Endogenous bursting due to altered sodium channel function in rat hippocampal CA1 neurons

BRAIN RESEARCH ELSEVIER

Brain Research 680 (1995) 164-172

Research report

Endogenous bursting due to altered sodium channel function in rat hippocampal CA1 neurons Lian-Ming Tian, Sameer Otoom, Karim A. Alkadhi * Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, Uniuersity of Houston, Houston, TX 77204-5515, USA

Accepted 7 February 1995

Abstract

Intracellular recordings were obtained from pyramidal neurons in the rat hippocampal CA1 area in order to investigate membrane mechanisms involved in veratridine-induced epileptiform activity. Veratridine (0.03-0.2 /zM) caused no changes in the passive membrane parameters including the resting potential, input resistance, and time constant. In the presence of small doses (0.03-0.1 /.LM) of veratridine, a single stimulus caused a relatively slow, large, synaptic-independent potential called the slow depolarizing after-potential (SDAP). When the hippocampal slice was treated with higher doses of veratridine (over 0.1 /zM), bursting, or seizure-like activity (SLA) occurred in response to a brief super threshold intraceilular stimulation. The duration of SLA bursting could be as long as ten seconds depending on the amplitude of SDAP, and was independent of the stimulus strength or duration. The frequency and configuration of SLA were sensitive to changes in membrane potential caused by applied DC current. At 0.3 /zM or higher, veratridine induced spontaneous rhythmic bursting that was also sensitive to membrane potential changes. The evoked or spontaneous bursting is characterized by being: (1) independent of synaptic transmission in that it persisted after complete blockade of evoked synaptic potential with kynurenic acid (0.5 raM), (2) sensitive to selective inhibition by low doses of the specific sodium channel blockers tetrodotoxin ('I"FX) or cocaine with no apparent influence on the evoked action potential. These results indicate that endogenous SLA bursting can be induced in hippocampal CA1 pyramidal neurons when certain properties of sodium channels are altered by veratridine. Keywords: Slow depolarizing after-potential; Epileptiform activity; Seizure-like activity; Kynurenic acid; Cocaine; Tetrodotoxin; Veratridine

1. Introduction

The hallmark of the human epileptic brain is the presence of abnormal electrical activity in the electroencephalogram (EEG) during non-seizure interval. The most obvious component of the abnormal electrical activity is called the interictal spikes. The cellular equivalent of the interictal spike in single cell recording is termed the paroxysmal depolarizing shift (PDS). The PDS is a prolonged sudden depolarization that triggers a high frequency bursting discharge [28,29]. The PDS-triggered bursting is regarded as epileptiform activity of neurons. The PDS is a common feature of neurons in a wide range of experimental and clinical

* Corresponding author. Fax: (1) (713) 743-1229, e-maih kalkadhi @uh.edu 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00258-8

epileptic foci, and therefore, considered as a cellular characteristic of neuronal epileptogenesis. After almost three decades since the discovery of PDS, there is substantial progress in the understanding of its origin. Synaptic mechanism of the PDS induction in the brain has been at the center of epilepsy research. Diminution of inhibitory synaptic transmission a n d / o r enhancement of excitatory synaptic transmission has been proposed [2,10,19,28-30,35]. However, in recent investigations the intrinsic nature of PDS has been emphasized. A number of studies in experimental animal models show that PDS can be induced without the involvement of chemical synaptic transmission [18,44,56]. When hippocampal slices are exposed to low Ca 2÷ solutions (0.1-0.2 mM), evoked synaptic transmission is blocked but spontaneous seizure-like activity appears in CA1, CA2 and CA3 areas. This low calcium-induced seizure-like activity bears a remarkable

L.-M. Tian et al. /Brain Research 680 (1995) 164-172

resemblance to focal seizure activity in vivo [23]. It has been reported that an increase in the number of sodium channels in the neurons of the genetically seizure-susceptible El mice is responsible for hyperexcitability of the central nervous system and may account for the predisposition to epileptic seizure [37,38]. An increase in the conductance of individual Na-channels in cerebral neurons was also found in another epileptic animal model, the tottering mouse [52]. Llinas and Sugimori [25] also found a long-lasting burst which could be attributed to a T/X-sensitive, non-inactivating conductance in rat cerebellar purkinje cells. Because of their importance in determining excitability of the neuronal membrane, sodium channels are likely to be the common primary mechanism for most, if not all, forms of epilepsy. Alteration of sodium channels could be the primary cause in genetic epilepsy, or the secondary in traumatic epilepsy. In any case, sodium channel mechanism could be the most relevant factor to epileptic seizure. The influence of veratridine on neuronal discharge pattern has been studied in peripheral excitable tissues. In single nerve axons of the lobster, repetitive after-discharge and plateau-shaped after-depolarization were induced by various ceveratrum alkaloids [16]. In frog skeletal muscle veratridine induced fast repetitive discharge which was very similar to the bursts of neuronal pacemakers [31]. In rat skeletal muscle, veratridine caused oscillations of membrane potential associated with rhythmic bursting of action potentials [5]. However, there was no study on the influence of veratridine on neuronal discharge behavior in freshly prepared mammalian brain tissues. The objective of this study was to test whether endogenous epileptiform activity can be induced in rat hippocampal CA1 pyramidal neurons as a result of altering sodium channel function with veratridine which has been widely used as a tool to study sodium channel properties [17,22,46]. 2. Materials and methods

