Toxicon 40 (2002) 149157

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Neurotoxic effects of fractions isolated from Tityus bahiensis scorpion venom (Perty, 1834)
Geane Antiques Lourenc o a,c, Ivo Lebrun b, Valquiria A. Coronado Dorce a,*
a o Paulo, 05503-900, Brazil Laboratory of Pharmacology, Butantan Institute, Sa o Paulo, 05503-900, Brazil Laboratory of Biochemistry and Biophysics, Butantan Institute, Sa c o Paulo University, Sa o Paulo, 05508-900, Brazil Laboratory of Pharmacology, School of Veterinary Medicine, Sa b

Received 17 April 2001; accepted 25 July 2001

Abstract Tityus serrulatus and Tityus bahiensis are considered to be the most venomous scorpions in Brazil and are responsible for most of the accidents that occur in our country. The main toxic agents in scorpion venoms are small basic polypeptides that act as neurotoxins. They cause a derangement of ion channels that result in abnormal release of neurotransmitters. In the present study we fractionated the venom of Tityus bahiensis and studied the effects of fractions P2, P3, P4, P5, P6 and P7, on the mammalian central nervous system. Intravenous injection of P5, P6 and P7 in rats induced spontaneous convulsion, intrahippocampal injection caused behavioural seizures, and P5 and P6 induced electrographic seizures. P5 caused neuronal damage in the CA1 area and P6 in the CA1, CA3 areas and hilus of the dentate gyrus (DG) of the hippocampus. Injection of P3 in the hippocampus did not induce convulsions or lesions. However, when injected intravenously in mice, this fraction reduced behavioural activity in an open eld test. Unilateral injection of P4 in the hippocampus caused neuronal damage in the contralateral CA3, but not in the ipsilateral hippocampus. These results suggest that scorpion toxins present in the venom are able to act directly on the central nervous system promoting behavioural and histopathological effects. q 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Scorpion venom; Neurotoxins; Tityus bahiensis; Seizure; Open eld; Neuronal damage; Electrographic record

1. Introduction Many investigators have shown that crude scorpion venoms or toxins puried from the venoms evoke complex effects on ion channels, with release of chemical mediators (Freire-Maia et al.,1989). Four different families of toxins have been described which specically interact with membrane-bound ion channels such as Na 1 channels (Catterall, 1988; Rochat et al., 1979), K 1 channels (Carbone et al., 1982), Cl 2 channels (De Bin et al., 1993; Lippens et al., 1995), and Ca 21 channels (Valdivia et al., 1992). At present, the best studied toxins are those that recognise Na 1 and K 1 channels. One of their main effects is neurotransmitter release from neuronal endings (Couraud and Jover, 1983). The two scorpion genera Tityus and
* Corresponding author. Tel.: 155-11-3726-7222, ext. 2133; fax: 155-11-3726-1505. E-mail address: vdorce@usp.br (V.A.C. Dorce).

Centruroides have been studied extensively, but the only Tityus species studied to some extent is Tityus serrulatus from Brazil. Recently four components have been isolated from Tityus bahiensis venom, i.e. the toxins g-bahiensis (g-b), III-8 bahiensis (III-8b), IV-5 bahiensis (IV-5b) and TbTX-VI (Trequattrini et al., 1995; Becerril et al., 1996). The amino acid sequence of toxin g-b is 95% identical to that of toxin g of Tityus serrulatus (Becerril et al., 1997), but little is known about the effect of this toxin. In our laboratory the Tityus serrulatus venom or toxins isolated from it were studied and we show that they produced an epileptiform tracing in the cerebral electric activity records, brain damage and convulsions after injection into different structures of the rat brain (Sandoval and Dorce, 1993; Dorce and Sandoval, 1994; Carvalho et al., 1998; 2000; Nencioni et al., 2000). Excitatory amino acid (EAA) release has been implicated in these effects (Carvalho et al., 1998; 2000; Nencioni et al., 2000). The participation of EAA neurotransmitters in epilepsy,

