Effects of a Toxin Isolated from Tityus Bahiensis Scorpion Venom on the Hippocampus of Rats

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Life Sciences 91 (2012) 230–236

Contents lists available at SciVerse ScienceDirect

Life Sciences

journal homepage: www.elsevier.com/locate/lifescie

Effects of a toxin isolated from Tityus bahiensis scorpion venom on the hippocampus of rats

Luciene ToshieTakeishi Ossanai a,c, Geane Antiques Lourenço a, Ana Leonor A. Nencioni a, Ivo Lebrun b, Norma Yamanouye a, Valquiria Abrão Coronado Dorce a,⁎

a Laboratory of Pharmacology, Butantan Institute, Av. Dr. Vital Brazil, 1500, 05503‐900, São Paulo, SP, Brazil b Laboratory of Biochemistry and Biophysics, Butantan Institute, Av. Dr. Vital Brazil, 1500, 05503‐900, São Paulo, SP, Brazil c Disease Control Coordinating Agency of São Paulo State Public Health Ministry, 01246‐900, São Paulo State, Brazil

article info abstract

Article history: Aims: The objective of the present study was to determine the effects of a toxin from T. bahiensis scorpion Received 29 August 2011 venom on the hippocampus of rats. This toxin, called Tb V-4, was chosen since it shows remarkable convul- Accepted 22 June 2012 sive activity. Main methods: Male Wistar rats weighing 250 g were used. The toxin (1.0 μg/μl) was injected into the hippo- Keywords: campus. The animals were then submitted to electroencephalographic and behavioral examinations or to mi- Scorpion toxin crodialysis to determine the levels of neurotransmitters. The location of the implanted guide cannulae and Tityus bahiensis electrodes was checked histologically. The number of cells in the CA1, CA3 and CA4 areas of the hippocampus Neurotransmitters Microdialysis was determined by light microscopy. Changes in the concentration of cytosolic free calcium were evaluated Cytosolic calcium by confocal microscopy. Key findings: The toxin evoked behavioral alterations such as wet dog shakes, myoclonus, yawning and orofacial automatisms. Electroencephalographic recordings exhibited alterations such as isolated or grouped spikes and epileptic-like discharges. Injection of the toxin augmented glutamate concentration in the extracellular fluid in some animals. There was also a decrease in the number of pyramidal cells, mainly in the CA1 and CA4 areas for some rats. In some slices of the hippocampus, an increase in intracellular calcium mobilization was seen. Significance: The present results suggest that the Tb V-4 toxin may be responsible for the epileptic and behavioral effects observed with the crude venom. We suggest that the convulsive and degenerative effects induced by the toxin could be due to the enhanced release of excitatory amino acids involved in the most important pathways of the hippocampus. © 2012 Elsevier Inc. Open access under the Elsevier OA license.

Introduction neurotoxins represent powerful tools to study the structure and function of these channels, affecting both permeation and gating proper- Scorpion venoms are composed of basic proteins, peptides, and ties of the channel (Cestèle and Catterall, 2000). minor compounds such as serotonin, histamine, salts and some en- The injection of scorpion venom in experimental animals pro- zymes (Cologna et al., 2009). They are rich sources of different classes duces systemic effects with signs and symptoms similar to those of peptides, such as neurotoxins, which represent useful tools for observed in human envenomation, such as fever, psychomotor biological research (Bertazzi et al., 2003). Neurotoxins disrupt the agitation, salivation, lachrymation, increased gastrointestinal tract normal function of excitable tissues, such as muscle and nerves, mobility, cardiac and respiratory arrhythmias, arterial hypertension through their interaction with Na+,K+,Ca2+ or Cl− channels (Tian followed by hypotension, cardiac failure, pulmonary edema and et al., 2008). One of their main effects is the release of neurotransmit- shock, among others (Freire-Maia and Campos, 1989). These manifes- ters from nerve endings (Couraud and Jover, 1984; Fukuhara et al., tations are explained by the ability of the toxins to act on sodium 2004). channels in nerve terminals (Freire-Maia and Campos, 1989). Addi- Sodium channels are the molecular target for several groups of tionally, clinical findings can be part of a systemic inflammatory neurotoxins, which strongly alter channel function by binding to response, which may lead to multiple organ dysfunction (Andrade specific receptor sites. Due to their high affinity and specificity, et al., 2004). Crude venom of Tityus serrulatus scorpion, as well its toxins, has ⁎ Corresponding author. Tel.: +55 11 37267222x2299; fax: +55 11 3726 1505. already been extensively studied (Arantes et al., 1989; Carvalho E-mail address: [email protected] (V.A.C. Dorce). et al., 1998, 2000; Conceição et al., 2005; Dorce and Sandoval, 1994;