2.1. Brain slice preparation Experiments were performed on male SpragueDawley rats (150-300 g). The animal was decapitated and the skull bone was removed with a rongeur. The brain was gently taken out with a stainless steel spatula and immersed in ice-cold oxygenated ( 0 2 95%, CO 2 5%) artificial cerebrospinal fluid (ACSF-NaC1, 124 mM; CaC12, 2.0 mM; KCI, 3.7 mM; MgC12, 1.2 mM; NaHCO3, 22 mM; NaH2PO 4, 1.2 mM; glucose, 11.0 mM). The two hemispheres were separated by dividing the brain midsagittally and a block of tissue containing the hippocampus was prepared by cutting the hemisphere across, posterior to the first branch of the superior cerebral vein and anterior to the last branch

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of this vein. The block was then fixed on the chuck of a vibroslice (Cam pden Instruments L T D ) using cyanoacrylate glue, and slices (approximately 500 /zm thick) were cut in a coronary plane. In this manner, from each hemisphere 4-5 slices were obtained which contain the CA1 region of the hippocampus. The slices were transferred to a beaker of ACSF solution continuously bubbled with gas (95% 0 2 - 5 % CO 2) and maintained at room temperature for at least 4 hours before recording.

2.2. Intracellular recording The recording chamber was fashioned from sylastic gel. It consisted of a circular bath of low volume ( < 1 ml) with gravity feed inlet and outlet. The base of the bath was fitted with two layers of nylon mesh glued onto a plastic plate. The whole recording chamber was mounted above the stage of a dissecting microscope. The bath was trans-illuminated using a fiber-optic illuminator. The entire recording system was housed in a grounded Faraday cage on a micro-G air suspension table. The ACSF, stored in a 500 ml bottle, was continuously dripped via a modified i.v. set connected to a three-way stopcock. Changes in the superfusing solutions were accomplished by switching this stopcock to an alternate reservoir. Polyethylene tubing was used to extend the connection from the stopcock and run through a jacket containing heated, circulating water which controlled the ACSF temperature. The ACSF in the polyethylene tubing was warmed as it passed through the water jacket, and fed into the inlet of the recording chamber. The water in the water jacket was heated and circulated with a circulating pump (Lauda C3, model T-l). The temperature of the ACSF in the recording bath was maintained at 32 + 1° C. The ACSF flowed out into a beaker through a gravity feed outlet after circulating through the bath. The slice was placed upon the nylon mesh on the bath floor submerged in the ACSF solution and was held in place on top with another nylon mesh glued on a plastic ring. Recording and stimulating electrodes were positioned using micromanipulators with the aid of a stereomicroscope. A bipolar stimulating electrode was placed on the stratum radiatum of CA2/CA3 region for orthodromic activation. A stimulator with two channels of output (Grass, model $88) was used, one channel to control constant current pulses for intracellular activation and the second channel for orthodromic stimulation via the bipolar stimulating electrode. The recording electrode was filled with 4 M K-acetate (80-120 MO) and visually positioned on the stratum pyramidale of CA1 region. A reference electrode (silver-silver chloride) was placed in the solution within the outlet of the chamber. An intracellular impalement was indicated by a shift in membrane potential in the hyperpolarizing direction detected on

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the oscilloscope. In one series of experiments extracetlular recordings of population responses were achieved by using microelectrodes filled with l m M sodium chloride (10-15 M J2). Cells were determined to be healthy based on the following criteria: (1) when they have a stable resting m e m b r a n e potential more negative than - 6 0 mV and (2) when passing a depolarization current through the recording electrode, an action potential with an amplitude of more than 70 m V should be generated. The low pass frequency was kept constant at 3 kHz. The sampled current and voltage signals were amplified (Axoclamp-2A, Axon Instrument) and stored on video tapes (PCM Data Recorder, A.R. Veter Co., Model 200) for later analysis. The current and voltage traces were displayed on digital storage oscilloscopes (Kikusui, DSS 5040; LeCroy, 9310). Hard copies of data were obtained by using a linear chart recorder (Graph Tec) or by a laser jet printer (LaserJet III Printer, Hewlet Packard) from a digital oscilloscope (LeCroy, 9310). 2.3. Drug Veratridine was dissolved in 0.1 mM HC1 solution. The stock solutions of veratridine (0.1 M, Sigma Chem) and T'I'X (1.0 mM, Sigma Chem) were prepared and kept in freezer. Cocaine (0.1 M) was prepared and stored in refrigerator. Kynurenic acid (Sigma, Chem) was dissolved in 0.5 N sodium hydroxide, adjusted to p H 7.4 with 0.1 N sodium phosphate buffer and diluted with ACSF to the appropriate concentration. The final concentrations of drugs were prepared by adding a calculated amount of stock solution to ACSF.