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Fig. 1. Chromatographic prole of Tityus bahiensis scorpion venom. Forty milligrams of the crude venom of Tityus bahiensis were extracted three times with 0.02 mM ammonium acetate, pH 4.7. In each extraction the material was centrifuged. Two hundred microliter of the nal supernatant were submitted to a gel ltration chromatography using a Merck TSK-3000 SW gel ltration column running with ammonium acetate solution 200 mM, pH 4.7 at a ow rate of 0.4 ml/1 min. Fractions of 2.5 ml were collected and read at 280 nm. Fractions corresponding to peaks were pooled. Each pool was lyophilised and protein concentration estimated.

seizure-induced brain damage, and glutamate excitotoxicity has been extensively investigated but is not well understood (Choi, 1987; Dingledine et al., 1990). Since the fractions of the venom are composed of toxins that must be responsible, at least in part, for the convulsive effect observed with whole venom and since these toxins are important not only for the pathophysiology of envenomation but also as neurological tools for studying convulsive activity, the present study was undertaken to investigate the ability of six of the fractions isolated from T. bahiensis venom to induce convulsive activity and their effects on the electrographic activity and neuronal integrity of the rat hippocampus. An open eld study was also used but only to quantify possible alterations in general activity.

at a ow of 0.4 ml/min and a running time of 2:20 h. Eight peaks were obtained, of which P2, P3, P4, P5, P6 and P7 were used for this study (Fig. 1). 2.1. Behavioural observation after intravenous injection Mice were divided at random into 22 groups injected intravenously through the caudal vein with 0.9% NaCl or with a venom fraction plus kainic acid (ka) dissolved in 0.9% NaCl. Immediately after injection, for direct behavioural observation, each mouse was placed individually in the center of an open eld arena and behavioural parameters were recorded for 10 min. The open eld used was based on that described by Broadhurst (1960), adapted for mice. Ambulation frequency (number of oor units entered), rearing frequency (number of times the animal stood on its hind legs), immobility time (total time, in seconds, without movement) and the appearance of convulsions were quantied. The open eld was washed with a wateralcohol (5%) solution before behavioural testing to eliminate possible bias due to odors left by previous subjects. To minimise possible circadian inuences on mouse open eld behaviour, all animals were observed between 9:00 and 11:00 h. 2.2. Behavioural and electrographic studies after intrahippocampal injections Rats were divided at random into eight groups and microinjected into the CA1 hippocampal region with 1.0 ml of Ringer (control group), or with ka solution (1.5 mg/ml), or with fractions P2, P3, P4, P5, P6, P7 (3.0 mg/ml) dissolved in ringer (n 6 for every groups). For surgery, rats were anaesthetised (4.0 ml/kg) with a

2. Materials and methods Male Swiss Webster mice, 2225 g, and male Wistar rats, 220250 g, were used. Upon their arrival to the laboratory, 7 days before the beginning of the experiments, the animals were housed six to a cage and maintained at 22 ^ 28C on a 12/12 h light/dark cycle (lights on from 7:00 to 19:00 h) with free access to chow and water. The animals used in this study were maintained in accordance with the guide o Paulo lines of the Department of Pathology at the Sa University School of Veterinary Medicine, which are based on the guidelines for animal care prepared by the Committee on Care and Use of Laboratory Animal Resources, National Research Council, USA. Dried Tityus bahiensis scorpion venom was obtained from the Arthropod Laboratory of the Butantan Institute and partially puried in our laboratory. By gel ltration HPLC the samples were eluted from a TSK 03000 SW column, with 200 mM ammonium acetate buffer, pH 4.7,

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Fig. 2. Examples of epileptiform parameters observed during electrographic activity recording after intrahippocampal injections of fractions of Tityus bahiensis scorpion venom. (A) normal activity; (B) spikes; (C) moderate seizure; (D) strong seizure. CX cortex; HPC hippocampus.

