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Veratridine-treated brain slices: a cellular model for epileptiform activity
Brain Research 789 Ž1998. 150–156
Research report
Veratridine-treated brain slices: a cellular model for epileptiform activity Sameer Otoom, Lian-Ming Tian, Karim A. Alkadhi
)
Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, UniÕersity of Houston, Houston, TX 77204-5515, USA Accepted 30 December 1997
Abstract This study introduces veratridine-treated brain slices as a new in vitro synaptic-independent model for epileptiform discharge. Studies were performed on the hippocampus in rat brain slices using conventional electrophysiological intracellular recording techniques. Veratridine Ž0.3 m M. produced a time-dependent blockade of synaptic transmission as indicated by inhibition of the evoked population spike in the region CA1 of the hippocampus. However, in the same slices, intracellularly-evoked single action potentials were converted to epileptiform bursting shortly after exposure to veratridine. Additionally, in the veratridine model, spontaneous epileptiform activity developed after prolonged Žmore than 45 min. superfusion. The model was utilized to examine the action of two antiepileptic drugs: a sodium channel dependent and a synaptic dependent antiepileptic agents. Therapeutic concentrations of valproic acid ŽVPA, 10–100 m M. inhibited both evoked and spontaneous bursting induced by veratridine. However, therapeutic concentrations of the synaptic-dependent antiepileptic drug phenobarbital Ž20–40 m M. failed to inhibit veratridine-induced bursting. These results demonstrate that the veratridine-treated brain slice is a simple and reliable model for studying mechanisms of action and for screening of potential sodium channel-dependent antiepileptic drugs. q 1998 Elsevier Science B.V. Keywords: Hippocampus; Veratridine; Valproic acid; Phenobarbital; Spontaneous bursting; Epilepsy; Anticonvulsants
1. Introduction The voltage-dependent sodium channel is believed to have at least six major active sites that have been characterized pharmacologically w6x. A variety of toxins bind to these sites on the sodium channel; some of them are used as biochemical markers and probes for channel function. Veratridine, a steroidal compound found in the alkaloids Veratrum, Zygadenus and Schoenocaulon w26x, is believed to binds to site 2 on the sodium channel. This toxin produces persistent activation of sodium channels at normal membrane potentials by blocking channel inactivation and shifting the voltage-dependence of channel activation to a more hyperpolarizing state w10x. Two modes of binding of veratridine to the sodium channel have been identified: a rapid binding to open channels and a slow binding to inactivated channels. The latter causes the channels to remain open at rest and at more depolarized potentials. The effects produced by veratridine are reversible upon removal of the toxin w29x. )
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Voltage-dependent sodium channels are considered important targets in the pathology of a number of diseases, including epilepsy, cardiac arrhythmia and ischemic brain damage w25x. Drugs that are believed to block sodium channels such as phenytoin, and valproic acid ŽVPA. are used in the treatment of clinical and experimental tonic– clonic and partial seizures w21,22x. Blocking sodium channels by quinidine or amiodarone is believed to be cardioprotective against arrhythmias w17x. Sodium channel blockers provide advantages over the use of calcium channel blockers or NMDA antagonists as cerebroprotective agents because they are used in small doses that do not produce toxic cardiovascular effects. Additionally, sodium channel blockers can protect mammalian central nervous system white matter from anoxia-induced injury w5x. Controlled clinical trials indicate promising results when lifarizine is used for treatment of stroke w30x. Another sodium channel blocker, riluzole, is useful for treatment of the progressive motor neuron disease amyotrophic lateral sclerosis w4x. Moreover, sodium channel blockers are effective in alleviation of pain in cases of trigeminal neuralgia and diabetic neuropathy w11x, and in decreasing regional cerebral edema resulting from percussive brain injury in rats w27x.
S. Otoom et al.r Brain Research 789 (1998) 150–156
Studies in this laboratory have shown that veratridine enhances the slowly inactivating sodium current in hippocampal pyramidal neurons w2x. This enhancement leads to induction of bursting and development of a negative slope resistance w32x. We also have shown that synaptic transmission is not required for induction of seizure-like activity by veratridine w32x. Although synaptic transmission is blocked by superfusion of the brain slice with kynurenic acid, intracellular stimulation evokes a repetitive discharge riding on top of a slow depolarization plateau w32x. In this study, we present further evidence that veratridine reversibly inhibits synaptic transmission in CA1 pyramidal neurons and at the same time converts intracellularly-induced single action potentials to epileptiform bursts. We propose the veratridine model as sodium channel-defective, synaptic-independent model for epileptiform discharge that can be used to screen for potential sodium channel-dependent antiepileptic drugs.
