus w5,17x. In recent studies, the same group of investigators pharmacologically manipulated the serotonergic transmission to further evaluate the link between brain serotonin levels and the inhibition of epileptiform activity w15,16x. They administer a combination of the serotonin precursor, 5-hydroxytryptophan Ž5-HTP. and transport inhibitor fluoxetine and found that the seizure severity declined in a dose-dependent manner. The combination of these two agents produced a greater reduction of convulsion than either agent alone. They also reported, that the anticonvulsant effect of fluoxetine was diminished with p-chlorophenylalanine ŽPCPA, an inhibitor of tryptophan hydroxylase
0006-8993r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 0 4 4 - X
D. Salgado-Commissariat, K.A. Alkadhir Brain Research 743 (1996) 212–216
which is the rate limiting enzyme in serotonin synthesis.. More recently, a decrease in the in vitro binding of the agonist 8-hydroxy-2-Ždi-n-propylamino.tetralin Žw 3 Hx8OH-DPAT. to serotonin 5-HT1A receptors and an increase in 5-HT1B sites were found in the GEPR-9 hippocampus w13x. In another epilepsy animal model, the DBAr2J mouse, Sparks and Buckholtz w12x have shown that fluoxetine was not so effective at inhibiting audiogenic seizures. However, when the mice were treated with a combination of fluoxetine and the MAO-A inhibitor, clorgyline, seizures were effectively inhibited. Thus, it is still unclear whether the antiepileptic effects are directly related to the action of serotonin. The purpose of this study was to determine susceptibility to epileptiform activity and evaluate the effects of serotonin in pyramidal neurons of the CA1 region of the GEPR-9 hippocampus using electrophysiological techniques.
2. Materials and methods The methods in this study were as described earlier w11x. Briefly, male rats ŽGEPR-9 or Sprague–Dawley; 200–300 g body weight. were decapitated and the brain quickly removed from the skull and placed in ice-cold artificial cerebrospinal fluid ŽACSF ŽmM.: NaCl, 127; CaCl 2 , 2.5; KCl, 4.7; MgCl 2 , 1.2; NaHCO 3 , 22 and NaH 2 PO 3 , 1.2; Glucose, 11.0: pH 7.4.. Brain slices Žapproximately 0.5 mm thick. were obtained from both hemispheres using a tissue slicer ŽVibroslice, Campden Instruments.. The slices were then transferred to a beaker containing ACSF at room temperature and continuously bubbled with a mixture of 95% O 2r5% CO 2 . After a recovery period, a slice was transferred to a recording chamber, immobilized between two nylon nets and continuously superfused with oxygenated ACSF at 328C. Conventional electrophysiological techniques for intracellular recording from pyramidal CA1 neurons were employed. The glass microelectrodes Ž1.0 mm, Kwik-fil, WPI., filled with 4 M potassium acetate, had tip resistance of 80–160 M V. Action potentials were evoked either directly Žintracellular positive current injection. or indirectly by stimulation of the presynaptic nerves at the Schaffer collateral pathway using a bipolar electrode. Electrical signals were amplified by Axoclamp 2A amplifier and displayed on an oscilloscope ŽKikusui, DSS 5040.. Signals were stored on video tapes using a PCM Data recorder ŽVetter Model 200.. Digitized tracings were obtained by capturing signals on an oscilloscope ŽLecroy 9310. and printing output via a laser printer ŽLaser Jet III, Hewlett Packard.. 2.1. Animals and drugs GEPR-9s were purchased from the University of Illinois, College of Medicine, Peroria, IL and Sprague–Daw-
213
ley rats from Harlan Sprague–Dawley. Drugs were purchased from Research Biochemicals International ŽRBI.. Bicuculline, serotonin and 8-OH-DPAT stock solutions were made with distilled water. 2.2. Statistical methods Membrane properties of two different groups of CA1 neurons Že.g. GEPR vs. Sprague Dawley. were compared using the unpaired t-test. When comparing membrane properties before and after drug treatment in the same neuron, we used paired t-test. Statistical significance was assumed when p - 0.05.
