Different firing patterns induced by veratridine and aconitine in injured dorsal root ganglion neurons

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1 Acta Physiologica Sinica, April 25, 2005, 57 (2): Research Paper Different firing patterns induced by veratridine and aconitine in injured dorsal root ganglion neurons DUAN Jian-Hong, XING Jun-Ling, YANG Jing, HU San-Jue * Institute of Neuroscience, The Fourth Military Medical University, Xi an , China Abstract: Ectopic spontaneous activity originated from the injured dorsal root ganglion (DRG) neurons in rats was recorded through single dorsal root fiber. The firing patterns induced by veratridine and aconitine, inhibitors of inactivation gate of sodium channel operating on different binding sites, were compared. In the same neuron, veratridine (1.5~5.0 µmol/l) caused slow wave oscillations of interspike intervals (ISIs), while aconitine (10~200 µmol/l) caused tonic firing. Moreover, even if the background firing patterns were various and the reagent concentrations used were different, veratridine and aconitine still induced slow wave oscillations and tonic firing patterns, respectively. The results suggest that veratridine and aconitine induce different firing patterns in injured DRG neurons, which may relate to their inhibitory effects on different binding sites of the sodium channel. Key words: dorsal root ganglion; firing patterns; veratridine; aconitine; sodium channels 藜芦碱和乌头碱在受损背根节神经元诱发不同的放电模式 段建红, 邢俊玲, 杨晶, 胡三觉 * 第四军医大学神经科学研究所, 西安 摘要 : 为了研究钠通道失活门阻断后受损背根节神经元放电模式的变化特征, 在大鼠背根节慢性压迫模型上采用单纤维技术记录 A 类神经元的自发放电 藜芦碱和乌头碱是钠通道失活门的抑制剂, 但二者作用于不同的位点, 前者结合于 D2-S6, 后者结合于 D3-S6 我们比较了这两种试剂引发的放电模式 结果发现, 在同一神经元, 藜芦碱 (1.5~5.0 µmol/l) 可以引起放电峰峰间期的慢波振荡, 即峰峰间期由大逐渐减小, 然后又逐渐增大, 形成重复的振荡波形, 每个振荡持续约数十秒至数分钟 ; 而乌头碱 (10~200 µmol/l) 则引起强直性放电, 即峰峰间期逐渐减小, 然后维持在一个稳定的水平 这两种不同的放电模式不因背景放电或试剂浓度的不同而发生明显的改变 实验结果表明, 藜芦碱和乌头碱在受损的背根节神经元可以引发不同的放电模式, 这可能与它们结合于钠通道上不同位点的抑制作用有关 关键词 : 背根节 ; 放电模式 ; 藜芦碱 ; 乌头碱 ; 钠通道中图分类号 : Q424 Voltage-gated sodium channels play a critical role in regulating the electrical excitability of neurons. The fundamental properties that enable sodium channels to carry out their physiological roles include rapid voltage-dependent activation and inactivation [1, 2]. Inactivation closes the channel and prevents it from reopening until there has been sufficient time for recovery, which contributes to determining the firing frequency and patterns [3, 4]. A large number of biological toxins exert their effects by modifying the properties of sodium channels in nerve, heart and skeletal muscle [5, 6]. Veratridine and aconitine, which exist in nature and are isolated from certain plants, are both inhibitors of inactivation gate of sodium channels [6]. These two toxins cause persistent activation of sodium channels at rest- Received Accepted This work was supported by the National Natural Science Foundation of China (No , ) and the National Basic Research Priorities Programme of China (No. G ). * Corresponding author. Tel: ; Fax: ; sjhu@fmmu.edu.cn

2 170 ing membrane potential by blocking the closure of the inactivation gate and shifting the voltage-dependence of the channel activation to a more negative membrane potential [7, 8]. However, their binding sites to sodium channels α-subunit are significantly different, the former is D2- S6 and the latter is D3-S6 [9]. It has been reported that endogenous seizure-like activity or bursting can be induced in hippocampal CA1 pyramidal neurons by veratridine [10, 11]. Moreover, the influence of veratridine on neuronal firing patterns has been studied in peripheral excitable tissues. For example, veratridine induced fast repetitive firing that was similar to the bursts of neuronal pacemakers in frog skeletal muscle [12]. While in rat skeletal muscle, veratridine caused rhythmic bursting associated with oscillations of membrane potential [13, 14]. In our previous study, veratridine induced slow wave oscillations of ISIs in injured DRG neurons [15, 16] and injured nerve fibers [17]. On the other hand, the influence of aconitine is different from that of veratridine [12, 18]. In frog skeletal muscle, the firing pattern induced by aconitine was tonic, while the firing rate with veratridine was progressively increasing. A possible explanation is