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air suspension table ŽTechnical Manufacturing.. After a recovery period, a single slice was transferred to the recording chamber and trapped between two nylon mesh rings. The brain slice was superfused with warm ACSF continuously bubbled with carbogen. Intracellular recordings were performed using microelectrodes filled with 4 M potassium acetate Žtip resistance: 80–120 M V .. The microelectrodes were made from capillary tubes Ž1.0 mm, Kwik-fil, WPI. pulled by an electrode puller ŽNarishige, Model PN-3.. The recording electrode was visually positioned by a micromanipulator over the CA1 cell layer of the hippocampus. A reference electrode Žsilver–silver chloride. was placed in the solution at the outlet of the chamber. A successful intracellular
2. Materials and methods Experiments were performed on male Sprague–Dawley rats ŽHarlan Sprague–Dawley. weighing 150–250 g. The rats were housed in cages Žno more than six ratsrcage. under a standard 12-h lightr12-h dark cycle. Animals were quickly decapitated by a small animal guillotine. The skull bone was removed by a small bone ronjours and the dura was carefully cut with small scissors. A stainless steel spatula was used to lift the brain out of the skull. The brain was transferred to a petri dish filled with cold Ž08C. oxygenated Ž95% O 2 , 5% CO 2 , carbogen. artificial cerebrospinal fluid ŽACSF.. The brain was divided midsagittally into two hemispheres. From each hemisphere, a transverse block containing hippocampal tissue was isolated. This block was affixed on the stage of a slicer using cyanoacrylate glue. Transverse slices, 500-m m thick Žapproximately four to five from each hemisphere., were cut using a vibroslice ŽCampden Instruments.. Prior to experimental manipulation, the slices were allowed to recover at room temperature in a beaker filled with ACSF solution continuously gassed with carbogen. The recording chamber was made from clear sylastic gel. It consisted of a circular bath of low volume Žless than 1 ml. with an inlet connected to a polyethylene tube that delivered the superfusate and an outlet for the drainage of the solution by gravity. Within the bath the brain slice was held between two nylon nets so that stable electrophysiological recordings could be obtained. Solution reached the circular bath through a polyethylene tube passed through a water jacket connected to a heater–circulator ŽLauda C3, model T-1.. The temperature of the ACSF in the tissue bath was maintained at 32 " 18C. The hippocampal slices were illuminated from below by a fiber-optic illuminator ŽColeParmer, model 9741-50.. The entire recording system was housed in a grounded Faraday cage mounted on a Micro-G
Fig. 1. The effect of veratridine on synaptic transmission in hippocampal slices. A: Population spike Žcontrol. evoked by stimulation of the Schaffer collaterals was completely abolished after superfusion with 0.3 m M veratridine Žmiddle trace.. At this stage Ž20 min after application., stimulation of the Schaffer collaterals failed to elicit a population spike. The veratridine effect was reversed after washout of the drug Žlower trace.. B: Represents the time-dependent decrease in the amplitude of the population spike. Bars are the means of four neurons, vertical lines are S.E.M., ) indicates significant difference from control Žone-way ANOVA, P - 0.05..
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impalement of a neuron was indicated by a resting membrane potential more negative than to 60 mV and an action potential amplitude of more than 60 mV. Population spikes were recorded extracellularly using a low tip resistance microelectrode Ž5–15 M V . filled with 1 M NaCl and positioned in the striatum pyramidale layer. A bipolar stimulating electrode was placed on the stratum radiatum of CA2rCA3 region for orthodromic stimulation. Potentials were amplified by an Axoclamp 2A amplifier ŽAxon Instruments. and stored on video tapes for later analysis. 2.1. Drugs and chemicals All the chemicals were obtained from Sigma Chemical. Stock solutions of VPA and phenobarbital were prepared by dissolving the drugs in distilled water. Veratridine was dissolved in 0.1 mM HCL. The final concentrations of drugs were prepared by adding a calculated amount of stock solution to ACSF. Drugs were delivered to the hippocampal slices by changing the superfusate from ACSF
Fig. 3. Spontaneous bursting induced by veratridine. A: Veratridine Ž0.1, 0.3 and 1 m M. was superfused and the time for the beginning of spontaneous bursting was recorded. Bursting onset was inversely proportional to veratridine concentrations. Each point is the mean of bursting onset Žfour neurons.. Vertical lines are S.E.M. B: The percentages of experiments in which veratridine induced spontaneous bursting within 1 h of superfusion. Each bar represents the mean Ž"S.E.M., vertical lines. of four experiments.
to that containing the appropriate drug. The composition of the ACSF was ŽmM.: NaCl, 127; CaCl 2 , 2.5; KCl, 4.7; MgCl 2 , 1.2; NaHCO 3 , 22 and NaH 2 PO4 , 1.2; Glucose, 11.0. The pH of this solution was 7.4. 2.2. Statistics Data were expressed as mean " S.E.M. Statistical analysis was performed using paired t-test and one-way ANOVA as appropriate. A value of P - 0.05 was considered statistically significant. Fig. 2. Veratridine induces seizure-like activity in rat hippocampal pyramidal neurons. A: Each of the intracellular current pulses Ž0.1 Hz, control; lower trace. evoked only one spike in control period in a CA1 neuron. Bursting resulted from the same pulses after 10 min in 1.0 m M veratridine, and disappeared after 40-min wash. The right panel shows one of the responses in left panel recorded at a faster speed. All traces in A are from the same neuron ŽRMP: y70 mV. representing four other neurons. B: Spontaneous bursting was observed after prolonged exposure to 0.3 m M veratridine in another neuron ŽRMP: y72 mV.. The spikes were truncated by the chart recorder.