3. Results 3.1. Basic membrane properties of GEPR-9 CA1 neurons Stable intracellular recordings were obtained from 20 pyramidal neurons in GEPR-9 hippocampal CA1 area. Neurons investigated had a stable resting membrane potential Žmean " S.E.M.: 72 " 6 mV. and input resistance Ž53 " 8 M V .. Neurons did not exhibit unusual spontaneous activity. The membrane resting potential and input resistance of the GEPR-9 CA1 neurons were not significantly different from those of the CA1 neurons of the Sprague–Dawley rats ŽFig. 1.. 3.2. Effect of serotonin on untreated GEPRS brain slices In the Sprague–Dawley rats, both directly and synaptically evoked discharge were inhibited in 100% of CA1
Fig. 1. Comparison of the membrane resting potential ŽA. and input resistance ŽB. in untreated hippocampal CA1 pyramidal neurons of Sprague–Dawley ŽS.D.. rats and genetically epilepsy-prone rats ŽGEPR.. Bars are the means of 5–8 neurons. Vertical lines are S.E.M. No significant difference was observed between the two groups Žunpaired t-test, P ) 0.05..
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D. Salgado-Commissariat, K.A. Alkadhir Brain Research 743 (1996) 212–216
Fig. 2. The effects of serotonin Ž20 mM. on hippocampal CA1 pyramidal neurons of GEPR-9. Panel A shows the inhibition of a burst of action potentials evoked by direct stimulation Žpositive current for 100 ms. recorded from a representative neuron. Similar results were seen in five neurons of nine total neurons tested. Panel B shows the inhibition by serotonin of an action potential evoked with synaptic stimulation in a representative CA1 pyramidal neuron. A bipolar electrode was placed on the CA2rCA3 region to stimulate the Schaffer collateral pathway. Similar results were obtained in three neurons out of a total of nine neurons tested.
neurons tested w11x. The actions of serotonin Ž20 mM. on GEPR-9 pyramidal CA1 neurons were qualitatively similar to those seen with the Sprague–Dawley rats. However,
serotonin reversibly inhibited the action potential bursts evoked by intracellular current injection Ždirect stimulation, Fig. 2A. in only 57% of the GEPR-9 neurons tested.
Fig. 3. A: changes in the membrane resting potential Žleft. and input resistance Žright. of GEPR-9 CA1 pyramidal neurons produced by serotonin Ž5-HT, 20 mM.. Bars are the means of seven neurons. Vertical lines are S.E.M. The asterisk indicates significant difference from the control Žpaired t-test, P - 0.05.. B: inhibition by serotonin Ž5-HT. of epileptiform bursts in bicuculline ŽBIC, 10 mM. pre-treated pyramidal CA1 neurons of GEPR-9 brain slices. The burst was evoked by a single subthreshold pulse to the Schaffer collateral pathway. Similar results were obtained in three other neurons.
D. Salgado-Commissariat, K.A. Alkadhir Brain Research 743 (1996) 212–216
215
3.4. Effect of 8-OH-DPAT on untreated GEPRS brain slices To ascertain the involvement of 5-HT1A receptors in the anti-epileptic effects of serotonin, we treated GEPR-9 brain slices with the selective 5-HT1A receptor agonist, 8-OHDPAT Ž20 mM.. The drug produced effects similar to those of serotonin. A comparison of the effects of 8-OHDPAT on membrane properties with those obtained in the presence of serotonin is shown in Table 1.
4. Discussion Fig. 4. Effect of serotonin on the I – V curve of a representative pyramidal CA1 neuron of hippocampus of GEPR. The reversal potential in the presence of serotonin is close to y80 mV in all four neurons tested.