Massensini et al., 1998; Nencioni et al., 2000, 2003; Possani et al., Electroencephalographic recordings and behavioral observations 1991; Revelo et al., 1996). On the other hand, few reports are available in the literature regarding the venom of the scorpion Tityus Rats were anesthetized with an i.p. injection of 3 ml/kg mixture of bahiensis, and the majority concern immunological effects for serum 1 g pentobarbitone and 4 g chloral hydrate in 100 ml of 0.9% NaCl, production (Nishikawa et al., 1994) or the purification and sequenc- and placed in a stereotaxic frame. For depth recordings, bipolar twisted ing of the toxins that compose it (Becerril et al., 1996, 1997; electrodes were positioned on the dorsal hippocampus and anchored to Trequattrini et al., 1995). the skull with dental acrylate. Coordinates towards the bregma were AP Recently, two toxins present in this venom were found to have −4.8, L +3.2, V −3.0 (Paxinos and Watson, 1998). Surface recordings anti-insect activity (Pimenta et al., 2001) and some semi-purified were obtained with jeweler screws positioned bilaterally over the oc- toxins were shown to act directly on the CNS, resulting in behavioral cipital cortex. An additional screw placed in the frontal sinus served as and histopathological effects (Lourenço et al., 2002). the reference electrode. After surgery, animals were housed individual- Thus, the aim of this work was to determine the effects of a toxin ly and were allowed to recover for a period of 1–2days. isolated from T. bahiensis scorpion venom on the CNS, by examining Electroencephalographic (EEG) recordings and behavioral obser- the concentration of some neurotransmitters or their metabolites in vations were carried out in a glass compartment placed in a Faraday the hippocampus as well as alterations in electroencephalographic cage. The bipolar twisted electrodes of each animal were connected recordings, neuronal integrity, and cytoplasmic calcium mobilization. to a PowerLab® recording apparatus, and after 15 min of acclimation to the test cage, the baseline electroencephalographic activity of the cortex and hippocampus was recorded for 15 min. The electrodes Material and methods were then disconnected, and the animals were given an injection of 1.0 μl of Ringer solution (control group, n=6) or 1.0 μg/μl Tb V-4 Subjects (n=9) in the hippocampus. Subsequently, continuous EEG recording was performed and animal behavior was observed for a period of 4 h. Male Wistar rats weighing 250 g were used. The animals were Fisher's test was used to analyze the data for all the study param- housed in standard cages (40×50×20 cm) under controlled condi- eters (pb0.05). tions during the study: 12:12 h photoperiod, 22±2 °C, and water and food provided ad libitum. All the experimental procedures were Determination of amino acid neurotransmitter concentrations by in agreement with the Ethical Principles in Animal Research adopted microdialysis by the Brazilian College of Animal Experimentation and were con- ducted with prior permission of the Ethical Committee for Animal The levels of neurotransmitters were determined in rats by sub- Research of Butantan Institute (protocol no. 462/08). mitting them to microdialysis. The animals were anesthetized, and Dried crude venom from the scorpion T. bahiensis was obtained guide cannulae were chronically implanted into one side of the dorsal from the Arthropod Laboratory and supplied by the Venom Commis- hippocampus and fixed to the skull with dental acrylate. Coordinates sion of the Butantan Institute (São Paulo, Brazil). towards the bregma were AP −5.3, L −4.0, V −2.0. The experiments were performed on freely moving rats 24 h after the implantation of the guide cannulae. Probes (CMA/11 microdialysis probes, Stockholm, Isolation of the fractions and purification of toxins Sweden; membrane length, 2 mm) were introduced into the guide cannulae. A syringe (2.5 ml) was connected to the inlet cannula of Briefly, for the separation of the fractions, 50 mg of dried venom the probe with a 40-cm length of polyethylene tubing. Ringer solution were dissolved in 500 μl of 50 mM ammonium bicarbonate buffer. was used as the perfusion fluid at a flow rate of 2.13 μl/min. After a The solution