3. Results 3.1. Effect of Õeratridine on synaptic transmission Stimulation of the striatum pyramidale elicited a population spike in CA1 pyramidal cell layer of the hippocampus ŽFig. 1A, control, four neurons.. This indicator of
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synaptic transmission w1x was completely inhibited after the application of veratridine Ž0.3 m M, middle trace.. Washout of veratridine with normal ACSF restored the evoked population spike Žlower trace.. Veratridine attenuated the amplitude of the population spike in a time-dependent manner. Fig. 1B shows that the amplitude of the population spike was abolished after 20 min of superfusion of the brain slice with veratridine Ž0.3 m M.. 3.2. Veratridine-induced epileptiform discharge After successful impalement, neurons were stimulated by injecting positive DC current pulses, each of which produced a single action potential ŽFig. 2A.. Treatment with veratridine Ž0.3 m M. converted some of these single action potentials into prolonged bursts within 30 min of exposure ŽFig. 2A.. This effect was reversed when veratridine was removed ŽFig. 2A.. With prolonged exposure to veratridine, a neuron may develop spontaneous bursting ŽFig. 2B.. To determine the
Fig. 5. A: Dose-dependent reduction of evoked busting by VPA. Intracellularly evoked bursting was obtained in 30 min of exposure to veratridine Ž0.3 m M.. Data were normalized from seven neurons, vertical lines are S.E.M. B: The inhibition of veratridine-induced spontaneous bursting activity by VPA. After treatment with 0.3 m M veratridine the neuron ŽRMP: y70 mV. exhibited rhythmic spontaneous bursting activity Žcontrol.. The application of 100 m M VPA completely blocked the bursting activity.
Fig. 4. The dose-dependent effect of VPA on veratridine-induced bursting activity. Traces were recorded from a single neuron ŽRMP: y72 mV. continuously superfused with veratridine Ž0.3 m M, Control.. The bursting activity was evoked by intracellular injection of DC current pulses as indicated by the lower traces. After prolonged wash Ž50 min. with veratridine ACSF, spontaneous bursting reappeared Žbottom trace..
concentration needed to produce spontaneous bursting in this model, three different concentrations of veratridine were tested Ž0.1, 0.3, 1 m M, four neurons for each concentration.. Each concentration was superfused until spontaneous bursting appeared. The onset of veratridine bursting Žthe length of time required for three or more action potentials to appeared outside the current pulse. was recorded. The onset of spontaneous bursting was inversely proportional to the veratridine concentration ŽFig. 3A.. We also calculated the percentage of experiments at each concentration where spontaneous bursting was produced within 1 h of veratridine application. Fig. 3B shows that 0.1 m M veratridine produced spontaneous bursting only in one experiment within 60 min of veratridine application. At 1 m M, veratridine produced spontaneous bursting within 20–30 min. However, the electrical stability of the impaled neurons deteriorated very quickly when this was used for a long period of time. Therefore, 0.3 m M of veratridine was used to induce spontaneous bursting.
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3.3. Effect of antiepileptic drugs on action potential bursts in the Õeratridine model 3.3.1. Valproic acid Inhibition by VPA of evoked action potential bursts in the veratridine model is illustrated in Fig. 4. The return of bursting was achieved only after prolonged washout of VPA Ž50 min, Fig. 4.. A significant and dose-dependent reduction of evoked bursts was achieved with VPA concentrations of 30 m M or higher ŽFig. 5A.. The effect of VPA Ž10–100 m M. on spontaneous bursting produced by veratridine was also studied. Inhibition of the veratridine-induced spontaneous bursting was achieved with all concentrations tested ŽFig. 5B, four neurons.. 3.3.2. Effect of phenobarbital The effectiveness of the synaptic-dependent antiepileptic drug phenobarbital in inhibiting seizure-like activity
induced by veratridine also was examined. The seizure-like bursts induced by veratridine Ž0.3 m M, Fig. 6. was not inhibited by the application of a therapeutic concentration of phenobarbital Ž40 m M, Fig. 6.. However, administration of a supratherapeutic concentration Ž500 m M. of the drug produced partial inhibition of veratridine induced seizurelike activity ŽFig. 6..
4. Discussion This study examines veratridine-treated brain slices as a model of experimental seizure. Small concentrations of veratridine blocked chemical synaptic transmission but induced seizure-like bursts of action potentials in response to a single intracellular stimulus. Moreover, spontaneous bursting was observed after prolonged superfusion of veratridine. The seizure-like activity induced by veratridine was sensitive to inhibition by therapeutic concentrations of sodium channel blockers such as VPA but less sensitive to non-sodium channel blockers such as phenobarbital. These characteristics make the veratridine model suitable for screening potential sodium-dependent antiepileptic drugs. 4.1. The model
Fig. 6. The effect of phenobarbital on veratridine-induced bursting. Digitized tracings were recording from a single neuron ŽRMP: y73 mV. in the CA1 pyramidal area of the hippocampus. The superfusion of phenobarbital Ž40 m M, five neurons. following the induction of seizurelike activity by veratridine Ž0.3 m M, upper trace. did not suppress the evoked epileptic activity Žmiddle trace.. However, superfusion of a large concentration of the drug Ž500 m M, five neurons. partially inhibited this seizure-like activity. The spikes were truncated by the chart recorder. Calibrations bars applied to all traces. RMP of the neuron was y77 mV.