Our experiments on CA1 pyramidal neurons of the GEPR-9 demonstrate that serotonin and its selective 5-HT1A receptor agonist, 8-OH-DPAT, produced inhibitory effects qualitatively similar to those reported in Sprague–Dawley rat brain slices w11x. As with Sprague–Dawley rats, the present results implicate the involvement of serotonin 5HT1A receptor in this anticonvulsant effect of serotonin. It has been demonstrated that 5-HT1A receptor activation leads to the opening of a potassium channel that shows inward rectification. The 5-HT1A mediated effects were reversed on treatment with the 5-HT1A potassium channel blocker BaCl 2 w1,10x. In the present experiments, the reversal potential of the GEPR-9 CA1 neurons in the presence of serotonin was close to y80 mV indicating an opening of a Kq channel population typical of activation of 5-HT1A receptors. The present results also suggest that a large number of GEPR-9 CA1 pyramidal neurons are not responsive to serotonin. This may be due to possible defects in these neurons such as paucity of serotonin receptors, deficiency in the receptor-signal transduction mechanism or alteration in the serotonin reuptake mechanism. The decrease in the responsiveness of the hippocampal CA1 region to serotonin was unexpected, particularly in light of the reported reduction in hippocampal serotonin content in the GEPR brain w4x. Ordinarily, such a reduction should have led to upregulation of serotonin receptors. However, a recent study w13x indicates a decrease in 8OH-DPAT binding sites in GEPR-9 hippocampus which supports the proposition of diminished 5-HT1A receptor sites. Our results indicate no difference in the membrane resting potential and input resistance between GEPR-9 and Sprague–Dawley CA1 pyramidal neurons. This is at vari-
Synaptically evoked action potentials were also reversibly blocked by serotonin ŽFig. 2B. but only in about 33% of the neurons tested. Where there was inhibition of action potentials by serotonin, concurrent membrane hyperpolarization and reduction of input resistance were also observed in these neurons ŽFig. 3A.. Using current clamp method Žconstant currents ranging from y1.0 to q0.2 nA were injected and the resultant changes in membrane potential were measured., current– voltage Ž I–V . curves were constructed for GEPR-9 pyramidal CA1 neurons. The reversal potential in the presence of serotonin was close to y80 mV ŽFig. 4.. 3.3. Effect of serotonin on bicuculline-treated GEPR-9 brain slices Synaptically evoked epileptiform activity was induced by treatment of GEPR-9 brain slices with bicuculline Ž10 mM. w2x. The bursts of action potentials evoked in the presence of bicuculline, were inhibited by serotonin in GEPR-9 pyramidal CA1 neurons ŽFig. 3B.. However, the degree of inhibition of the epileptiform bursts was less in the GEPR-9 neurons than in neurons from Sprague–Dawley rats Žnumber of action potentialsrburst in Sprague– Dawley: 3.3 " 0.5, after serotonin 0.3 " 0.3, n s 4; in GEPR-9: 3.3 " 0.9, after serotonin 1.3 " 0.3, n s 3.. Here also, the inhibition of epileptiform activity was accompanied by hyperpolarization and reduction in membrane input resistance.
Table 1 Changes in resting membrane potential ŽRMP. and membrane input resistance in CA1 pyramidal neurons of GEPR-9 induced by 5-HT and 8-OH-DPAT DrugŽ20 mM.
5-HT 8-OH-DPAT
ResistanceŽM V .
RMPŽmV. Control
After drug
y72.5 " 4.4 y71.9 " 2.5
y78.1 " 3.5 y76.5 " 2.9
) )
Control
After drug
47.7 " 4.6 36.4 " 7.2
31.5 " 3.2 30.1 " 6.9
The slices were treated with either serotonin Ž5-HT, 20 mM, seven neurons. or 8-OH-DPAT Ž20 mM, four neurons.. a Indicates significant difference from the control Žpaired t-test; P - 0.05, " indicates S.E.M. values..
) )
216
D. Salgado-Commissariat, K.A. Alkadhir Brain Research 743 (1996) 212–216
ance with a report by Verma-Ahuja and Pencek w14x who, although found no difference in resting membrane potential, reported an increase in membrane resistance in GEPR9 CA1 pyramidal neurons as compared to those of the Sprague–Dawley rats. The reason for this discrepancy is unclear. It may reflect different methodology and test conditions. An alternative explanation may be provided by the results of several studies that indicate abnormalities in GABAergic transmission in the GEPR brain w7,8x. It is possible that fewer GABA receptor-linked chloride channels are open at the resting state hence the higher membrane input resistance. However, it is rather surprising that such a large difference in the membrane input resistance between Sprague Dawley and GEPR CA1 neurons can exist with no detectable difference in the resting membrane potential. In the GEPR-9 model of epilepsy, sensory stimuli initiate generalized seizures w6x. Although GEPRs may experience spontaneous seizures w3,9x, the epileptiform activity in these animals is usually triggered by stimuli such as sound, hyperthermia, electroshock and kindling. In our experiments, we treated GEPR-9 brain slices with bicuculline to evoke epileptiform bursts. Our results are in accord with those from intact animal studies w5,15,16x. It was demonstrated that fluoxetine or a combination of fluoxetine and 5-HTP but not PCPA produced anticonvulsant activity in GEPRs w15,16x. Coincidental with the anticonvulsant activity of these agents, an increase in thalamic serotonin levels was detected w15x. Thus in an indirect fashion, these investigators have shown that the serotonin system may be involved in anticonvulsant activity in the brain of these animals. The present study provides more direct evidence for the involvement of serotonin in the inhibition of epileptiform activity by demonstrating the inhibitory effects of exogenously applied serotonin and the selective 5-HT1A agonist 8-OH-DPAT on the hippocampal region of the GEPR-9 brain.