3. Results 3.1. Induction of slow depolarizing (SDAP) and seizure-like activity (SLA)

after-potential

Stable recordings were obtained from 93 neurons in rat hippocampal CA1 area with resting m e m b r a n e potential (RMP) of - 69.9 + 0.3 m V and action potential amplitude of 95.7 + 0.7 mV. These neurons had input

resistance of 38.3 + 2.7 M/2. Spontaneous discharge was observed in 18 out of a total of 93 neurons when the m e m b r a n e potential was stabilized after impalement. Table 1 summarizes the passive m e m b r a n e parameters measured immediately before and 30 minutes after addition of veratridine. No significant differences were detected in any p a r a m e t e r when compared in presence and absence of various concentrations of veratridine (paired t-test, P > 0.05). The application of a single depolarizing current pulse to neurons evoked only an action potential spike in normal hippocampal CA1 pyramidal neurons (Fig. 1A, control). After treatment with veratridine (0.03-1.0 Iz M), the same current pulses evoked slow depolarizing after-potential (SDAP). Once the SDAP reached spike firing threshold, a long-lasting burst was triggered (Fig. 1A, veratridine). This burst was independent of the stimulus strength or duration. The bursting volleys, called the seizure-like activity (SLA), were composed of individual fast action potentials arising spontaneously from the SDAP phase. An after-hyperpolarization with an amplitude of 5 to 10 mV followed the bursting activity. During this period a threshold stimulation could only evoke a depolarizing plateau. As the after-hyperpolarization diminished, the threshold stimulation triggered another bursting activity. The bursting was reversed after prolonged wash-out of veratridine (Fig. 1A, wash). Higher doses of veratridine (0.3 /zM or higher) induced spontaneous m e m b r a n e potential oscillation which triggered spontaneous bursting volleys (Fig. 1B). The duration of individual bursts varied in different neurons and with different times of veratridine exposure. However, the bursting pattern was rather consistent in a given neuron at a given time. The firing frequency and the burst duration were independent of the stimulus intensity as long as the stimulus reached the threshold. The repetitive firing activity within an individual burst normally lasted no longer than 10 s. After prolonged exposure (60 min) to high doses (0.3 /zM or higher) of veratridine, the depolarizing plateau could last as long as several minutes during which no action potential or only a small spike could be induced. We did not see spontaneous recovery of the m e m b r a n e

Table 1 Effect of veratridine on passive membrane parameters a (mean -+ S.E.M.) Veratridine (~M) Vm (mV) n R m (MO) Control Drug Control 1.0 - 70.1 _+1.0 - 69.2 + 1.5 10 39.0 _+7.0 0.3 -70.2+0.7 -69.7+1.1 29 34.9-+3.0 0.2 -68.5_+2.0 -67.1+2.3 11 43.3-+6.0 0.1 -68.1 _+2.2 -67.2 + 2.2 6 42.5 -+ 10.0 0.03 - 70.0 -+ 1.3 - 70.8 5- 1.8 5 NT

n Drug 37.5 _+5.0 35.7_+3.1 36.2_+7.0 46.5 -+ 13.1 NT

5 9 5 4

t m (ms) Control NT 11.9-+0.8 13.7-+0.8 NT NT

n Drug NT 11.9_+0.2 11.3+_1.4 NT NT

4 5

a Membrane potential (Vm), membrane resistance ( R m ) , and membrane time c o n s t a n t (t m) were measured right before and 30 min after the introduction of veratridine. 'n' is the number of neurons tested. NT: not tested.

L.-M. Tian et al. / Brain Research 680 (1995) 164-172

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illustrated in Fig. 2. After 40 rain of treatment with veratridine (0.2 ~M) spontaneous slow depolarizing plateau and bursting appeared in addition to the evoked bursting. In these slices, subthreshold stimulation of the Schaffer collateral input induced a normal EPSP. The duration of the spontaneous depolarizing plateau was much longer than that of evoked EPSP (Fig. 2A, left). However, a brief intracellular stimulation via the recording electrode caused a prolonged depolarization which triggered multiple bursts (Fig. 2A, right). Spontaneous prolonged depolarizing plateaus with no spikes similar to the SDAP in amplitude and duration were recorded in three neurons treated with veratridine (Fig. 2B). Consistent with previous studies [16], this result suggests that the first spike elicited by the intracellular current pulse may not be a prerequisite for the SDAP. In these neurons, a superthreshold Schaffer