was homogenized, centrifuged (5000 g) and the super- 30- to 45-min equilibration period, the outflow was collected every natant stored at 4 °C. The precipitate was resuspended in the same 60 min. After three collections, which were used to determine the buffer, homogenized and centrifuged again. The resulting supernatant baseline of extracellular neurotransmitter levels in the hippocampus, was added to the first one, resulting in a total volume of 1.0 ml, and the toxin was injected into the hippocampus (1.0 μg/μl; n=6 ani- the final precipitate was discarded. The final supernatant was applied mals), and five more microdialysis samples were collected until the to a Sephadex G50 column (0.5 cm in diameter and 78 cm in height), end of the experiment. Extracellular amino acid levels were deter- with a total volume of 245 ml and 49 ml of dead volume. Fractions mined in the dialysate and expressed as μg/μl. For determination of were collected after discarding the dead volume, using a Bio-Rad au- glutamate levels, only the dialysate of four animals was used. tomatic fraction collector. Chromatography was performed with a The perfusates were dried in a vacuum lyophilizer immediately after flow rate of 9.0 ml/h, collecting fractions of 1.5 ml, and six fractions their collection and maintained at −80 °C until the analysis. All samples were obtained, according to their optical density monitored spectro- were assayed using an HPLC (Shimadzu, UFLC Prominence) with an an- photometrically at 280 nm. The fractions were then lyophilized and alytical ODS-Shim Pak column (100 mm×3 mm) with detection at stored at −20 °C until use. 254 nm after derivatization with phenyl-isothiocyanate. Next, 50 μlof For the purification of toxins, the fractions of interest were ultrapure water and 150 μl of phenyl-isothiocyanate were added. resuspended in 1.5 ml of ultrapure water and applied to a high per- After mixing, half of the solution (200 μl) was injected into the HPLC formance liquid chromatography system (HPLC‐HP 1100 series) column. The gradient profile was determined to identify each amino equipped with a C18 analytical column (Beckman Ultrasphere 5 μm, acid sample by comparison with the retention time of an amino acid 250 mm×4.6 mm) with detection with a spectrophotometer (HP) standard solution containing 1 mM glutamate, 1 mM γ-aminobutyric at 214 nm, using a polar aqueous solution of 0.1% trifluoroacetic acid (GABA), and 1 mM glycine. Each sample was run for 20 min, and acid in ultrapure water (solvent A) and a nonpolar phase of 90% ace- aspartate, glutamate, glycine and GABA were identified in this order. tonitrile and 10% solvent A (solvent B). The material applied to the Statistical significance was evaluated by one-way analysis of variance HPLC was subjected to a 30-min run with a linear gradient of 10 to (ANOVA) for repeated measures and Tukey's post-hoc test (pb0.05). 70% solvent B. The amount of material in each peak was estimated by the peak area. The different peaks of material were collected, Histology freeze-dried, and stored at −20 ° C until use. In this work, the fourth peak from pool V was utilized, called Tb The location of the implanted guide cannulae and electrodes was V-4. checked histologically. Animals in which the target area was missed 232 L.T. Ossanai et al. / Life Sciences 91 (2012) 230–236 were excluded. Seven days after toxin injection, all animals were with excitation at the argon ion laser line of 488 nm and an emission sacrificed. They were completely anesthetized with CO2 and perfused wavelength of 505 nm. Photo-multiplier gain and laser power were through the heart (left ventricle) with phosphate-buffered saline kept constant throughout each experiment. (PBS) followed by 10% formalin solution. The brains were removed, Thirty images were obtained at 5-s intervals. In each experiment stored in formalin for at least 1 week, and then embedded in maximum fluorescence (total intracellular Ca2+) was measured by Paraplast®. Coronal brain sections of 10 μm were cut from a 700 μm rupturing the cells with 0.1 mM digitonin, and minimum fluorescence brain block in the region of the hippocampus (bregma AP −3.6). was measured by adding 2 mM MnCl2 followed by the addition of The slices