Electrophysiological studies have shown that there are three general factors which influence the neuronal bursting leading to development of epileptogenic activity w19,28x. These include neuronal bursting resulting from intrinsic capacity of neurons, diminution in the potency of inhibitory pathways and increase in the recruitment of excitatory synapses. In hyperexcitable membrane, blocking sodium channels stabilizes the membrane w15x. This suggests a possible involvement of sodium channels in the genesis of abnormal activities in epilepsy. In fact, there is evidence implicating the sodium channel as a relevant factor in the pathogenesis of human and experimental epileptic seizure. Studies in our laboratory indicate that endogenous seizure-like activity can be produced in hippocampal CA1 pyramidal neurons when sodium channels are altered by veratridine w32x. Furthermore, we have shown that veratridine augments a slowly-inactivating subthreshold sodium current I NaŽs. that leads to the generation of negative slope resistance observed in bursting neurons w2x. Synaptic transmission is not required to induce seizurelike activity by veratridine. Veratridine inhibition of synaptic transmission in hippocampal pyramidal CA1 neurons is indicated by the disappearance of the population spike seen in response to stimulation of the Schaffer collaterals. This inhibition may be related to veratridine-induced depolarization of the presynaptic terminal. Although synaptic transmission was blocked, veratridine was still effective in converting a single intracellularly evoked action potential
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to bursting discharge. These results are supported by other studies conducted in this laboratory. Although synaptic transmission was blocked in the CA1 region of the rat hippocampus by superfusion of the brain slice with kynurenic acid, intracellular stimulation evoked a repetitive discharge riding on a slow depolarization plateau w32x. A number of studies investigated the mechanisms involved in the propagation of seizure activity in the absence of synaptic transmission. It is believed that ephaptic interaction is the most acceptable explanation for synchronization in CA1 pyramidal neurons w18,31x. The fact that CA1 pyramidal neurons are densely packed and have a low extracellular conductivity favors a strong ephaptic interaction w16x. Depolarization of the neural membrane by extracellular potassium which increases during seizure is also another mechanism for synchronization in hippocampal pyramidal neurons w14x. When hippocampal slices are exposed to low-calcium solution, synaptic transmission is blocked and seizure-like activity appears in area CA1 of the hippocampus w36x. The mechanism of seizure induction by lowering calcium is not fully understood. Studies suggest that reducing extracellular calcium may enhance neuronal excitability by affecting sodium channel gating w37x. Furthermore, external calcium has been shown to stabilize the resting conformation of sodium channels and to block these channels at negative membrane potential w3x. The disadvantages of the low calcium model are that synaptic transmission may not be completely blocked, inhibition of this kind of seizure is only achieved at high concentrations of antiepileptic drugs and the mechanism by which seizures in this model are generated is not fully clear w13x. Compared to other synaptic-independent models of epilepsy, the veratridine model provides the following advantages: Ž1. a small concentration is needed to generate a long-lasting seizure-like activity that persists after washing; Ž2. based on the well-known mechanism of action of veratridine, the model involves alteration of sodium channel function, a major mechanism in the pathogenesis of many types of epilepsy; Ž3. since veratridine blocks synaptic transmission, seizure-like activity can be induced without the involvement of chemical synaptic transmission; Ž4. it is a reliable model for the assessment of both anti- and pro-convulsant activity of certain Žsodium channel-dependent. drugs and Ž5. it is a useful model for generating spontaneous seizure-like activity. 4.2. Effect of antiepileptic drugs on seizure-like actiÕity induced by Õeratridine Intracellular recording from mouse primary dissociated central neurons showed that VPA at therapeutic concentrations blocked sustained high frequency repetitive firing w24x. Even at supratherapeutic concentration levels, VPA did not affect GABA postsynaptic responses in the majority of neurons tested w8,24x. VPA has been used success-
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fully in the treatment of absence, generalized tonic–clonic and partial seizures w9x. Moreover, VPA has been shown to be effective in the treatment of status epilepticus w12x and in seizure prophylaxis in alcohol withdrawal w34x. This broad spectrum of antiepileptic activity of VPA may be related to its dose-dependent multiple mechanisms of action. Our study indicates that therapeutic concentrations of VPA inhibit evoked and spontaneous action potential bursting induced by veratridine. In comparison, the low calcium model requires larger concentrations of VPA to inhibit bursting w13x. This indicates the high sensitivity of the veratridine model to sodium channel-dependent antiepileptic drugs. The antiepileptic drug phenobarbital is used to treat both partial and generalized tonic–clonic seizures w23x. Its major antiepileptic mechanism of action is to increase GABA A receptor-chloride channel mean open time by binding to an allosteric regulatory site on the GABA A receptor-chloride channel complex w33x. This effect, which is produced at therapeutic concentrations of the drug, leads to an increase in chloride permeability and hyperpolarization of the postsynaptic membrane w22x. Results from the present study indicate that therapeutic concentrations of this synaptic-dependent drug failed to inhibit seizure-like activity induced by veratridine. Therapeutic concentrations of phenobarbital require an intact synaptic transmission to be effective, therefore no inhibitory effect is seen because synaptic transmission is blocked in the veratridine model. However, a much larger concentration of phenobarbital partially inhibits seizure-like activity induced by veratridine. Phenobarbital possesses direct neuronal membrane effects at supratherapeutic concentrations. At these concentrations, the drug inhibits batrachotoxin-stimulated 22 Naq influx w35x, reduces the sodium-dependent sustain highfrequency repetitive firing of action potentials w20x and blocks sodium channels in skeletal muscles and myelinated nerve preparations w7x.
5. Conclusion In rat hippocampal CA1 pyramidal neurons, veratridine inhibits synaptic transmission but is still effective in inducing seizure-like activity in response to a single intracellular stimulus. The seizure-like activity induced by this model is inhibited by therapeutic concentrations of the sodium channel blocker VPA but not by the synaptic-dependent antiepileptic drug phenobarbital. These features make the veratridine model a valuable tool to study direct membrane effects of sodium channel-dependent antiepileptic drugs.