that aconitine-modified Na + channels in skeletal muscle inactivated in voltage-dependent manner while veratridine-modified Na + channels failed to close via their fast inactivation machinery within the entire voltage range studied. [12]. Moreover, aconitine slowly and reversibly decreased the population spike recorded in the CA1 pyramidal cell layer [19]. These results suggest that different firing patterns induced by veratridine and aconitine may be related to their different binding sites to sodium channels. However, these experiments were performed on different tissue and animals. Therefore, it is an intriguing question whether the two toxins induce different firing patterns in the same preparation and cells. In order to answer this question, in the present study, we compared the effects of veratridine and aconitine with different concentrations on specific firing patterns in the same injured DRG neurons with different background conditions. 1 MATERIALS AND METHODS 1.1 Animal model preparation The model of chronic compression of DRG was prepared according to methods described previously [20]. In brief, sterilized surgical procedures were carried out under pentobarbital sodium anesthesia (40 mg/kg, i.p.) on adult Sprague-Dawley rats (200~300 g) of both sexes. The L5 intervertebral foramen on one side was clearly exposed and an L-shape stainless steel rod (4 mm in length, 0.6 Acta Physiologica Sinica, April 25, 2005, 57 (2): ~ 0.8 mm in diameter) was inserted into the L5 intervertebral foramen to produce a steady compression on the L5 DRG. Another group of rats served as unoperated controls. 1.2 Surgical exposure of DRG and microfilament recording Electrophysiological recordings were conducted 3~10 d after the insertion of stainless steel rod. A lumbar laminectomy was performed under chloralose-urethane anesthesia (40 mg/kg, i.p.) and at both L1-L2 and L4-L5 small pools were prepared. In the L4-L5 pool, the L5 DRG on injured side was sufficiently exposed and the compressing steel rod was taken out. The spinal nerve was transected about 5~10 mm distal to DRG so that spontaneous activity recorded from dorsal root fibers originated primarily from the ganglion and not from receptors in peripheral tissues. During the experiment, the L4-L5 pool was filled with 1 ml of warm Kreb s solution (35~37 ºC) containing: (in mmol/l) NaCl 150, KCl 5, CaCl 2 2, MgCl 2 1, glucose 10 and Tris 5, at a ph of 7.4. In the L1-L2 pool, the dorsal root of the injured L5 DRG was identified and covered with warm paraffin oil (35~37 ºC). Under a microscope, a microfilament of about 30~50 µm in diameter was teased away from the dorsal root and its distal end was placed on a fine platinum electrode (30 µm in diameter) for electrophysiological recording of spontaneous activity of single myelinated fibers from the injured DRG neurons. The myelinated fibers were identified by their conduction velocity (CV) and waveform [20]. The conduction velocity was measured via electrical stimulation of the sciatic nerve through an isolator. Only A-type neurons with CV of 3~62.5 m/s were studied in the present experiment. The original spike trains were monitored with a memory oscilloscope to make sure that the recordings was of a single unit, the time series histogram of rate and the ISI sequence were recorded and calculated on a computer via an A/D converter. The temporal distribution of the ISI sequence was displayed by scatter plot to discriminate the firing patterns. 1.3 Test of the effect of reagents on DRG neurons Veratridine (Sigma) was dissolved in 0.1 mmol/l HCl solution. Aconitine (Sigma) was dissolved in ethanol. The stock solutions of varatridine (100 µmol/l) and aconitine (20 mmol/l) were prepared and kept in freezer. The final concentrations of reagents were prepared by adding a calculated amount of stock solution to Kreb s solution. The ganglion capsule was carefully slit open with fine forceps so that the Kreb s solution, containing a given reagent in the L4-L5 pool would reach the neuronal somata within the DRG. Before applying the agent, the pool was