were mounted on a glass slide and stained with cresyl vio- 4 mM ethylene-glycol-bis-(β-aminoethylether)-N,N,N′,N′-tetraacetic let. Similar areas from five slices of each rat were analyzed. The num- acid (EGTA) to chelate cytosolic Ca2+ to validate the experiment. ber of cells in the CA1, CA3 and CA4 areas ipsilateral (i) and Only data from validated slides were used. Data were expressed as contralateral (c) to injection was determined by light microscopy the percentage of baseline fluorescence. using a 40× objective. A two-dimensional cell count was performed using a 100×100 μm reticule. Pyramidal neurons, located inside the Results reticule area, with a visible nucleus and nucleolus were considered intact and those with pyknotic nucleus and shrunken cytoplasm Electroencephalographic recordings and behavioral observations were considered dead. Only intact cells were counted. The data were represented as the sum of the number of cells of each slide. The toxin evoked behavioral alterations in the animals, including Of all the animals submitted to histological processing for cannula WDS in 88.9%, myoclonus (88.9%), yawning (77.7%), and orofacial au- localization, twelve were randomly selected for estimation of cell tomatisms (77.8%). Increased secretion was also observed in 33.3% of numbers. the animals (Table 1). ANOVA followed by Tukey's test was used for statistical analysis EEG recordings exhibited alterations such as isolated or grouped (pb0.05). spikes (88.9%) and epileptic-like discharges varying in intensity from short (77.8%) to medium (44.4%) or strong (11.1%). Each animal Analysis of calcium mobilization by confocal microscopy had at least one kind of epileptic behavior or discharge (Table 1), which began immediately after the injection (latency for the first ep- Adult male Wistar rats (n=8) were anesthetized in a CO cham- 2 isode, 91.2±44.9 s) and persisted during the entire recording. ber before rapid decapitation. Brains were rapidly removed and coro- As a control, Ringer solution was injected into the hippocampus, nal slices of the hippocampus (200 μm) were cut with a vibratome and these animals showed no changes in behavior or electroenceph- (St. Louis, MO, USA) into a cold oxygenated artificial CSF solution alographic recordings (Table 1). with the following composition (in mM): 4.7 KCl, 2.5 CaCl2, 1.2 NaHPO4, 1.2 KH2PO4, 1.2 MgSO4, 11.0 D-glucose, and 225.0 sucrose (pH 7.4). Slices were placed in an interface-type chamber at room Evaluation of amino acid neurotransmitters temperature (22–24 °C) gassed with 95% O2–5% CO2 for at least 1 h to allow them to recover prior to treatment. The slices were then in- Extracellular levels of glutamate, GABA and glycine were not af- dividually glued with cyanoacrylate onto coverslips and loaded with fected after injection of the toxin Tb V-4 into the hippocampus 20 μM Fluo-3 AM (Molecular Probes, Inc., Eugene, OR, USA) in the (Fig. 1A). However, individual analysis of glutamate levels (Fig. 1B) presence of 0.04% Pluronic F127 (20% in DMSO, Sigma-Aldrich, St. demonstrated an increase of 182% for one animal (rat 4) compared Louis, MO, USA) for 30 min at room temperature. The slices were to the baseline level immediately after the toxin injection, which per- washed 4 times with CSF solution and then placed in the confocal sisted up to the end of the experiment. The same animal displayed be- chamber with 500 μl of the same solution. havioral alterations including agitation, WDS, myoclonus, increased The CA1 area was located with a LD 20× objective for the capture lacrimal and nasal secretions and respiratory difficulty for 2 h after of the first image. Next, 10 μl of Tb V-4 solution (1 μg/μl) were added toxin injection. Two other animals (rats 2 and 3) had increased gluta- to the chamber to obtain a concentration of 0.02 μg/μl. Each slice used mate levels, 88.6% and 46.9%, respectively, over baseline levels. These came from a single rat. animals remained agitated and showed some WDS, yawning, Fluorescent measurement of cytosolic Ca2+ was performed using grooming and increased oral and nasal secretions. Rat 1 showed no a laser confocal scanning microscope (LSM 410, Zeiss, Jena, Germany) changes in glutamate levels or behavior.