References w1x Andrade, R. and Nicoll, R.A., A G-protein couples serotonin and GABA B receptors to the same channels in hippocampus, Science, 234 Ž1986. 1261–1265.
w2x Curtis, D.R., Duggan, A.W., Felix, D. and Johnston, G.A.R., GABA, bicuculline and central inhibition, Nature, 226 Ž1970. 1222–1224. w3x Dailey, J.W., Riegel, C.E., Mishra, P.K. and Jobe, P.C., Neurobiology of seizure predisposition in the genetically epilepsy-prone rat, Epilepsy Res., 3 Ž1989. 3–17. w4x Dailey, J.W., Mishra, P.K., Ko, K.H., Penny, J.E. and Jobe, P.C., Serotonergic abnormalities in the central nervous system of seizurenaive genetically epilepsy-prone rats, Life Sci., 50 Ž1992. 319–326. w5x Dailey, J.W., Yan, Q.S., Mishra, P.K., Burger, R.C. and Jobe, P.C., Effects of fluoxetine on convulsions and on brain serotonin as detected by microdialysis in genetically epilepsy-prone rats, J. Pharmacol. Exp. Ther., 260 Ž1992. 533–540. w6x Faingold, C.L., The genetically epilepsy-prone rat, Gen. Pharmacol., 19 Ž1988. 331–338. w7x Faingold, C.L., Gehlbach, G. and Caspary, D.M., Decreased effectiveness of GABA-mediated inhibition in the inferior colliculus of the genetically epilepsy-prone rat, Exp. Neurol., 93 Ž1986. 145–159. w8x Faingold, C.L., Gehlbach, G., Tavis, M.A. and Caspary, D.M., Inferior colliculus neuronal response abnormalities in genetically epilepsy prone rats: evidence for a deficit of inhibition, Life Sci., 39 Ž1986. 869–878. w9x Jobe, P.C. Mishra, P.K. and Dailey, J.W., Genetically epilepsy prone rats: actions of antiepileptic drugs and monoaminergic neurotransmitter. In C.L. Faingold and G. Fromm ŽEds.., Drugs for the Control of Epilepsy: Actions on Neuronal Networks InÕolÕed in Seizures, CRC Press, Boca Raton, FL, 1992, pp. 235–275. w10x Okuhara, D.Y. and Beck. S.G., 5-HT1A receptor linked to inwardrectifying potassium current in hippocampal CA3 pyramidal Cells, J. Neurophysiol., 71 Ž1994. 2161–2167. w11x Salgado, D. and Alkadhi, K.A., Inhibition of epileptiform activity by serotonin in rat CA1 neurons, Brain Res., 669 Ž1995. 176–182. w12x Sparks, D.L. and Buckholtz, N.S., Combined inhibition of serotonin uptake and oxidative deamination attenuates audiogenic seizures in DBAr2J mice, Pharmacol. Biochem. BehaÕ., 23 Ž1985. 753–757. w13x Statnick, M.A., Dailey, J.W., Jobe, P.C. and Browning, R.A., Abnormalities in 5-HT1A and 5-HT1B receptor binding on severe-seizure genetically epilepsy-prone rats ŽGEPR-9s., Neuropharmacology, 35 Ž1996. 111–118. w14x Verma-Ahuja, S. and Pencek, T.L., Hippocampal CA1 neuronal properties in genetically epilepsy-prone rats: evidence for increased excitation, Epilepsy Res., 18 Ž1994. 205–215. w15x Yan, Q.-S., Jobe, P.C. and Dailey, J.W., Evidence that a serotonergic mechanism is involved in the anticonvulsant effect of fluoxetine in genetically epilepsy-prone rats, Eur. J. Pharmacol., 252 Ž1994. 105–112. w16x Yan, Q.-S., Jobe, P.C., Cheong, J.H., Ko, K.H. and Dailey, J.W., Role of serotonin in the anticonvulsant effect of fluoxetine in genetically epilepsy-prone rats, Naunyn-Schmiedeberg’s Arch. Pharmacol., 350 Ž1994. 149–151. w17x Yan, Q.-S., Jobe, P.C. and Dailey, J.W., Further evidence of anticonvulsant role for 5-hydroxytryptamine in genetically epilepsyprone rats, Br. J. Pharmacol., 115 Ž1995. 1314–1318.