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Fig. 1. Veratridine induces seizure-like activity in rat hippocampal CA1 pyramidal neurons. A: intracellular current pulses were given at 0.1 Hz. Each of these current pulses induced only one spike in control period before the introduction of veratridine (Control). A slow depolarizing after-potential (SDAP) was induced after 22 min in 0.3/zM veratridine, and this SDAP triggered bursting 18 min later. The bursting disappeared 60 rain after 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. B: Spontaneous bursting was observed after prolonged exposure to 0.3/zM veratridine in another neuron. The spikes were truncated by the chart recorder. Calibration sign on left is for left panel and right for right panel.

potential with this long, persistent depolarization. This aggravated prolonged depolarization (longer than 5 min) resulted in irreversible loss of membrane potential. However, persistent depolarization lasting as long as 5 min could be reversed when a negative current was injected through the recording electrode.

3.2. The SDAP is different from the EPSP The difference between the SDAP and EPSP is

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Fig. 2. The SDAP is not the same as the excitatory postsynaptic potentials (EPSPs). The neuron was treated with 0.3/~M veratridine. A: Subthreshold presynaptic stimulation (arrow) evoked an EPSP. A spontaneous slow depolarizing after-potential and burst (*) also appears in the same record. A brief intracellular stimulation evoked a slow depolarizing after-potential with multiple bursts (right) in the same neuron. B: a spontaneous slow depolarizing plateau ( " ) without initial spike also occurred. Threshold synaptic activation evoked a single action potential (arrow). C: synaptic transmission was completely blocked after the addition of 0.5 mM kynurenic acid (left) without influence on the intracellularly evoked slow depolarization (right) which triggered high frequency repetitive discharge. Similar results were obtained in three other experiments. All traces are from the same neuron.

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no measurable effect on the population responses, however at 0.3-0.4 mM, the drug completely blocked these responses.

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3.3. Characteristics of seizure-like activity (SLA) (~ 20

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Veratridine (laM) Fig. 3. The delay of veratridine-induced bursting activity. The bursting onset time was measured from the moment of veratridine introduction to that when five or more spikes appear in addition to the one induced by the current pulse. The amplitude of the current pulses was maintained constantly throughout of the experiments. N u m b e r in parentheses indicates n u m b e r of experiments at the particular veratridine concentration.

The SLA appeared in one out of eight neurons treated with 0.1 g M veratridine (60 min) and in 20 out of 23 neurons with 0.3 /~M veratridine (60 min). The delay of bursting induction (from the time of veratridine introduction to that when 5 action potentials appear outside the current pulses) was highly dose-dependent within the concentration range used in this study (Fig. 3). It was noted that neurons initially showing spontaneous discharge developed into bursting activity with much shorter delay after the introduction of

A collateral activation of CA1 pyramidal neurons evoked a single action potential, but did not induce a slow depolarizing plateau (Fig. 2B, arrow). To further ascertain the non-synaptic nature of the SDAP, excitatory synaptic transmission was blocked by the glutamate receptor antagonist, kynurenic acid. While the addition of kynurenic acid (0.5 mM) completely blocked the EPSP evoked by the Schaffer collateral stimulation (Fig. 2C, left), a long-lasting repetitive discharge, riding on top of a slow depolarization plateau, could still be evoked with intracellular stimulation (Fig. 2C, right). Additionally, in a series of 4 experiments the effect of veratridine on extracellular population response of the CA1 neurons evoked by stimulation of the Schaffer collaterals was studied. Veratridine at 0.1-0.2 mM had 70 mV

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Fig. 4. Voltage-dependency of the veratridine-induced SDAP. A sharp decrease of S D A P amplitude, measured at 200 ms after pulse (arrow at inset), occurs with hyperpolarization of the m e m b r a n e potential. The hyperpolarization is achieved by intracellular injection of anodal current. This figure is from one neuron treated with 0.2 ~ M veratridine representing over 30 other neurons with similar response. Each point is the m e a n + S E M of 9 measurements. The insets are digitized traces of responses.

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Membrane potential (mV) Fig. 5. The dependency of evoked bursting frequency on m e m b r a n e potential. Panel A shows traces from a single neuron recorded in the presence of veratridine (0.1 /xM) at the levels of the resting ( - 6 8 mV) and hyperpolarized ( - 7 8 mV) potentials. At resting level each threshold stimulation triggered a burst; at hyperpolarized level threshold stimulations triggered only occasional burst with longer duration as compared to resting level. Panel B shows the percentage change of burst frequency in response to 10 mV hyperpolarization. Each sign represents one neuron.