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Research report
Veratridine-treated brain slices: a cellular model for epileptiform activity Sameer Otoom, Lian-Ming Tian, Karim A. Alkadhi
)
Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, UniÕersity of Houston, Houston, TX 77204-5515, USA Accepted 30 December 1997
Abstract This study introduces veratridine-treated brain slices as a new in vitro synaptic-independent model for epileptiform discharge. Studies were performed on the hippocampus in rat brain slices using conventional electrophysiological intracellular recording techniques. Veratridine Ž0.3 m M. produced a time-dependent blockade of synaptic transmission as indicated by inhibition of the evoked population spike in the region CA1 of the hippocampus. However, in the same slices, intracellularly-evoked single action potentials were converted to epileptiform bursting shortly after exposure to veratridine. Additionally, in the veratridine model, spontaneous epileptiform activity developed after prolonged Žmore than 45 min. superfusion. The model was utilized to examine the action of two antiepileptic drugs: a sodium channel dependent and a synaptic dependent antiepileptic agents. Therapeutic concentrations of valproic acid ŽVPA, 10–100 m M. inhibited both evoked and spontaneous bursting induced by veratridine. However, therapeutic concentrations of the synaptic-dependent antiepileptic drug phenobarbital Ž20–40 m M. failed to inhibit veratridine-induced bursting. These results demonstrate that the veratridine-treated brain slice is a simple and reliable model for studying mechanisms of action and for screening of potential sodium channel-dependent antiepileptic drugs. q 1998 Elsevier Science B.V. Keywords: Hippocampus; Veratridine; Valproic acid; Phenobarbital; Spontaneous bursting; Epilepsy; Anticonvulsants
1. Introduction The voltage-dependent sodium channel is believed to have at least six major active sites that have been characterized pharmacologically w6x. A variety of toxins bind to these sites on the sodium channel; some of them are used as biochemical markers and probes for channel function. Veratridine, a steroidal compound found in the alkaloids Veratrum, Zygadenus and Schoenocaulon w26x, is believed to binds to site 2 on the sodium channel. This toxin produces persistent activation of sodium channels at normal membrane potentials by blocking channel inactivation and shifting the voltage-dependence of channel activation to a more hyperpolarizing state w10x. Two modes of binding of veratridine to the sodium channel have been identified: a rapid binding to open channels and a slow binding to inactivated channels. The latter causes the channels to remain open at rest and at more depolarized potentials. The effects produced by veratridine are reversible upon removal of the toxin w29x. )
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Voltage-dependent sodium channels are considered important targets in the pathology of a number of diseases, including epilepsy, cardiac arrhythmia and ischemic brain damage w25x. Drugs that are believed to block sodium channels such as phenytoin, and valproic acid ŽVPA. are used in the treatment of clinical and experimental tonic– clonic and partial seizures w21,22x. Blocking sodium channels by quinidine or amiodarone is believed to be cardioprotective against arrhythmias w17x. Sodium channel blockers provide advantages over the use of calcium channel blockers or NMDA antagonists as cerebroprotective agents because they are used in small doses that do not produce toxic cardiovascular effects. Additionally, sodium channel blockers can protect mammalian central nervous system white matter from anoxia-induced injury w5x. Controlled clinical trials indicate promising results when lifarizine is used for treatment of stroke w30x. Another sodium channel blocker, riluzole, is useful for treatment of the progressive motor neuron disease amyotrophic lateral sclerosis w4x. Moreover, sodium channel blockers are effective in alleviation of pain in cases of trigeminal neuralgia and diabetic neuropathy w11x, and in decreasing regional cerebral edema resulting from percussive brain injury in rats w27x.
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Studies in this laboratory have shown that veratridine enhances the slowly inactivating sodium current in hippocampal pyramidal neurons w2x. This enhancement leads to induction of bursting and development of a negative slope resistance w32x. We also have shown that synaptic transmission is not required for induction of seizure-like activity by veratridine w32x. Although synaptic transmission is blocked by superfusion of the brain slice with kynurenic acid, intracellular stimulation evokes a repetitive discharge riding on top of a slow depolarization plateau w32x. In this study, we present further evidence that veratridine reversibly inhibits synaptic transmission in CA1 pyramidal neurons and at the same time converts intracellularly-induced single action potentials to epileptiform bursts. We propose the veratridine model as sodium channel-defective, synaptic-independent model for epileptiform discharge that can be used to screen for potential sodium channel-dependent antiepileptic drugs.