mixture of pentobarbitone (1.0 g) and chloralhydrate (4.0 g) in 100 ml of 0.9% NaCl, and positioned in a stereotaxic frame. For the injections, a stainless steel guide cannula was chronically implanted into one side of the dorsal hippocampus. For depth recordings, bipolar twisted electrodes were positioned in the dorsal hippocampus and surface recordings were obtained with jeweler screws positioned bilaterally over the occipital cortex. Coordinates were derived from the Atlas of Paxinos and Watson (1982) (AP 24.8; L 3.5; V 3.5). An additional screw placed in the frontal sinus served as reference (indifferent electrode). The system was anchored to the skull with dental acrylate. After surgery animals were housed individually and were allowed to recover for a period of 57 days. Electrographic recording and behavioural observations were carried out in a glass compartment. After 15 min of habituation to the test cage the basal electrographic activity was recorded for 15 min and the animals were then microinjected. After injection, electrographic activity records and observations of animal behaviour were performed intermittently for 3 h. The electrographic parameters observed and considered to indicate epileptiform activity were: spikes, moderate seizure, and strong seizure (Fig. 2). 2.3. Neuropathological studies The neuronal loss and the correct location of implanted

deep electrodes and cannulae were checked histologically. The morphological analysis was performed 7 days after toxin injection. The animals were anaesthetised with ether and perfused and xed by intracardiac administration of phosphate buffered saline followed by 10% formalin solution. The brains were removed, stored in formalin and embedded in parafn. Coronal brain sections of 10 mm were cut from a 700 mm brain block including the cannula track. Every seventh slice along a 300 mm length from either side of the track was mounted on a glass slide and stained with cresyl violet. The number of cells was counted in a 100 mm 2 area of the pyramidal cell layers of the CA1, CA3 and hilus of dentate gyrus (DG) hippocampal regions. Only cells showing normal morphological characteristics were counted. The mean number of neurons from each area was pooled and expressed as mean (^SEM) neurons per brain area. The pooled mean cell number counted in the treated animals was compared with the corresponding number counted in control animals. 2.4. Statistical analysis For the open eld behaviour studies, ANOVA and Dunnett's test were used to analyze the results after the Bartlett test showed that the data were homogeneous. For the electrographic and neuropathological studies ANOVA

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followed by the Turkey Kramer test was used. Differences were considered to be signicant when p , 0.05. 3. Results 3.1. Behavioural observation after intravenous injection The results of the total time of observation of the animals submitted to the open eld are listed in Table 1. Fractions P3, P5, P6, P7 and ka decreased locomotion and rearing frequencies. Fraction P2 only decreased rearing frequency. The immobility time was signicantly increased by fractions P5, P6, P7 and ka. All fractions produced dyspnea, which was moderate after P2, P3 and P4, and intense after P5, P6, P7 and ka. These last fractions also produced an increase in breathing secretion, and the animals that received the highest dose of fraction P7 presented intense haemorrhagic exudate. P5, P6 and P7 at the two largest doses also elicited myoclonus and clonus of the limbs and tonicclonic seizures at the highest dose. Ka only produced these behavioural alterations in the animals treated with the highest dose. Fraction P4 caused no signicant alterations in the behaviour of the animals submitted to the open eld. 3.2. Behavioural and electroencephalographic studies after intrahippocampal injections After hippocampal injection, both ka and fractions P5, P6 and P7 produced behavioural manifestations related to convulsions such as wet dog shakes, myoclonus and complete immobility, and the animals that received fractions P5 and P7 also presented clonus of the limbs. Besides the behavioural changes, P5 and P6 induced electrographic alterations such as moderate and intense discharges, occurring concomitantly with immobility (Table 2). Although it caused convulsive behaviour, fraction P7 did not modify signicantly the electrographic record but wet dog shake and myoclonus occurred at the same time that isolated spikes. Only two animals that received fraction P3 manifested behavioural and electrographic alterations such as wet dog shakes and myoclonus with discharges. Fractions P2 and P4 did not elicit behavioural or electrographic changes when injected into the hippocampus. 3.3. Neuropathological studies Analysis of the hippocampal sections by light microscopy showed neurodegeneration in the CA1 pyramidal cell area ipsilateral to the injection site in animals treated with fractions P5, P6 and ka (Figs. 3 and 4) and in the contralateral CA1 area in animals treated with fraction P6 (experimental group: 41.0 ^ 8.3 cells; control group: 61.0 ^ 1.0 cells). Neurodegeneration was observed also in the CA3 pyramidal cell area ipsilateral to the injection site in animals injected with fraction P6 and ka; and in the contralateral CA3 area in animals treated with fraction P4 (experimental group:

23.3 ^ 1.2 cells; control group: 37.8 ^ 1.3 cells). Fractions P2, P3 and P7 did not cause cell damage in the hippocampal areas studied.

4. Discussion Few reports are available in the literature regarding the scorpion Tityus bahiensis, and these are mainly about the immunological effects of the venom for serum production (Nishikawa et al., 1994) or for purication and sequencing of the toxins that compose it, but without explaining their mechanism of action (Trequattrini et al., 1995; Becerril et al., 1996; 1997). We rst determined the LD50 of the crude venom and observed its effects as well as its capacity to produce convulsion after systemic injection. After intravenous injection of the venom the animals presented dyspnea, increased secretions and convulsive symptoms. These symptoms were dose dependent, appearing seconds after injection and worsening with time, progressing to running with clonus, and limbic seizures. The LD50 of the crude venom injected intravenously in mice was 1.18 mg/kg (data not shown). Then, we used the peptide pools obtained by HPLC from the total venom to determine the convulsive agents. Besides fractions P2, P3, P4, P5, P6 and P7, we also used kainic acid (ka) as positive control since previous studies from our laboratory suggested that fractions of Tityus serrulatus venom act intrahippocampally through excitatory amino acids (Carvalho et al., 2000; Nencioni et al., 2000). Kainic acid, an analog of the putative excitatory neurotransmitter glutamate, has a prominent toxic effect on the hippocampus characterised by motor and electrographic convulsion and lesion of cell bodies (Ben Ari et al., 1980a,b; Fisher, 1989). The frequency of the parameters quantied in the open eld such as locomotion, rearing and immobility time expresses the general activity of the animals and provides information about the state of activation of the neural systems linked to motor function. Fractions P5, P6 and P7 decreased the locomotion frequencies and rearing and increased the time of immobility of the animals. This decrease of general activity was probably caused by the convulsive symptoms of the animals. Thus, these three fractions induced myoclonus, clonus of the limbs, dyspnea and increased secretions during the observation period. P7 and the whole venom induced intense haemorrhagic exudate that characterises pulmonary edema (Freire-Maia and Campos, 1989). The pulmonary edema could be explained by a heart failure induced by a hypertensive effect (Freire-Maia and Campos, 1989) and by chemical mediators release (Amaral and Rezende, 2000). Fractions P2 and P3 decreased the frequencies of rearing and P3 also decreased the frequency of locomotion, without any effect of either fraction on the time of immobility of the animals. In spite of this decrease in general activity, these animals still showed good motor function because they did not present incoordination or