3 DUAN Jian-Hong et al: Firing Patterns Induced by Veratridine and Aconitine 171 washed twice with Kreb s solution. The application time for each reagent was about 8~10 min and the interval between the applications of each reagent was more than 10 min. 2 RESULTS 2.1 Firing pattern induced by veratridine With the application of veratridine (1.5~5 µmol/l) on L5 DRG, significant transformation of firing patterns was observed on neurons with spontaneous activity. In some cases (8/10, 80%), the pattern of ISIs converted from bursting into slow wave oscillations after a latency of about 30 s~10 min (Fig.1A). During an oscillation, the ISIs changed from thin to dense and then to thin again. When the concentration of veratridine was kept constant, the oscillatory pattern persisted several hours. The duration of oscillation ranged from 10 to 40 s and the inter-oscillation interval ranged from 20 to 80 s. The minimum ISI was about 2 ms while the maximum was 40 ms, which means that the firing rate changed from 25 to 500 Hz (Fig.1B). In addition, DRG neurons from control rats also exhibited slow wave oscillations in response to veratridine. However, the incidence of slow wave oscillations in controls was significantly lower than that in the injured neurons. Of 9 DRG neurons with spontaneous activity from control rats, 4 (44%) exhibited a transformation of firing patterns in response to veratridine. Moreover, slow wave oscillations were also observed in 4 silent units of 19 DRG neurons (21%) from control rats. 2.2 Firing pattern induced by aconitine After application of aconitine (10~200 µmol/l), in the ma- Fig. 1. Firing pattern induced by veratridine in injured DRG neurons. A: The scatter plot of ISI series (each point represents an interval between two action potentials) shows that spontaneous burst firing was converted into slow wave oscillation firing after the neuron was treated with veratridine (4 µmol/l, approximately 20 min, indicated by the bar) and the bursting recovered gradually after washout of the reagent. VER is the abbreviation for veratridine. B: Original recording of burst firing before the application of veratridine at time point of b as shown in (A). C: An original recording of slow wave oscillation after the application of veratridine at time point of c as shown in (A). D: Segments of the recording at time points 1~3 shown in (C) were displayed on a larger scale to illustrate the course of a slow wave oscillation.

4 172 jority of injured DRG neurons (8/10, 80%), the pattern of ISIs gradually converted from burst into tonic, i.e. the ISIs was equal. Fig.2 shows a typical recording in the same neuron as shown in Fig.1. Three of 15 DRG neurons (20%) Acta Physiologica Sinica, April 25, 2005, 57 (2): with spontaneous activity from control rats also exhibited a transformation of firing patterns in response to aconitine. However, no spike generation was observed in 12 silent neurons. Fig. 2. Firing pattern induced by aconitine in rat injured DRG neurons. A: The scatter plot of ISIs shows that the bursting gradually became tonic firing after the addition of aconitine (30 µmol/l, approximately 15 min, indicated by the bar) and reappeared after washout of the reagent. ACT is the abbreviation for aconitine. B: Original recording of burst firing before the application of aconitine at time point of b as shown in (A). C: Original spike trains of tonic firing after the application of aconitine at time point of c as shown in (A). Note that the recordings are from the same neuron as shown in Fig 1. Fig. 3. Effects of veratridine and aconitine with different concentrations on the burst firing in injured DRG neurons. A: Oscillation firing was induced shortly after the application of 2.5, 5 µmol/l veratridine and disappeared after washout. 1 µmol/l was too low to evoke any response. Note that under the treatment with veratridine no other transformations of firing patterns were observed. B: After the application of 50, 100 µmol/l aconitine, the background firing pattern gradually developed into tonic firing. 5 µmol/l was too low to evoke any response. The immersion time for each concentration was about 8~10 min and the interval among the applications of each concentration was more than 20 min. 2.3 Effects of veratridine and aconitine with different concentrations on firing pattern In the injured DRG neurons, if the concentration was sufficiently high, veratridine with different concentrations changed the firing pattern from the bursting into slow wave oscillations (n=3, Fig.3A). In contrast, the bursting gradually developed into tonic firing after the treatment with certain concentration of aconitine (n=5, Fig.3B). For each