Table 1 Individual effects of injection of Tityus bahiensis scorpion venom toxin Tb V-4 into the hippocampus on behavior and on electroencephalographic recordings determined in the same area. The rats had cannulae and electrodes implanted in the CA1 area of the hippocampus. The recordings were made up to 4 h after injection.

Yawning Increased Wet dog Myoclonus Orofacial Spikes Short Medium Strong secretions shakes automatisms discharges discharges discharges

Ringer (n=6) ––––– –––– Tb V-4 Rat 1 –– xx x x– x – 2x – xx x xx –– 3x – xx x x––– 4x – xx x – x –– 5x x x x x x x –– 6xxxxx xxxx 7x ––x – xx x – 8x – x –– xx –– 9 – xxxx xxx – ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ Total percent 77.8 33.3 88.9 88.9 77.8 88.9 77.8 44.4 11.1

–Not observed. x Observed. ⁎ Signiﬁcantly different from control group treated with Ringer solution; pb0.05, Fisher's exact test. L.T. Ossanai et al. / Life Sciences 91 (2012) 230–236 233

AB 1.0 glutamate

0.8

0.6 injection 0.4

0.2 Concentration (µg/µl)

12345678 rat 1 rat 2 rat 3 rat 4 (hours)

glycine 0.5

0.4

0.3

injection 0.2

0.1 Concentration (µg/µl) 12345678 rat 1 rat 2 rat 3 rat 4 rat 5 (hours)

GABA 0.6

0.5 injection 0.4

0.3

0.2 Concentration (ng/µl) 12345678 rat 1 rat 2 rat 3 rat 4 rat 5 rat 6 (hours)

Fig. 1. Amino acid neurotransmitter levels. A: Extracellular levels of glutamate, glycine and GABA in μg/μl as determined by microdialysis in the CA1 area of the hippocampus in conscious rats before (up to 3 h) and after injection of 1.0 μg/μl toxin Tb V-4 in the same area. Error bars show S.E.M. p>0.05 by ANOVA and Tukey's test. Arrows indicate venom injection. B: The set of points means the extracellular levels of glutamate, glycine, and GABA reached by each rat after injection of the toxin (from 3 to 8 h). Horizontal bars indicate the mean baseline level of the neurotransmitter before injection.

Histological analysis was different in slice 2, 4 and 5, peaking at 40 s (90.5%), 130 s (127.7%) and 95 s (110.5%), respectively, relative to baseline. The The intracerebral injection of Tb V-4 evoked a decrease in the other slices displayed minimal variations, ranging from −6to3% number of pyramidal cells, mainly in the CA1 and CA4 areas, but not from baseline (Fig. 3). to a significant extent (Fig. 2). Many dark staining neurons were observed, consequently resulting in a decrease in intact cells counted. Discussion Three animals had strong neuronal degeneration in CA1i, one animal in CA1c and two animals in CA4c. The other animals had mild The most dangerous scorpion group belongs to the family neuronal degeneration in CA1i, CA1c, CA3i, CA4i and CA4c (Fig. 2). Buthidae, and in Brazil, it is represented by the genus Tityus, which Injection of Ringer solution into the hippocampus, as a control, did is widely distributed all over the country. T. serrulatus is the best stud- not affect the number of cells in the hippocampus, and the numbers ied, and most of the scorpion data in the literature are about its of intact cells in the ipsilateral and contralateral areas were similar venom and toxins. Almost nothing is known about the venom of (Fig. 2). No other regions in the brain of treated rats showed neuronal other Brazilian scorpions from the family Buthidae (Severino et al., loss. 2009). Concerning T. bahiensis scorpion venom, Lourenço et al. (2002) reported that some of its fractions caused convulsion, even Calcium mobilization more intense than that evoked by T. serrulatus venom when injected directly into the hippocampus. Eight slices of the hippocampus from different animals were used The present study demonstrated for the first time the effects of a in this experiment. Tb V‐4 (0.02 μg/μl) increased cytosolic calcium toxin isolated from T. bahiensis scorpion venom, when directly only in three slices. The kinetics of cytosolic calcium accumulation injected into the hippocampus. This toxin, called Tb V-4, was purified 234 L.T. Ossanai et al. / Life Sciences 91 (2012) 230–236