Table 1 Effects of the intravenous injection of fractions of the venom of scorpion Tityus bahiensis on open eld behaviour of mice (n 6) (values are reported as means ^ SEM; *p , 0.05 (ANOVA and Dunnett's test)) Treatment Cont Dose (mg/kg) NaCl 0.9% Ka 4.0 6.0 18.0 26.9* ^ 8.1 0.0* ^ 0.0 P2 1.0 250.7 ^ 25.1 100.9 ^ 6.0 0.0 ^ 0.0 3.0 258.3 ^ 48.0 54.4* ^ 13.1 40.8 ^ 24.1 6.0 318.7 ^ 36.2 77.5* ^ 18.2 4.3 ^ 4.0 P3 0.6 1.0 3.0 P4 0.5 1.0 314.5 ^ 33.0 137.5 ^ 12.0 3.3 ^ 3.2 2.0 P5 0.5 1.0 2.0 73.2* ^ 8.4 2.1* ^ 1.1 P6 0.6 241.1 ^ 26.2 67.8* ^ 8.3 0.0 ^ 0.0 1.0 53.8* ^ 11.0 2.0* ^ 1.1 3.0 P7 0.6 1.0 3.0 44.8* ^ 9.0 8.4* ^ 3.0

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Locomotion 327.2 102.5* 43.3* ^ ^ ^ 25.1 13.6 26.3 Rearing 119.4 ^ 8.3 0.0 ^ 0.0 4.6* ^ 1.2 0.0* ^ 0.0

256.2 107.8* 112.2* 266.3 ^ ^ ^ ^ 21.4 15.2 37.3 29.1 62.7* ^ 13.2 0.0 ^ 0.0 32.2* ^ 14.1 0.0 ^ 0.0 43.3* ^ 14.1 0.0 ^ 0.0 92.4 ^ 13.2 6.4 ^ 4.2

336.7 200.8* 73.9* ^ ^ ^ 37.1 35.2 10.5 132.7 ^ 19.3 0.0 ^ 0.0 46.2* ^ 12.3 29.7 ^ 17.5 14.0* ^ 5.1

45.6* 183.5* 43.3* ^ ^ ^ 13.3 11.1 13.2 4.2* ^ 3.0 42.4* ^ 11.2 3.2* ^ 2.1

Immobility Time (seg)

314.2* 373.7* 413.9* ^ ^ ^ 53.2 43.5 24.4

223.8* 322.1* ^ ^ 33.2 19.4

167.4* 233.6* 100.3* 290.7* 114.5* ^ ^ ^ ^ ^ 42.3 26.1 45.5 46.2 39.1

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Table 2 : Electrographics alterations of the hippocampus and/or cortex of rats after intrahippocampal injection of the fractions from the venom of the scorpion Tityus bahiensis (3 mg/1 ml) or ka (1.5 mg/1 ml). The data refer to 3 h of non-serial registration (values are reported as means ^ SEM; *p , 0.05 (ANOVA and Tukey Kramer test)) Treatment Control P2 P3 P4 P5 P6 P7 Ka Moderate seizure frequency 0^0 0^0 3.3 ^ 2.1 0^0 9.4 ^ 2.7 13.2 ^ 2.4* 8.5 ^ 3.4 7.6 ^ 3.9 Strong seizure frequency 0^0 0^0 1.6 ^ 0.6 0^0 3.0 ^ 1.0 5.8 ^ 2.7 0^0 0^0 Total time (s) of moderate seizure 0^0 0^0 60.0 ^ 54.1 0^0 247.7 ^ 71.5* 168.7 ^ 24.1* 115.0 ^ 42.2 102.3 ^ 36.4 Total time (s) of strong seizure 0^0 0^0 24.0 ^ 23.0 0^0 131.6 ^ 28.0* 210.6 ^ 97.7* 0^0 0^0

Fig. 3. Photomicrographs of the hippocampus showing a typical pattern of neurodegeneration 7 days after microinjection. ARinger (CA1 ipsilateral), B1.5 mg of the ka (CA1 ipsilateral), C3.0 mg of the fraction P5 (CA1 ipsilateral), D3.0 mg of the fraction P6 (CA1 ipsilateral), ERinger (CA3 ipsilateral), F3.0 mg of the fraction P4 (CA3 contralateral). In B, C, D and F dark staining neurons (pycnotic cells) are present throughout the entire section with few neurons appearing normal. The control hippocampus appears to be unaffected (A, E). Cresil violet stain. Magnication: X140.