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air suspension table ŽTechnical Manufacturing.. After a recovery period, a single slice was transferred to the recording chamber and trapped between two nylon mesh rings. The brain slice was superfused with warm ACSF continuously bubbled with carbogen. Intracellular recordings were performed using microelectrodes filled with 4 M potassium acetate Žtip resistance: 80–120 M V .. The microelectrodes were made from capillary tubes Ž1.0 mm, Kwik-fil, WPI. pulled by an electrode puller ŽNarishige, Model PN-3.. The recording electrode was visually positioned by a micromanipulator over the CA1 cell layer of the hippocampus. A reference electrode Žsilver–silver chloride. was placed in the solution at the outlet of the chamber. A successful intracellular
2. Materials and methods Experiments were performed on male Sprague–Dawley rats ŽHarlan Sprague–Dawley. weighing 150–250 g. The rats were housed in cages Žno more than six ratsrcage. under a standard 12-h lightr12-h dark cycle. Animals were quickly decapitated by a small animal guillotine. The skull bone was removed by a small bone ronjours and the dura was carefully cut with small scissors. A stainless steel spatula was used to lift the brain out of the skull. The brain was transferred to a petri dish filled with cold Ž08C. oxygenated Ž95% O 2 , 5% CO 2 , carbogen. artificial cerebrospinal fluid ŽACSF.. The brain was divided midsagittally into two hemispheres. From each hemisphere, a transverse block containing hippocampal tissue was isolated. This block was affixed on the stage of a slicer using cyanoacrylate glue. Transverse slices, 500-m m thick Žapproximately four to five from each hemisphere., were cut using a vibroslice ŽCampden Instruments.. Prior to experimental manipulation, the slices were allowed to recover at room temperature in a beaker filled with ACSF solution continuously gassed with carbogen. The recording chamber was made from clear sylastic gel. It consisted of a circular bath of low volume Žless than 1 ml. with an inlet connected to a polyethylene tube that delivered the superfusate and an outlet for the drainage of the solution by gravity. Within the bath the brain slice was held between two nylon nets so that stable electrophysiological recordings could be obtained. Solution reached the circular bath through a polyethylene tube passed through a water jacket connected to a heater–circulator ŽLauda C3, model T-1.. The temperature of the ACSF in the tissue bath was maintained at 32 " 18C. The hippocampal slices were illuminated from below by a fiber-optic illuminator ŽColeParmer, model 9741-50.. The entire recording system was housed in a grounded Faraday cage mounted on a Micro-G
Fig. 1. The effect of veratridine on synaptic transmission in hippocampal slices. A: Population spike Žcontrol. evoked by stimulation of the Schaffer collaterals was completely abolished after superfusion with 0.3 m M veratridine Žmiddle trace.. At this stage Ž20 min after application., stimulation of the Schaffer collaterals failed to elicit a population spike. The veratridine effect was reversed after washout of the drug Žlower trace.. B: Represents the time-dependent decrease in the amplitude of the population spike. Bars are the means of four neurons, vertical lines are S.E.M., ) indicates significant difference from control Žone-way ANOVA, P - 0.05..
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impalement of a neuron was indicated by a resting membrane potential more negative than to 60 mV and an action potential amplitude of more than 60 mV. Population spikes were recorded extracellularly using a low tip resistance microelectrode Ž5–15 M V . filled with 1 M NaCl and positioned in the striatum pyramidale layer. A bipolar stimulating electrode was placed on the stratum radiatum of CA2rCA3 region for orthodromic stimulation. Potentials were amplified by an Axoclamp 2A amplifier ŽAxon Instruments. and stored on video tapes for later analysis. 2.1. Drugs and chemicals All the chemicals were obtained from Sigma Chemical. Stock solutions of VPA and phenobarbital were prepared by dissolving the drugs in distilled water. Veratridine was dissolved in 0.1 mM HCL. The final concentrations of drugs were prepared by adding a calculated amount of stock solution to ACSF. Drugs were delivered to the hippocampal slices by changing the superfusate from ACSF
Fig. 3. Spontaneous bursting induced by veratridine. A: Veratridine Ž0.1, 0.3 and 1 m M. was superfused and the time for the beginning of spontaneous bursting was recorded. Bursting onset was inversely proportional to veratridine concentrations. Each point is the mean of bursting onset Žfour neurons.. Vertical lines are S.E.M. B: The percentages of experiments in which veratridine induced spontaneous bursting within 1 h of superfusion. Each bar represents the mean Ž"S.E.M., vertical lines. of four experiments.
to that containing the appropriate drug. The composition of the ACSF was ŽmM.: NaCl, 127; CaCl 2 , 2.5; KCl, 4.7; MgCl 2 , 1.2; NaHCO 3 , 22 and NaH 2 PO4 , 1.2; Glucose, 11.0. The pH of this solution was 7.4. 2.2. Statistics Data were expressed as mean " S.E.M. Statistical analysis was performed using paired t-test and one-way ANOVA as appropriate. A value of P - 0.05 was considered statistically significant. Fig. 2. Veratridine induces seizure-like activity in rat hippocampal pyramidal neurons. A: Each of the intracellular current pulses Ž0.1 Hz, control; lower trace. evoked only one spike in control period in a CA1 neuron. Bursting resulted from the same pulses after 10 min in 1.0 m M veratridine, and disappeared after 40-min wash. The right panel shows one of the responses in left panel recorded at a faster speed. All traces in A are from the same neuron ŽRMP: y70 mV. representing four other neurons. B: Spontaneous bursting was observed after prolonged exposure to 0.3 m M veratridine in another neuron ŽRMP: y72 mV.. The spikes were truncated by the chart recorder.