5 DUAN Jian-Hong et al: Firing Patterns Induced by Veratridine and Aconitine 173 Fig. 4. Effects of veratridine and aconitine on tonic firing. A: The scatter plot of ISIs series shows that spontaneous tonic firing was converted into slow wave oscillation firing after the neuron was treated with veratridine (1.5 µmol/l, about 5 min, indicated by the bar). B: The scatter plot of ISIs displays that the background tonic firing gradually became higher frequency tonic pattern after the addition of aconitine (10 µmol/l, 10 min, indicated by the bar). reagent, no other firing patterns were found when different concentrations were used in this experiment. 2.4 Effects of veratridine and aconitine in different background firing patterns There were several kinds of background firing patterns, such as tonic, bursting and irregular, can be observed in the injured neurons. Each would convert into slow wave oscillation firing pattern after the application of veratridine (n =17, tonic: 5/10; bursting: 7/9; irregular: 5/8). In the similar way neurons would convert their basic firing patterns into higher frequency tonic activity when aconitine was applied (n =21, tonic: 11/12; bursting: 8/10; irregular: 2/3). Fig.4 is an example displaying that how the firing pattern changed when the neuron fired tonically at the beginning. 3 DISCUSSION Previous investigations have reported the affirmative effect of veratridine on spontaneous activity of neurons whether it is of the central or peripheral [11,12,14,15]. Aconitine, as a similar chemical modifying the inactivation of sodium channel, was reported to have an effect on hippocampal slices by attenuating the population spike [19] and the effect on nerve by reducing the maximal inward current [5]. However, there is little study of the effect of aconitine on firing patterns, and the result about the comparison of the effects of these two reagents on firing patterns in the same neuron has not been reported yet. The major finding of the present study is that veratridine and aconitine can induce different firing patterns in the same injured DRG neurons. Veratridine induced slow wave oscillations of ISIs. In contrast, aconitine evoked a tonic pattern. It was found that different firing patterns were induced by these two reagents in the same neuron as shown in Fig.1 and Fig.2, which allowed us to rule out the possibility that the distinct firing patterns could be explained by individual differences in neurons. In addition, slow wave oscillation firing was exclusively induced by veratridine even with different concentrations, while tonic pattern can only be observed under the effect of different concentrations of aconitine, which excluded the probability that different concentrations of reagents may underlie the different firing patterns. Moreover, in the present study, such phenomena were also found that after application of veratridine the different background firing patterns, including bursting, irregular and tonic, converted to the slow wave oscillations. Correspondingly, different background firing patterns also gradually converted to tonic after the treatment with aconitine. These findings have led us to propose that the inactivation gate of sodium channels may play a critical role for determining firing patterns, and different firing patterns induced by these two reagents may reflect distinct intrinsic mechanisms underlying these responses. There is emerging evidence that S6 segments of sodium channels are prime target sites for lipid-soluble neurotoxins that elicit specific gating changes [9, 21]. D2-S6 and D3- S6 located within the α-subunit of sodium channels have been reported to be the binding sites of veratridine and aconitine, respectively. It has been reported that aconitine can cause arrhythmia in ventricular and Purkinje fiber preparations from the heart and repetitive afterdischarge following stimulation of the nerve [6]. Veratridine has a broad spectrum of pharmacological activity causing muscle contracture, repetitive firing of nerves and irregular heart rhythms [6]. Our present study revealed that these two toxins could induce different firing patterns in the same injured DRG neurons. These experimental results suggest that different effects of aconitine and veratridine on the neuronal firing patterns may be related to their different binding sites to sodium channels. In addition, it was observed that the various range of the interval of oscillations