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Fig. 4. Number of intact cells in the CA1 (A), CA3 (B) areas and hilus of DG (C) of the hippocampus of rats injected with Ringer (cont), ka (1.5 mg) and the different fractions of the venom of the scorpion Tityus bahiensis (3.0 mg). Values are reported as means ^ SEM. *p , 0.05 (ANOVA and Tukey Kramer test).

locomotion difculty. The intravenous administration of ka decreased locomotion and rearing and increased the time of immobility of the animals in the open eld. Ka also produced clonus that progressed to widespread tonic clonic convulsion 10 min after injection. The effects of fractions started 23 min after injection. The convulsive dose of the ka was 18.0 mg/kg and of fractions were 2.0 mg/kg (P5) and 3.0 mg/kg (P6 and P7). Although not conclusive these data suggest a different mechanism for ka and for fractions. As expected, the animals that received the intrahippocampal injection of the vehicle showed no alteration in the electrographic record or in behaviour, but ka at the concentration of 1.5 mg, a dose determined according to the literature (Ben Ari et al., 1980a,b; 1985;Cavalheiro, et al., 1982), induced the appearance of spikes in the cortex/hippocampus during practically the entire recording time, independent of animal behaviour, as well as the appearance of a few episodes of moderate discharges. The behaviour of the animals changed, with the appearance of face myoclonus, clonus of the forelimbs, loss of postural control and circling behavioural before the electrographic

alterations. The lesion produced by ka injection was extensive in the CA1 and CA3 areas and hilus of the DG of the side ipsilateral to the injection. The injection of P2 and P4 did not produce any alteration in the electrographic registration and the animals behaved like the controls. The count of intact hippocampal neurons showed no change in the number of neurons of animals that received P2; however, P4 produced signicant neuronal loss in the CA3 area contralateral to the injection. In the P4 treated animals the neuronal damage was not caused by convulsion since none of then showed behavioural or electrographic seizure. The contralateral lesion probably occurred by activation of some neuronal system. According with Ben Ari et al. (1980a,b) a small amount of substance injected into a brain area cannot spread to another area and it is not probable that a ventricular diffusion may explain effect occurring in the contralateral side without a damage in ipisilateral side. There is no evidence that CA1 can directly activate the contralateral CA3 area, but it is not clear how P4 produces this lesion. Of the six animals injected with P3, two just showed alterations in the registration such as spikes and

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discharges accompanied by wet dog shakes and myoclonos respectively. Histopathological examination showed no lesion in the hippocampal neurons of these animals. Fraction P3 had a lower convulsive potential compared with fractions P5, P6 and P7. Fraction P5 produced moderate discharges more frequently than intense discharges. The rst discharges only occurred in the hippocampus and extended to the cortex during the recording. Myoclonus preceded the discharges and did not occur simultaneously with them. During the discharges, frequent wet dog shakes or complete immobility of the animal were observed. Cell counts showed that P5 signicantly damaged the CA1 area ipsilateral to the injection, showing that this fraction acts on the area where it is injected, and it is not activating other neurons around it. The presence of hippocampal discharges agrees with the lesion data because the convulsive focus was generated in the hippocampus itself. When P6 was injected into the hippocampus the cortical recording followed the hippocampal one, with synchronised and intense discharges and behavioural immobility. Several episodes of wet dog shakes were observed during recording, and behaviours such as myoclonus occurred without any alteration in the record, indicating they were induced by activation of other cerebral areas. The animals injected with P6 had extensive neuronal lesion in CA1, CA3 and hilus of the DG on the ipsilateral side to the injection, and small although signicant lesion in the contralateral CA1 area. Fraction P7 produced spikes and moderate discharges, which were more frequent in the hippocampus, but they were not signicant compared to control. These changes in the record occurred at the same time as behavioural alterations such as wet dog shake and myoclonus, respectively. It is important to point out that none of the fractions obtained from Tityus serrulatus venom was capable to generate this synchronism between behavioural and electrographic alterations (Carvalho et al., 2000; Nencioni et al., 2000). The spikes occurred in the eletrographic record 24 h after the administration of P7, and the animal did not present further behavioural alteration. The neuronal cell count showed no signicant lesion compared to control. It is interesting to note that P7 elicited peripherical symptoms like salivary and bronchial secretions increase and respiratory disorders following intrahippocampal injection. Despite evidence of the possible mechanisms of action by which the fractions would be acting, it will be necessary to investigate the fractions by other experimental methods in order to obtain conclusive data. However, what can be afrmed is that any fraction, injected systemically or intrahippocampally, was able to produce effects different from those produced by ka, showing that not only glutamate participate of these effects. Comparing the present data with the results obtained previously in our laboratory with Tityus serrulatus venom (Carvalho et al., 2000; Nencioni et al., 2000), we can say that remarkable differences exist in the effects of the venoms of these two scorpions. The effects caused by toxins from Tityus serrulatus venom were due to