Research report
Effects of serotonin on induced epileptiform activity in CA1 pyramidal neurons of genetically epilepsy prone rats Delanthi Salgado-Commissariat, Karim A. Alkadhi
)
Department of Pharmacological and Pharmaceutical Sciences UniÕersity of Houston, Houston, TX 77204-5515, USA Accepted 20 August 1996
Abstract The seizure susceptibility in genetically epilepsy prone rats ŽGEPRs. is reported to be caused by abnormalities in several neurotransmitter systems including the serotonergic system. Among the reported abnormalities is a decrease in brain serotonin content. Therefore, we examined the effects of exogenous serotonin on brain slices from the severe seizure strain of GEPRs ŽGEPR-9s.. We employed conventional electrophysiological techniques to record from CA1 pyramidal neurons of hippocampi of GEPR-9s. The membrane resting potential and input resistance of the GEPR-9 CA1 pyramidal neurons were not different from those of the Sprague–Dawley rats. Serotonin Ž20 mM. inhibited the directly and synaptically evoked action potentials in GEPR-9 CA1 neurons, as it did in the Sprague Dawley neurons, but only in some and not all of the neurons tested Žblocked the directly evoked potentials in 57% and synaptically evoked potentials in 33.3% of the total neurons.. This inhibition was also accompanied by hyperpolarization and reduction of membrane input resistance. In the bicuculline-treated brain slices of the GEPR-9, serotonin inhibited the epileptiform bursts causing concurrent hyperpolarization and reduction in membrane input resistance. The effects of the selective serotonin 5-HT1A receptor agonist, 8-OH-DPAT Ž20 mM. on GEPR-9 pyramidal CA1 neurons were similar to those of serotonin, except the magnitude of hyperpolarization and reduction of membrane input resistance were less than those produced by serotonin. We conclude that the apparent decrease in sensitivity of the GEPR-9 CA1 pyramidal neurons to serotonin may represent a deficiency of serotonin 5-HT1A receptor. Keywords: Genetically epilepsy-prone rat strain 9 ŽGEPR-9.; Epilepsy; Serotonin; Electrophysiology
1. Introduction The genetically epilepsy-prone rats ŽGEPRs., described by Dailey et al. w3x, were derived from Sprague–Dawley stock by a selective inbreeding process that resulted in a line of animals susceptible to audiogenic seizures. In this manner, two strains of GEPRs were developed: a moderate seizure strain ŽGEPR-3. and severe seizure strain ŽGEPR9.. They undergo seizures spontaneously and in response to sound, hyperthermia and many other seizure-provoking stimuli. Dailey et al. w4x measured serotonin levels in various regions of the GEPR brain and found a significant decrease in serotonin content in the hippocampus and five other discrete brain areas. They also reported that the GEPR-9 strain has significantly lower serotonin content in the striatum and cerebellum than GEPR-3 or control. Although this study suggests that the level of serotonin in the striatum may determine seizure severity, it does not indi) Corresponding author. Fax: q1 Ž713. 743-1229; E-mail: [email protected]
cate how this reduction in serotonin content relates to seizure susceptibility in the GEPRs. Furthermore, it is unclear in what specific areas in the brain the lack of serotonin content is most responsible for seizure susceptibility. The same group reported that, in the GEPR, serotonin transport inhibitors prevented audiogenic seizures in a dose-dependent manner. The antiepileptic actions of the transport inhibitors were accompanied by a corresponding increase in extracellular serotonin in the thalamus w5,17x. In recent studies, the same group of investigators pharmacologically manipulated the serotonergic transmission to further evaluate the link between brain serotonin levels and the inhibition of epileptiform activity w15,16x. They administer a combination of the serotonin precursor, 5-hydroxytryptophan Ž5-HTP. and transport inhibitor fluoxetine and found that the seizure severity declined in a dose-dependent manner. The combination of these two agents produced a greater reduction of convulsion than either agent alone. They also reported, that the anticonvulsant effect of fluoxetine was diminished with p-chlorophenylalanine ŽPCPA, an inhibitor of tryptophan hydroxylase
0006-8993r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 0 4 4 - X
D. Salgado-Commissariat, K.A. Alkadhir Brain Research 743 (1996) 212–216
which is the rate limiting enzyme in serotonin synthesis.. More recently, a decrease in the in vitro binding of the agonist 8-hydroxy-2-Ždi-n-propylamino.tetralin Žw 3 Hx8OH-DPAT. to serotonin 5-HT1A receptors and an increase in 5-HT1B sites were found in the GEPR-9 hippocampus w13x. In another epilepsy animal model, the DBAr2J mouse, Sparks and Buckholtz w12x have shown that fluoxetine was not so effective at inhibiting audiogenic seizures. However, when the mice were treated with a combination of fluoxetine and the MAO-A inhibitor, clorgyline, seizures were effectively inhibited. Thus, it is still unclear whether the antiepileptic effects are directly related to the action of serotonin. The purpose of this study was to determine susceptibility to epileptiform activity and evaluate the effects of serotonin in pyramidal neurons of the CA1 region of the GEPR-9 hippocampus using electrophysiological techniques.