L.-M. Tian et al. / Brain Research 680 (1995) 164-172

veratridine (8 + 2 min for 0.3 /~M, 4 neurons). The bursting discharge occurred within 2 min after the introduction of veratridine in one spontaneously active neuron. The amplitude of the SDAP underlying the SLA was highly voltage dependent (Fig. 4). It decreased with membrane hyperpolarization and increased with depolarization, and its amplitude and the membrane voltage appeared to be non-linearly correlated. After prolonged exposure to veratridine, the SDAP plateau reached threshold and triggered bursting activity. In all the neurons treated with veratridine, hyperpolarizing the membrane potential by 5 mV caused a decrease in the amplitude and duration of the SDAP (Fig. 4, inset). In bursting neurons a slight hyperpolarization led to the elimination of bursting activity obviously as a result of reduction of the SDAP plateau. The rate and configuration of veratridine-induced bursts were sensitive to changes in membrane potential caused by applied DC current. A hyperpolarization of 10 mV caused a marked reduction of the rate of bursting (Fig. 5A). Complete cessation of bursting occurred in some of the neurons after they were hyperpolarized by over 10 mV. The change in burst frequency with applied current for three neurons is summarized in Fig. 5B. When T T X (30 riM) was used in neurons treated with veratridine (0.3 ~M), the SLA was eliminated, apparently due to a decrease of SDAP because the evoked action potential was not affected (Fig. 6A). Similarly, the application of cocaine (100 ~ M ) caused elimination of after-spike discharge with no measurable effect on the evoked action potential (Fig. 6B).

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4. Discussion

The data presented here clearly show that small concentrations of veratridine change the neuronal discharge pattern in rat hippocampal CA1 pyramidal neurons. This change results in seizure-like events. Chemical synaptic transmission is not required for the veratridine-induced activity. The sodium channel blockers, T T X and cocaine, are able to inhibit SDAP and thus abolish the epileptiform activity without affecting the evoked fast action potential. This study indicates that hippocampal CA1 pyramidal neurons possess an intrinsic mechanism for endogenous bursting activity that can be brought about by disturbance of the normal function of sodium channels. The significance of this study is that this epileptogenic mechanism may be the etiology of at least certain forms of clinical epilepsy such as absence and partial seizures with or without secondary generalization. Evidence that the hippocampal CA3 neurons possess an intrinsic capability for endogenous bursting has been provided by a variety of experiments from mammalian species [12,20,53,55]. However, it is still uncertain whether the epileptiform bursting activity can be initiated intrinsically in other regions of mammalian brain. To demonstrate that bursting activity is the result of an intrinsic membrane function, Hablitz and Johnston [12] have adopted a set of criteria from invertebrate neurons in which the intrinsic nature of bursting activity has been well documented [7]. These criteria include that: (1) burst frequency is a sensitive function of membrane potential; (2) bursts can be triggered by brief events; and (3) bursts can be ob-

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Fig. 6. The inhibitory effect of TTX and cocaine on veratridine-inducedseizure-like activity.A seizure-like activitywas induced in the presence of 0.3 #M veratridine (Control) by a single intracellular current pulse, and inhibited by 30 nM TTX (A) and by 100 #M cocaine (B). The bursting activity reappeared after washout of TTX (A) or cocaine (B). Note that TTX or cocaine failed to block the evoked single action potential while totally blocking the seizure-like activity. Records in A and B are from two different neurons.

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tained in the absence of propagated action-potentialdependent synaptic input. In our model of veratridinetreated rat hippocampal preparation, the evoked as well as spontaneous bursts in hippocampal CA1 neurons satisfy these criteria. Our study clearly shows that rat hippocampal CA1 pyramidal neurons possess the intrinsic nature for sudden, paroxysmal depolarizations which hallmark epileptogenesis in single neurons. Studies have shown that the discharge pattern of mammalian central neurons can be altered by a number of convulsants such as bicuculline, penicillin and pentylenetetrozal. However, veratridine offers a number of advantages over those convulsants. First, in small doses it allows the genesis of epileptiform bursts with no apparent effect on the inhibitory or excitatory synaptic transmission. Second, it is a well-known specific agent for the activation of sodium channels, thus, induction of epileptiform and SLA by small doses of veratridine can be attributed to alteration of sodium channel function. Third, the epileptiform activity is long-lasting, as it still in effect even 60 min after wash-out of the drug. Fourth, as the sodium channel is the 'common pathway' for the factors that alter the neuronal excitability, veratridine-induced epileptiform activity may be the best representative model for most experimental epilepsy research. Two possible explanations for the induction of SDAP can be offered. The first is that veratridine modifies a portion of the existing normal sodium channels responsible for the action potential [47]. Studies have shown that during repetitive depolarization in neurons treated with veratridine, the fast transient sodium current decreases in size and a maintained, slower one becomes larger [24,43]. These results presume that veratridine induces a secondary sodium conductance by slowing down the activation and inactivation processes of a portion of normal sodium channels [8,14,16,25]. This hypothesis has been supported by patch clamp studies at the single channel level in rat blastoma neurons, an inexcitable cell line derived from a rat brain tumor [4], as well as in rat cultured cardiac myocytes [48]. The results from these studies indicate that voltage-dependent activation of veratridine-modified channels is shifted to a more negative membrane potential, and inactivation is slowed or sometimes eliminated. The second explanation is based on the assumption of the existence of a 'silent' sodium channel where veratridine vitalizes this low- or non-conducting channel subtype (slow sodium channel). This vitalization can be the result of lowering the activation threshold or increasing the conductance. It is becoming increasingly evident that neither the presence of a sodium channel-dependent mechanism of excitation, nor excitability is a prerequisite for veratrum alkaloids to cause the appearance of persistently activating sodium channels. Veratridine opens sodium channels in the pancreatic/3-cell