glutamate release in hippocampus (unpublished data). The venom of Tityus bahiensis seems to represent a potential tool for the study of ion channels, neural hodology, and epilepsy models. Acknowledgements This work was supported by a grant from FAPESP Pesquisa do Estado de Sa o de Amparo a o Paulo) (Fundac a 95/5815-0 and 99/12869-2. This study is part of a Master's Thesis presented by Geane Antiques Lourenc o to the School o of Veterinary Medicine, Department of Pathology, Sa Paulo University. References
Amaral, C.F.S., Rezende, N.A., 2000. Treatment of scorpion envenoming should include both a potent specic antivenom and support of vital functions. Toxicon 38, 10051007. Becerril, B., Corona, M., Coronas, F.I.V., Zamudio, F., CalderonAranda, E.S., Fletcher, P.L., Martins, B.M., Possani, L.D., 1996. Toxic peptides and genes encoding toxins g of the Brazilian scorpions Tityus bahiensis and Tityus stigmurus. Biochemical Journal 313, 753760. Becerril, B., Marangoni, S., Possani, L.D., 1997. Toxins and genes isolated from scorpions of the genus Tityus. Toxicon 35 (6), 821835. Ben-Ari, Y., Tremblay, E., Ottersen, O.P., 1980. Injections of kainic acid into the amigdaloid complex of the rat: an electrographic, clinical and histological study in relation to the pathology of epilepsy. Neuroscience 5, 515528. Ben-Ari, Y., Tremblay, E., Ottersen, O.P., Meldrum, B.S., 1980. The role of epileptic activity in hippocampal and `remote' cerebral lesion induced by kainic acid. Brain Research 191, 7997. Ben-Ari, Y., 1985. Limbic seizure and brain damage syndrome produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy. Neuroscience 14, 375403. Broadhurst, P., 1960. Experiments in psychogenetics. In: Eisenk, E. (Ed.). Experiments in Personality. Routlege and Kegan Paul, London. Carbone, C., Wanke, E., Prestipino, G., Possani, L.D., Maelicke, A., 1982. Selective blockage of voltage-dependent potassium channel by a novel scorpion toxin. Nature 296 (4), 9091. Carvalho, F.F., Nencioni, A.L.A., Lebrun, I., Sandoval, M.R.L., Dorce, V.A.C., 1998. Behavioural, eletroencephalographic and histopathologic effects of a neuropeptide isolated from Tityus serrulatus scorpion venom in rats. Pharmacology, Biochemistry and Behaviour 60 (1), 714. Carvalho, F.F., Nencioni, A.L.A., Lebrun, I., Dorce, V.A.C., Sandoval, M.R.L., 2000. Convulsive effects of some isolated venom fractions of the Tityus serrulatus scorpion: behavioural, electroencephalographic, and neuropathological aspects. The Journal of Venomous Animals and Toxins 6 (2), 238260. Catterall, W.A., 1988. Structure and function of voltage-sensitive ion channels. Science 241, 5061. Cavalheiro, E.A., Richie, D.A., Le Gal La Salle, G., 1982. Long-term effects of intrahippocampal kainic acid injection in