3. Results 3.1. Effect of Õeratridine on synaptic transmission Stimulation of the striatum pyramidale elicited a population spike in CA1 pyramidal cell layer of the hippocampus ŽFig. 1A, control, four neurons.. This indicator of
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synaptic transmission w1x was completely inhibited after the application of veratridine Ž0.3 m M, middle trace.. Washout of veratridine with normal ACSF restored the evoked population spike Žlower trace.. Veratridine attenuated the amplitude of the population spike in a time-dependent manner. Fig. 1B shows that the amplitude of the population spike was abolished after 20 min of superfusion of the brain slice with veratridine Ž0.3 m M.. 3.2. Veratridine-induced epileptiform discharge After successful impalement, neurons were stimulated by injecting positive DC current pulses, each of which produced a single action potential ŽFig. 2A.. Treatment with veratridine Ž0.3 m M. converted some of these single action potentials into prolonged bursts within 30 min of exposure ŽFig. 2A.. This effect was reversed when veratridine was removed ŽFig. 2A.. With prolonged exposure to veratridine, a neuron may develop spontaneous bursting ŽFig. 2B.. To determine the
Fig. 5. A: Dose-dependent reduction of evoked busting by VPA. Intracellularly evoked bursting was obtained in 30 min of exposure to veratridine Ž0.3 m M.. Data were normalized from seven neurons, vertical lines are S.E.M. B: The inhibition of veratridine-induced spontaneous bursting activity by VPA. After treatment with 0.3 m M veratridine the neuron ŽRMP: y70 mV. exhibited rhythmic spontaneous bursting activity Žcontrol.. The application of 100 m M VPA completely blocked the bursting activity.
Fig. 4. The dose-dependent effect of VPA on veratridine-induced bursting activity. Traces were recorded from a single neuron ŽRMP: y72 mV. continuously superfused with veratridine Ž0.3 m M, Control.. The bursting activity was evoked by intracellular injection of DC current pulses as indicated by the lower traces. After prolonged wash Ž50 min. with veratridine ACSF, spontaneous bursting reappeared Žbottom trace..
concentration needed to produce spontaneous bursting in this model, three different concentrations of veratridine were tested Ž0.1, 0.3, 1 m M, four neurons for each concentration.. Each concentration was superfused until spontaneous bursting appeared. The onset of veratridine bursting Žthe length of time required for three or more action potentials to appeared outside the current pulse. was recorded. The onset of spontaneous bursting was inversely proportional to the veratridine concentration ŽFig. 3A.. We also calculated the percentage of experiments at each concentration where spontaneous bursting was produced within 1 h of veratridine application. Fig. 3B shows that 0.1 m M veratridine produced spontaneous bursting only in one experiment within 60 min of veratridine application. At 1 m M, veratridine produced spontaneous bursting within 20–30 min. However, the electrical stability of the impaled neurons deteriorated very quickly when this was used for a long period of time. Therefore, 0.3 m M of veratridine was used to induce spontaneous bursting.
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3.3. Effect of antiepileptic drugs on action potential bursts in the Õeratridine model 3.3.1. Valproic acid Inhibition by VPA of evoked action potential bursts in the veratridine model is illustrated in Fig. 4. The return of bursting was achieved only after prolonged washout of VPA Ž50 min, Fig. 4.. A significant and dose-dependent reduction of evoked bursts was achieved with VPA concentrations of 30 m M or higher ŽFig. 5A.. The effect of VPA Ž10–100 m M. on spontaneous bursting produced by veratridine was also studied. Inhibition of the veratridine-induced spontaneous bursting was achieved with all concentrations tested ŽFig. 5B, four neurons.. 3.3.2. Effect of phenobarbital The effectiveness of the synaptic-dependent antiepileptic drug phenobarbital in inhibiting seizure-like activity
induced by veratridine also was examined. The seizure-like bursts induced by veratridine Ž0.3 m M, Fig. 6. was not inhibited by the application of a therapeutic concentration of phenobarbital Ž40 m M, Fig. 6.. However, administration of a supratherapeutic concentration Ž500 m M. of the drug produced partial inhibition of veratridine induced seizurelike activity ŽFig. 6..
4. Discussion This study examines veratridine-treated brain slices as a model of experimental seizure. Small concentrations of veratridine blocked chemical synaptic transmission but induced seizure-like bursts of action potentials in response to a single intracellular stimulus. Moreover, spontaneous bursting was observed after prolonged superfusion of veratridine. The seizure-like activity induced by veratridine was sensitive to inhibition by therapeutic concentrations of sodium channel blockers such as VPA but less sensitive to non-sodium channel blockers such as phenobarbital. These characteristics make the veratridine model suitable for screening potential sodium-dependent antiepileptic drugs. 4.1. The model
Fig. 6. The effect of phenobarbital on veratridine-induced bursting. Digitized tracings were recording from a single neuron ŽRMP: y73 mV. in the CA1 pyramidal area of the hippocampus. The superfusion of phenobarbital Ž40 m M, five neurons. following the induction of seizurelike activity by veratridine Ž0.3 m M, upper trace. did not suppress the evoked epileptic activity Žmiddle trace.. However, superfusion of a large concentration of the drug Ž500 m M, five neurons. partially inhibited this seizure-like activity. The spikes were truncated by the chart recorder. Calibrations bars applied to all traces. RMP of the neuron was y77 mV.