membrane which normally produces impulses by activation of calcium channels [11]. The action potential of chick embryonic heart cells in an early stage of development is due to the activation of T/X-insensitive, slow sodium-calcium channels. Veratridine depolarizes these cells and the effect is blocked by T T X [42]. Furthermore, veratridine opens T-f X-sensitive sodium channels in rat blastoma neurons [36], produces a TTX-sensitive depolarization in non-spiking sensory dendrites of the crab [27], and at small concentrations, activates silent sodium channels in nonexcitable embryonic cockroach neurons [1]. These experiments indicate that veratridine is effective in activating sodium channels other than the channels responsible for the fast action potential. Voltage clamp and patch clamp studies show that at least two, perhaps three, kinetically and pharmacologically distinct sodium channels are present on a variety of excitable cells including skeletal muscle [34,49] (for review see Barchi [3]), dorsal root ganglion neurons [6,40], and cerebral neurons [9,15,33,39,41]. The differences in inactivation and activation kinetics, and toxin sensitivity of sodium channels can be associated with primary sequence differences revealed by sodium channel cDNA analysis [13,50]. With molecular cloning, three subtypes of sodium channels (RI, RII, and R i l l ) have been isolated from rat brain [21,32,45]. These cloning experiments provide the strongest evidence for the presence of multiple subtypes of sodium channel in rat brain neurons. The functional significance for the existence of multiple sodium channel subtypes is not clear. Molecular studies of Westenbroek et al. [51] show that two subgroups of sodium channels have different preferential cellular localizations; with subtype RII in the neuronal s o m a / a x o n and RI in the dendrites. It is possible that the distinction between the rapidly activating/inactivating and 'persistent' sodium currents detected in electrophysiological studies may parallel the differential localization of sodium channel subtypes identified in the molecular cloning studies. Electrophysiological studies have demonstrated that the dendritic area could be the site for the PDS initiation [54], which implies that the type RI sodium channel might relate to the PDS initiation. Exaggeration of the slow depolarization in dendrites as a result of overexpression of slow sodium channel might contribute to the epileptogenesis. Indeed, it has recently been reported that altered expression of two brain sodium channel mRNAs may have been responsible for the etiology of epilepsy in patients [26]. Two significant conclusions are drawn from this study. (1) Seizure-like activity can be induced in individual neurons without involvement of chemical synaptic transmission; and (2) alteration of sodium channel function contributes to the cause of seizure-like activity because neuronal discharge pattern is blocked selec-

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tively by small doses of TI'X and cocaine. These results indicate that normal neuronal firing behavior can be changed to epileptiform activity by slight modification of sodium channel properties.