2. Materials and methods The methods in this study were as described earlier w11x. Briefly, male rats ŽGEPR-9 or Sprague–Dawley; 200–300 g body weight. were decapitated and the brain quickly removed from the skull and placed in ice-cold artificial cerebrospinal fluid ŽACSF ŽmM.: NaCl, 127; CaCl 2 , 2.5; KCl, 4.7; MgCl 2 , 1.2; NaHCO 3 , 22 and NaH 2 PO 3 , 1.2; Glucose, 11.0: pH 7.4.. Brain slices Žapproximately 0.5 mm thick. were obtained from both hemispheres using a tissue slicer ŽVibroslice, Campden Instruments.. The slices were then transferred to a beaker containing ACSF at room temperature and continuously bubbled with a mixture of 95% O 2r5% CO 2 . After a recovery period, a slice was transferred to a recording chamber, immobilized between two nylon nets and continuously superfused with oxygenated ACSF at 328C. Conventional electrophysiological techniques for intracellular recording from pyramidal CA1 neurons were employed. The glass microelectrodes Ž1.0 mm, Kwik-fil, WPI., filled with 4 M potassium acetate, had tip resistance of 80–160 M V. Action potentials were evoked either directly Žintracellular positive current injection. or indirectly by stimulation of the presynaptic nerves at the Schaffer collateral pathway using a bipolar electrode. Electrical signals were amplified by Axoclamp 2A amplifier and displayed on an oscilloscope ŽKikusui, DSS 5040.. Signals were stored on video tapes using a PCM Data recorder ŽVetter Model 200.. Digitized tracings were obtained by capturing signals on an oscilloscope ŽLecroy 9310. and printing output via a laser printer ŽLaser Jet III, Hewlett Packard.. 2.1. Animals and drugs GEPR-9s were purchased from the University of Illinois, College of Medicine, Peroria, IL and Sprague–Daw-
213
ley rats from Harlan Sprague–Dawley. Drugs were purchased from Research Biochemicals International ŽRBI.. Bicuculline, serotonin and 8-OH-DPAT stock solutions were made with distilled water. 2.2. Statistical methods Membrane properties of two different groups of CA1 neurons Že.g. GEPR vs. Sprague Dawley. were compared using the unpaired t-test. When comparing membrane properties before and after drug treatment in the same neuron, we used paired t-test. Statistical significance was assumed when p - 0.05.
3. Results 3.1. Basic membrane properties of GEPR-9 CA1 neurons Stable intracellular recordings were obtained from 20 pyramidal neurons in GEPR-9 hippocampal CA1 area. Neurons investigated had a stable resting membrane potential Žmean " S.E.M.: 72 " 6 mV. and input resistance Ž53 " 8 M V .. Neurons did not exhibit unusual spontaneous activity. The membrane resting potential and input resistance of the GEPR-9 CA1 neurons were not significantly different from those of the CA1 neurons of the Sprague–Dawley rats ŽFig. 1.. 3.2. Effect of serotonin on untreated GEPRS brain slices In the Sprague–Dawley rats, both directly and synaptically evoked discharge were inhibited in 100% of CA1
Fig. 1. Comparison of the membrane resting potential ŽA. and input resistance ŽB. in untreated hippocampal CA1 pyramidal neurons of Sprague–Dawley ŽS.D.. rats and genetically epilepsy-prone rats ŽGEPR.. Bars are the means of 5–8 neurons. Vertical lines are S.E.M. No significant difference was observed between the two groups Žunpaired t-test, P ) 0.05..