6 174 induced by veratridine was larger in the present study. It is possible that during an oscillation, Ca 2+ ions enter with each action potential; [Ca 2+ ] i gradually rises, K(Ca) channels become activated. The enhancement of I K(Ca) hyperpolarize the neuron, shutting off activity and Ca 2+ entry; as [Ca 2+ ] i falls again and the K(Ca) channels shut, permitting a new cycle of oscillation [2]. It suggests that the formation of inter-oscillation interval depends on Ca 2+ entry and K + outflow. In rat hippocampal slices veratridine also decreased the [Ca 2+ ] o and increased the [K + ] o [19]. So the change of firing patterns induced by veratridine may be the result from alteration of several channels function. The next challenge is to prove the close relationship between the firing patterns and receptor sites on sodium channels and to investigate the effects of different channels on determining the firing patterns. REFERENCES 1 Catterall WA. Structure and function of voltage-sensitive ion channels. Science 1988; 242 (4875): Hille B. Modifiers of gating in ionic channels of excitable membrane. 2nd ed. Sinauer Associates Inc. Publisher Sunderland Massachusetts, 1992, Goldin AL. Mechanisms of sodium channel inactivation. Curr Opin Neurobiol 2003; 13 (3): Ogata N, Ohishi Y. Molecular diversity of structure and function of the voltage-gated Na + channels. J Pharmacol 2002; 88 (4): Ulbricht W. Effects of veratridine on sodium currents and fluxes. Rev Physiol Biochem Pharmacol 1998; 133: Catterall WA. Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes. Annu Rev Pharmacol Toxicol 1980; 20: Barnes S, Hille B. Veratridine modifies open sodium channels. J Gen Physiol 1988; 91 (3): Garber SS, Miller C. Single Na + channels activated by veratridine and batrachotoxin. J Gen Physiol 1987; 89 (3): Wang SY, Wang GK. Voltage-gated sodium channels as primary targets of diverse lipid-soluble neurotoxins. Cell Signal 2003; 15 Acta Physiologica Sinica, April 25, 2005, 57 (2): (2): Otoom S, Tian LM, Alkadhi KA. Veratridine-treated brain slices: a cellular model for epileptiform activity. Brain Res 1998; 789 (1): Tian LM, Otoom S, Alkadhi KA. Endogenous bursting due to altered sodium function in rat hippocampal CA1 neurons. Brain Res 1995; 680 (1-2): Nanasi PP, Kiss T, Danko M, Lathrop DA. Different actions of aconitine and veratrum alkaloids on frog skeletal muscle. Gen Pharmacol 1990; 21(6): Leibowitz MD, Sutro JB, Hille B. Voltage-dependent gating of veratridine-modified Na channels. J Gen Physiol 1986; 87 (1): Sutro JB. Kinetics of veratridine action on Na Channels of skeletal muscle, J Gen Physiol 1986; 87 (1): Duan JH ( 段建红 ), Duan YB, Han S, Hu SJ. Slow wave oscillation induced by veratridine in injured dorsal root ganglion neurons. Acta Biophys Sin ( 生物物理学报 ) 2002; 18 (1): (Chinese, English abstract). 16 Duan JH ( 段建红 ), Duan YB, Xing JL, Hu SJ. Triggered oscillation in type A dorsal root ganglion neurons induced by veratridine. Acta Physiol Sin ( 生理学报 ) 2002; 54 (3): (Chinese, English abstract). 17 Duan YB ( 段玉斌 ), Hu SJ, Jian Z, Duan JH. Three types of induced bursting rhythm in rat injured nerves. Acta Physiol Sin ( 生理学报 ) 2002; 54 (4): (Chinese, English abstract). 18 Ameri A. The effect of aconitum alkaloids on the central nervous system. Prog Neurbiol 1998; 56 (2): Ameri A, Shi Q, Aschoff J, Peters T. Electrophysiological effects of aconitine in rat hippocampal slices. Neuropharmacology 1996; 35 (1): Hu SJ, Xing JL. An experimental model for chronic compression of dorsal root ganglion produced by intervertebral foramen stenosis in the rat. Pain 1998; 77 (1): Yarov-Yarovoy V, Brown J, Sharp EM, Clare JJ, Scheuer T, Catterall WA. Molecular determinants of voltage-dependent gating and binding of pore-blocking drugs in transmembrane segment ⅢS6 of the Na + channel alpha subunit. J Biol Chem 2001; 276 (1):