References [1] Amar, M., Pichon, Y. and Inoue, I., Micromolar concentrations of veratridine activate sodium channels in embryonic cockroach neurones in culture, Pfliigers Arch., 417 (1991) 500-508. [2] Ayala, G.F., Dichter, M.A., Gumnit, R.J., Matsumoto, H. and Spencer, W.A., Genesis of epileptic interictal spikes. New knowledge of cortical feedback systems suggests a neurophysiological explanation of brief paroxysms, Brain Res., 52 (1973) 1-17. [3] Barchi, R.L., Probing the molecular structure of the voltage-dependent sodium channel, Annu. Rev. Neurosci., 11 (1988) 455495. [4] Barnes, S. and Hille, B., Veratridine modifies open sodium channels, J. Gen. Physiol., 91 (1988) 421-443. [5] Brodie, C. and Sampson, S.R., Veratridine-induced oscillations in membrane potential of cultured rat skeletal muscle: role of the Na-K pump, Cell. Mol. Neurobiol., 10 (1990) 217-226. [6] Caffrey, J.M., Eng, D.L., Black, J.A., Waxman, S.G. and Kocsis, J.D., Three types of sodium channels in adult rat dorsal root ganglion neurons, Brain Res., 592 (1992) 283-297. [7] Carnevale, N.T. and Wachtel, H., Two reciprocating current components underlying slow oscillations in Aplysia bursting neurons, Brain Res., 2 (1980) 45-68. [8] Catterall, W.A., Studies of voltage-sensitive sodium channels using ligand binding methods. In Yamamura et al. (Eds), Neurotrammitter Receptor binding, 2nd edn., Raven Press, New York 1985, pp. 103-121. [9] Corbett, A.M. and Krueger, B.K., Isolation of two saxitoxin-sensitive sodium channel subtypes from rat brain with distinct biochemical and functional properties, J. Memb. Biol., 117 (1990) 163-176. [10] Dichter, M. and Spencer, W.A., Penicillin induced interictal discharges from cat hippocampus. II. Mechanisms underlying origin and restriction, Neurophysiology, 32 (1969) 663-687. [11] Donatsch, p., Lowe, Richardson, A. and Tylor, B.P., The functional significance of sodium channels in pancreatic beta cell membranes, J. Physiol.,, 267 (1977) 357-376. [12] Hablitz, J.J. and Johnston, D., The endogenous nature of spontaneous bursts in hippocampal pyramidal neurons, Cell. Mol. Neurobiol., 1 (1981) 325-334. [13] Hartshorne, R.P., Keller, B.U., Talvenheimo, J.A., Catterall, W.A. and Montal, M., Functional reconstitution of purified channels from brain in planar lipid bilayers, Ann. NYAcad. Sci., 479 (1986) 293-305. [14] Hille, B., Modifiers of gating. In B. Hille (Ed.), Ionic Channels of Excitable Membrane 2nd edn., Sunderland, MA, 1992, pp. 445-471, [15] Hoehn, K., Watson, T.W. and MacVicar, B.A., A novel tetrodotoxin-insensitive, slow sodium current in striatal and hippocampal neurons, Neuron, 10 (1993) 543-552. [16] Honerjager, P., Electrophysiological effects of various ceveratrum alkaloids on single nerve axons, Naunyn Schmiedeberg Arch. Pharmacol., 280 (1973) 391-416. [17] Honerjager, P., Cardioactive substances that prolong the open state of sodium channels, Rev. Physiol. Biochem. Pharmacol.,, 92 (1982) 1-74. [18] Jefferys, J.G.R. and Haas, H.L., Synchronized bursting of CA1

171

hippocampal pyramidal cells in the absence of synaptic transmission, Nature, 300 (1982) 448-450. [19] Johnston, D. and Brown, T.H., Giant synaptic potential hypothesis for epileptiform activity, Science, 211 (1981) 289-297. [20] Kandel, E.R. and Spencer, W.A., Electrophysiology of hippocampal neurons. II. Afterpotentials and repetitive firing, J. Neurophysiol., 24 (1961) 243-259. [21] Kayano, T., Noda, M., Flockerzi, V., Takahashi, H. and Numa, S., Primary structure of rat brain sodium channel III deduced from the cDNA sequence, FEBS Lett., 228 (1988) 187-194. [22] Khodorov, B.I., Batrachotoxin as a tool to study voltage-sensitive sodium channels of excitable membranes, Prog. Biophys. Mol. Biol., 45 (1985) 57-148. [23] Konnerth, A., Heinemann, U. and Yaari, Y., Nonsynaptic epileptogenesis in the mammalian hippocampus in Vitro. I. Development of seizurelike activity in low extracellular calcium, J. Neurophysiol., 56 (1986) 409-423. [24] Leibowitz, M.D., Sutro, J.B. and Hille, B., Voltage-dependent gating of veratridine-modified Na channels, J. Gen. Physiol., 87 (1986) 25-46. [25] Liinas, R. and Sugimori, M., Electrophysiologic properties of in vitro purkinje cell somata in mammalian cerebellar slices, J. Physiol., 305 (1980) 171-195. [26] Lombardo, A.J., Lu, C.M., Kuzniecky, R. and Brown, G.B., Altered relative expression of two human brain sodium channel mRNAs in patients with epilepsy, Neurosci. Abstr., 18 (1992) 1134. [27] Lowe, D.A., Bush, B.M.H. and Ripley, S.H., Pharmacological evidence for 'fast' sodium channels in nonspiking neurones, Nature, 274 (1978) 289-290. [28] Matsumoto, H. and Ajmone-Marsan, C., Cortical cellular phenomena in experimental epilepsy: interictal manifestation, Exp. Neurol., 9 (1964) 286-304. [29] Matsumoto, H. and Ajmone-Marsan, C., Cortical cellular phenomena in experimental epilepsy: ictal manifestation Exp. Neurol., 9 (1964) 305-326. [30] Matsumoto, H., Ayala, G.F. and Gumnit, R.J., Neuronal behavior and trigering mechanisms in cortical epileptic focus, J. Neurophysiol., 32 (1969) 688-703. [31] Nanasi, P.P., Kiss, T., Danko, M., Lathrop, D.A., Different actions of aconitine and veratrum alkaloids on frog skeletal muscle, Gen. Pharmacol., 21 (1990) 863-868. [32] Noda, M., Ikeda, T., Kayano, T., Suzuki, H., and Takeshima, H., Existence of distinct sodium channel messenger RNAs in rat brain, Nature, 320 (1986) 88-192. [33] Noda, M., Ikeda, T., Suzuki, H., Takeshima, H., Takeshima, T., Kuno, M. and Numa, S., Expression of functional sodium channels from cloned cDNA, Nature, 322 (1986) 826-828. [34] Pappone, P., Voltage-clamp experiments in normal and denervated mammalian skeletal muscle fibers, Z Physiol.,, 305 (1980) 377-410. [35] Prince, D.A., The depolarization shift in 'epileptic' neurons, Exp. Neurol., 21 (1968) 467-485. [36] Romey, G., Jacques, Y., Schweitz, H., Fosset, M. and Lazdunski, M., The sodium channel in non-impulsive cells. Interaction with specific neurotoxins, Biochem. Biophys. Acta, 556 (1979) 344-353. [37] Sashihara, S., Yanagihara, N., Kobayashi, H., Izumi, F., Tsuji, S., Murai, Y. and Mita, T., Overproduction of voltage-dependent Na+ channels in the developing brain of genetically seizure-susceptible E1 mice, Neuroscience, 48 (1992) 285-291. [38] Sashihara, S., Aramaki, I., Murai, Y. and Mita, T., Biochemical abnormalities in developing E1 mouse, Jpn. J. Psychiat. Neurol., 45 (1991) 365-367. [39] Scheuer, T., Auld, V.J., Boyd, S., Offord, J., Dunn, R. and Catterall, W.A., Functional properties of rat brain sodium channels expressed in a somatic cell line, Science, 247 (1990) 854-858.