214
D. Salgado-Commissariat, K.A. Alkadhir Brain Research 743 (1996) 212–216
Fig. 2. The effects of serotonin Ž20 mM. on hippocampal CA1 pyramidal neurons of GEPR-9. Panel A shows the inhibition of a burst of action potentials evoked by direct stimulation Žpositive current for 100 ms. recorded from a representative neuron. Similar results were seen in five neurons of nine total neurons tested. Panel B shows the inhibition by serotonin of an action potential evoked with synaptic stimulation in a representative CA1 pyramidal neuron. A bipolar electrode was placed on the CA2rCA3 region to stimulate the Schaffer collateral pathway. Similar results were obtained in three neurons out of a total of nine neurons tested.
neurons tested w11x. The actions of serotonin Ž20 mM. on GEPR-9 pyramidal CA1 neurons were qualitatively similar to those seen with the Sprague–Dawley rats. However,
serotonin reversibly inhibited the action potential bursts evoked by intracellular current injection Ždirect stimulation, Fig. 2A. in only 57% of the GEPR-9 neurons tested.
Fig. 3. A: changes in the membrane resting potential Žleft. and input resistance Žright. of GEPR-9 CA1 pyramidal neurons produced by serotonin Ž5-HT, 20 mM.. Bars are the means of seven neurons. Vertical lines are S.E.M. The asterisk indicates significant difference from the control Žpaired t-test, P - 0.05.. B: inhibition by serotonin Ž5-HT. of epileptiform bursts in bicuculline ŽBIC, 10 mM. pre-treated pyramidal CA1 neurons of GEPR-9 brain slices. The burst was evoked by a single subthreshold pulse to the Schaffer collateral pathway. Similar results were obtained in three other neurons.
D. Salgado-Commissariat, K.A. Alkadhir Brain Research 743 (1996) 212–216
215
3.4. Effect of 8-OH-DPAT on untreated GEPRS brain slices To ascertain the involvement of 5-HT1A receptors in the anti-epileptic effects of serotonin, we treated GEPR-9 brain slices with the selective 5-HT1A receptor agonist, 8-OHDPAT Ž20 mM.. The drug produced effects similar to those of serotonin. A comparison of the effects of 8-OHDPAT on membrane properties with those obtained in the presence of serotonin is shown in Table 1.
4. Discussion Fig. 4. Effect of serotonin on the I – V curve of a representative pyramidal CA1 neuron of hippocampus of GEPR. The reversal potential in the presence of serotonin is close to y80 mV in all four neurons tested.
Our experiments on CA1 pyramidal neurons of the GEPR-9 demonstrate that serotonin and its selective 5-HT1A receptor agonist, 8-OH-DPAT, produced inhibitory effects qualitatively similar to those reported in Sprague–Dawley rat brain slices w11x. As with Sprague–Dawley rats, the present results implicate the involvement of serotonin 5HT1A receptor in this anticonvulsant effect of serotonin. It has been demonstrated that 5-HT1A receptor activation leads to the opening of a potassium channel that shows inward rectification. The 5-HT1A mediated effects were reversed on treatment with the 5-HT1A potassium channel blocker BaCl 2 w1,10x. In the present experiments, the reversal potential of the GEPR-9 CA1 neurons in the presence of serotonin was close to y80 mV indicating an opening of a Kq channel population typical of activation of 5-HT1A receptors. The present results also suggest that a large number of GEPR-9 CA1 pyramidal neurons are not responsive to serotonin. This may be due to possible defects in these neurons such as paucity of serotonin receptors, deficiency in the receptor-signal transduction mechanism or alteration in the serotonin reuptake mechanism. The decrease in the responsiveness of the hippocampal CA1 region to serotonin was unexpected, particularly in light of the reported reduction in hippocampal serotonin content in the GEPR brain w4x. Ordinarily, such a reduction should have led to upregulation of serotonin receptors. However, a recent study w13x indicates a decrease in 8OH-DPAT binding sites in GEPR-9 hippocampus which supports the proposition of diminished 5-HT1A receptor sites. Our results indicate no difference in the membrane resting potential and input resistance between GEPR-9 and Sprague–Dawley CA1 pyramidal neurons. This is at vari-