G.A. Lourenc o et al. / Toxicon 40 (2002) 149157 rats: A method for inducing spontaneous recurrent seizures. Eletroencephalographic. Clinical and Neurophysiology 53, 581589. Choi, D.W., 1987. Ionic dependence of glutamate neurotoxocity. Journal of Neuroscience 7, 369379. Couraud, F., Jover, E., 1983. Mechanism of action of scorpion toxins. In: Tu, T.A., Dekker, M. (Eds.). Handbook of Natural Toxins. Insect Poisons, Allergens and Other Invertebrate Venoms. , pp. 659678. De Bin, J.A., Maggio, J.E., Strichartz, G.R., 1993. Purication and characterization of chlorotoxin a chloride channel ligand from the venom of the scorpion. American Journal of Physiology 264, C361C369. Dingledine, R., McBain, C.J., McNamara, J.O., 1990. Excitatory amino acid receptor in epilepsy. Trends in Pharmacology Science 11, 334338. Dorce, V.A.C., Sandoval, M.R.L., 1994. Effects of Tityus serrulatus crude venom on the GABAergic and dopaminergic systems on the rat brain. Toxicon 32 (12), 16411647. Fisher, R.S., 1989. Animal models of the epilepsies. Brain Research Reviews 14, 245278. Freire-Maia, L., Campos, J.A., 1989. Pathophysiology and treatment of scorpion poisoning. In: Ownby, C.L., Odell, G.V. (Eds.). Natural Toxins. Characterization, Pharmacology and Therapeutics. Pergamon Press, Oxford, pp. 139159. Lippens, G., Najib, J., Wodak, S.J., Tatar, A., 1995. NMR sequential assignments and solution structure of chlorotoxin, a small scorpion toxin that blocks chloride channels. Biochemistry 34 (1), 1321.

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Nencioni, A.L.A., Carvalho, F.F., Lebrun, I., Sandoval, M.R.L., Dorce, V.A.C., 2000. Neurotoxic effects of three fractions isolated from Tityus serrulatus scorpion venom. Pharmacology and Toxicology 86 (4), 149155. Nishikawa, A.K., Caricati, C.P., Lima, M.L.S.R., Dos Santos, M.C., Kipnis, T.L., Eickstedt, V.R.W., Knysac, I., Da Silva, M.H., Higashi, H.G., Dias Da Silva, W., 1994. Antigenic crossreactivity among the venoms from several species of Brazilian scorpions. Toxicon 32 (8), 989998. Paxinos, G., Watson, C., 1982. The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney. Rochat, H., Bernard, P., Couraud, F., 1979. Scorpion toxins: chemistry and mode of action. In: Ceccarelli, B., Clementi, F. (Eds.). Neurotoxins: Tools in Neurobiology. Raven Press, New York, pp. 325334. Sandoval, M.R.L., Dorce, V.A.C., 1993. Behavioural and eletroencephalographic effects of Tityus serrulatus scorpion venom in rats. Toxicon 31 (9), 205212. Trequattrini, C., Zamudio, F.Z., Petris, A., Prestipino, G., Possani, L.D., Franciolini, F., 1995. Tityus bahiensis toxin IV-5b selectively affects Na channel inactivation in chick dorsal root ganglion neurons. Comparactive Biochemistry and Physiology 112A (1), 2128. Valdivia, H.H., Kirby, M.S., Lederer, W.J., Coronado, R., 1992. Scorpion toxins targeted against the sarcoplasmic reticulum calcium-release channel of skeletal and cardiac muscle. Proceedings of the National Academy of Sciences USA 89, 1218512189.