172

L.-M. Tian et al. /Brain Research 680 (1995) 164-172

[40] Schwartz, A., Palti, Y. and Meiri, H., Structural and developmental differences between three types of Na channels in dorsal root ganglion cells of new born rats, J. Membr. Biol., 116 (1990) 117-128. [41] Sheridan, R.E., Electrophysiological characterization of sodium channel types in the HCN-1A human cortical cell line, Brain Res. Bull., 30 (1993) 577-583. [42] Sperelakis, N. and Pappano A.J., Increase in PNa and PK of cultured heart cells produced by veratridine, J. Gen. Physiol., 53 (1969) 97-114. [43] Sutro, J.B., Kinetics of veratridine action on Na channels of skeletal muscle, J. Gen. Physiol., 87 (1986) 1-24. [44] Taylor, C.P. and Dudek, F.E., Synchronous neuronal afterdischarges in rat hippocampal slices without chemically active synapses, Science, 218 (1982) 810-812. [45] Trimmer, J.S. and Agnew, W.S., Molecular diversity of voltagesensitive Na channels, Annu. Reu. Physiol., 51 (1989) 401-418. [46] Ulbricht, W., The effect of veratridine on excitable membranes of nerve and muscle, Retd. Physiol. Biolchem. Exp. Pharmacol., 61 (1969) 18-71. [47] Ulbricht, W., The inactivation of sodium channels in the node of Ranvier and its chemical modification, Ion channels, 2 (1990) 123-168. [48] Wang, G., Dugas, M., Armah, B.I. and Honerjager, P., Sodium channel comodification with full activator reveals veratridine reaction dynamics, Mol. Pharmacol., 37 (1990) 144-148.

[49] Weiss, R.E. and Horn, R., Functional differences between two classes of sodium channels in developing rat skeletal muscle, Science, 233 (1986) 361-364. [50] West, J.W., Scheuer, T., Maechler, L. and Catterall, W.A., Efficient expression of rat brain type I I A N a ÷ channel a subunits in a somatic cell line, Neuron, 8 (1992) 59-70. [51] Westenbroek, R.E., Merrick, D.K. and Catterall, W.A., Differential subcellular localization of the RI and RII Na ÷ channel subtypes in central neurons, Neuron, 3 (1989) 695-704. [52] Willow, M., Taylor, S.M., Catteral, W.A. and Finnel, R.H., Down regulation of sodium channels in nerve terminals of spontaneously epileptic mice, Cell. Mol. Neurobiol., 6 (1986) 213-220. [53] Wong, R.S. and Prince, D.A., Participation of calcium spikes during intrinsic burst firing in hippocampal neurons, Brain Res., 159 (1978) 385-390. [54] Wong, R.S. and Prince, D.A., Dendritic mechanisms underlying penicillin-induced epileptiform activity, Science, 204 (1979) 1228-1231. [55] Wong, R.K.S. and Prince, D.A., Afterpotential generation in hippocampal pyramidal cells, J. Neurophysiol., 45 (1981) 86-97. [56] Yaari, Y., Konnerth, A. and Heinemann, U., Spontaneous epileptiform activity of CA1 hippocampal neurons in low extracellular calcium solutions, Exp. Brain Res., 51 (1983) 153-156.