Synaptically evoked action potentials were also reversibly blocked by serotonin ŽFig. 2B. but only in about 33% of the neurons tested. Where there was inhibition of action potentials by serotonin, concurrent membrane hyperpolarization and reduction of input resistance were also observed in these neurons ŽFig. 3A.. Using current clamp method Žconstant currents ranging from y1.0 to q0.2 nA were injected and the resultant changes in membrane potential were measured., current– voltage Ž I–V . curves were constructed for GEPR-9 pyramidal CA1 neurons. The reversal potential in the presence of serotonin was close to y80 mV ŽFig. 4.. 3.3. Effect of serotonin on bicuculline-treated GEPR-9 brain slices Synaptically evoked epileptiform activity was induced by treatment of GEPR-9 brain slices with bicuculline Ž10 mM. w2x. The bursts of action potentials evoked in the presence of bicuculline, were inhibited by serotonin in GEPR-9 pyramidal CA1 neurons ŽFig. 3B.. However, the degree of inhibition of the epileptiform bursts was less in the GEPR-9 neurons than in neurons from Sprague–Dawley rats Žnumber of action potentialsrburst in Sprague– Dawley: 3.3 " 0.5, after serotonin 0.3 " 0.3, n s 4; in GEPR-9: 3.3 " 0.9, after serotonin 1.3 " 0.3, n s 3.. Here also, the inhibition of epileptiform activity was accompanied by hyperpolarization and reduction in membrane input resistance.
Table 1 Changes in resting membrane potential ŽRMP. and membrane input resistance in CA1 pyramidal neurons of GEPR-9 induced by 5-HT and 8-OH-DPAT DrugŽ20 mM.
5-HT 8-OH-DPAT
ResistanceŽM V .
RMPŽmV. Control
After drug
y72.5 " 4.4 y71.9 " 2.5
y78.1 " 3.5 y76.5 " 2.9
) )
Control
After drug
47.7 " 4.6 36.4 " 7.2
31.5 " 3.2 30.1 " 6.9
The slices were treated with either serotonin Ž5-HT, 20 mM, seven neurons. or 8-OH-DPAT Ž20 mM, four neurons.. a Indicates significant difference from the control Žpaired t-test; P - 0.05, " indicates S.E.M. values..
) )
216
D. Salgado-Commissariat, K.A. Alkadhir Brain Research 743 (1996) 212–216
ance with a report by Verma-Ahuja and Pencek w14x who, although found no difference in resting membrane potential, reported an increase in membrane resistance in GEPR9 CA1 pyramidal neurons as compared to those of the Sprague–Dawley rats. The reason for this discrepancy is unclear. It may reflect different methodology and test conditions. An alternative explanation may be provided by the results of several studies that indicate abnormalities in GABAergic transmission in the GEPR brain w7,8x. It is possible that fewer GABA receptor-linked chloride channels are open at the resting state hence the higher membrane input resistance. However, it is rather surprising that such a large difference in the membrane input resistance between Sprague Dawley and GEPR CA1 neurons can exist with no detectable difference in the resting membrane potential. In the GEPR-9 model of epilepsy, sensory stimuli initiate generalized seizures w6x. Although GEPRs may experience spontaneous seizures w3,9x, the epileptiform activity in these animals is usually triggered by stimuli such as sound, hyperthermia, electroshock and kindling. In our experiments, we treated GEPR-9 brain slices with bicuculline to evoke epileptiform bursts. Our results are in accord with those from intact animal studies w5,15,16x. It was demonstrated that fluoxetine or a combination of fluoxetine and 5-HTP but not PCPA produced anticonvulsant activity in GEPRs w15,16x. Coincidental with the anticonvulsant activity of these agents, an increase in thalamic serotonin levels was detected w15x. Thus in an indirect fashion, these investigators have shown that the serotonin system may be involved in anticonvulsant activity in the brain of these animals. The present study provides more direct evidence for the involvement of serotonin in the inhibition of epileptiform activity by demonstrating the inhibitory effects of exogenously applied serotonin and the selective 5-HT1A agonist 8-OH-DPAT on the hippocampal region of the GEPR-9 brain.
References w1x Andrade, R. and Nicoll, R.A., A G-protein couples serotonin and GABA B receptors to the same channels in hippocampus, Science, 234 Ž1986. 1261–1265.
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