Electrophysiological studies have shown that there are three general factors which influence the neuronal bursting leading to development of epileptogenic activity w19,28x. These include neuronal bursting resulting from intrinsic capacity of neurons, diminution in the potency of inhibitory pathways and increase in the recruitment of excitatory synapses. In hyperexcitable membrane, blocking sodium channels stabilizes the membrane w15x. This suggests a possible involvement of sodium channels in the genesis of abnormal activities in epilepsy. In fact, there is evidence implicating the sodium channel as a relevant factor in the pathogenesis of human and experimental epileptic seizure. Studies in our laboratory indicate that endogenous seizure-like activity can be produced in hippocampal CA1 pyramidal neurons when sodium channels are altered by veratridine w32x. Furthermore, we have shown that veratridine augments a slowly-inactivating subthreshold sodium current I NaŽs. that leads to the generation of negative slope resistance observed in bursting neurons w2x. Synaptic transmission is not required to induce seizurelike activity by veratridine. Veratridine inhibition of synaptic transmission in hippocampal pyramidal CA1 neurons is indicated by the disappearance of the population spike seen in response to stimulation of the Schaffer collaterals. This inhibition may be related to veratridine-induced depolarization of the presynaptic terminal. Although synaptic transmission was blocked, veratridine was still effective in converting a single intracellularly evoked action potential
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to bursting discharge. These results are supported by other studies conducted in this laboratory. Although synaptic transmission was blocked in the CA1 region of the rat hippocampus by superfusion of the brain slice with kynurenic acid, intracellular stimulation evoked a repetitive discharge riding on a slow depolarization plateau w32x. A number of studies investigated the mechanisms involved in the propagation of seizure activity in the absence of synaptic transmission. It is believed that ephaptic interaction is the most acceptable explanation for synchronization in CA1 pyramidal neurons w18,31x. The fact that CA1 pyramidal neurons are densely packed and have a low extracellular conductivity favors a strong ephaptic interaction w16x. Depolarization of the neural membrane by extracellular potassium which increases during seizure is also another mechanism for synchronization in hippocampal pyramidal neurons w14x. When hippocampal slices are exposed to low-calcium solution, synaptic transmission is blocked and seizure-like activity appears in area CA1 of the hippocampus w36x. The mechanism of seizure induction by lowering calcium is not fully understood. Studies suggest that reducing extracellular calcium may enhance neuronal excitability by affecting sodium channel gating w37x. Furthermore, external calcium has been shown to stabilize the resting conformation of sodium channels and to block these channels at negative membrane potential w3x. The disadvantages of the low calcium model are that synaptic transmission may not be completely blocked, inhibition of this kind of seizure is only achieved at high concentrations of antiepileptic drugs and the mechanism by which seizures in this model are generated is not fully clear w13x. Compared to other synaptic-independent models of epilepsy, the veratridine model provides the following advantages: Ž1. a small concentration is needed to generate a long-lasting seizure-like activity that persists after washing; Ž2. based on the well-known mechanism of action of veratridine, the model involves alteration of sodium channel function, a major mechanism in the pathogenesis of many types of epilepsy; Ž3. since veratridine blocks synaptic transmission, seizure-like activity can be induced without the involvement of chemical synaptic transmission; Ž4. it is a reliable model for the assessment of both anti- and pro-convulsant activity of certain Žsodium channel-dependent. drugs and Ž5. it is a useful model for generating spontaneous seizure-like activity. 4.2. Effect of antiepileptic drugs on seizure-like actiÕity induced by Õeratridine Intracellular recording from mouse primary dissociated central neurons showed that VPA at therapeutic concentrations blocked sustained high frequency repetitive firing w24x. Even at supratherapeutic concentration levels, VPA did not affect GABA postsynaptic responses in the majority of neurons tested w8,24x. VPA has been used success-
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fully in the treatment of absence, generalized tonic–clonic and partial seizures w9x. Moreover, VPA has been shown to be effective in the treatment of status epilepticus w12x and in seizure prophylaxis in alcohol withdrawal w34x. This broad spectrum of antiepileptic activity of VPA may be related to its dose-dependent multiple mechanisms of action. Our study indicates that therapeutic concentrations of VPA inhibit evoked and spontaneous action potential bursting induced by veratridine. In comparison, the low calcium model requires larger concentrations of VPA to inhibit bursting w13x. This indicates the high sensitivity of the veratridine model to sodium channel-dependent antiepileptic drugs. The antiepileptic drug phenobarbital is used to treat both partial and generalized tonic–clonic seizures w23x. Its major antiepileptic mechanism of action is to increase GABA A receptor-chloride channel mean open time by binding to an allosteric regulatory site on the GABA A receptor-chloride channel complex w33x. This effect, which is produced at therapeutic concentrations of the drug, leads to an increase in chloride permeability and hyperpolarization of the postsynaptic membrane w22x. Results from the present study indicate that therapeutic concentrations of this synaptic-dependent drug failed to inhibit seizure-like activity induced by veratridine. Therapeutic concentrations of phenobarbital require an intact synaptic transmission to be effective, therefore no inhibitory effect is seen because synaptic transmission is blocked in the veratridine model. However, a much larger concentration of phenobarbital partially inhibits seizure-like activity induced by veratridine. Phenobarbital possesses direct neuronal membrane effects at supratherapeutic concentrations. At these concentrations, the drug inhibits batrachotoxin-stimulated 22 Naq influx w35x, reduces the sodium-dependent sustain highfrequency repetitive firing of action potentials w20x and blocks sodium channels in skeletal muscles and myelinated nerve preparations w7x.
5. Conclusion In rat hippocampal CA1 pyramidal neurons, veratridine inhibits synaptic transmission but is still effective in inducing seizure-like activity in response to a single intracellular stimulus. The seizure-like activity induced by this model is inhibited by therapeutic concentrations of the sodium channel blocker VPA but not by the synaptic-dependent antiepileptic drug phenobarbital. These features make the veratridine model a valuable tool to study direct membrane effects of sodium channel-dependent antiepileptic drugs.
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