400 CA1i CA1c

300

200

100 number of cells

0 control Tb V-4 control Tb V-4

400 CA3i CA3c

300

200

100 number of cells

0 control Tb V-4 control Tb V-4

400 CA4i CA4c

300

200

100 number of cells

0 control Tb V-4 control Tb V-4

Fig. 2. Histological analyses. Histological analyses of brain sections of rats sacrificed 7 days after injection of 1.0 μg/μl toxin Tb V-4 within the hippocampus. Individual numbers of intact cells of the pyramidal layer of the CA1, CA3 and CA4 areas of treated (ipsilateral) and contralateral sides are shown. The sum of the number of neurons from 5 slides of each rat was pooled and expressed as mean (±S.E.M.) neurons per area. from fraction V, which in a previous screening of the venom was in neural activity (Vornov, 1991). Any disturbance that favors excit- demonstrated to have the same convulsive effects described by ability may cause excessive and synchronized neuronal activity simi- Lourenço et al. (2002). The toxin was chosen for further study since lar to epileptiform discharges (Gerfin-Moser et al., 1995). It is known it showed remarkable convulsive activity. that toxins from scorpion venoms act by enhancing neurotransmitter Classically, seizures are associated with neurotransmitter alter- release in the central and peripheral nervous system (Coutinho-Netto ations. Pathways containing glutamate or related excitatory amino et al., 1980; Nencioni et al., 2003, 2009; Sampaio et al., 1997). acids are definitely involved in the generation and propagation of ep- Some studies have demonstrated that a toxin isolated from ileptic seizures (Cunningham et al., 1994; Fisher, 1991; Umeda et al., T. serrulatus scorpion venom releases glutamate from cortical synap- 2007). The relationship between excitation and inhibition of neural tosomes (Fletcher et al., 1996; Massensini et al., 1998) and from the activity is very important because seizures result from an imbalance hippocampus of rats after local injection. In this case, it induces electroencephalographic and histopathological alterations (Carvalho 150 et al., 1998). sl 1 Similarly, after the injection of the toxin Tb V-4, some behavioral sl 2 signs related to epileptiform activity, such as WDS, myoclonus, 100 sl 3 orofacial automatisms, and yawning, and electroencephalographic sl 4 outcomes such as spikes and epileptiform discharges with variable intensity were observed. Thus, it may be suggested that this toxin sl 5 50 changes the levels of neurotransmitters in the central nervous system. %variation sl 6 To confirm our hypothesis that Tb V-4 evokes alterations in gluta- sl 7 mate release, rats were submitted to microdialysis to determine the sl 8 0 amino acid neurotransmitter levels. Indeed, our experiments demonstrated a great increase in the extracellular levels of glutamate in 0 15 30 45 60 75 90 105 120 135 150 some animals—one rat had high levels during the whole time after time (in seconds) toxin injection and two of them had a variable increase between 50 to 100% over baseline (Fig. 1B). However, some animals showed no alter- Fig. 3. Cytosolic free calcium. Percent variation of cytosolic free calcium after exposure of hippocampal slices of rats to the toxin Tb V-4, determined by confocal laser scanning ation, and if the data are analyzed as a whole, only a small increase in microscopy of the CA1 area. glutamate levels is observed without reaching statistical significance L.T. Ossanai et al. / Life Sciences 91 (2012) 230–236 235

(Fig. 1A). It is interesting to note that the rats that had an increase in glu- and Fundação Butantan. This research is part of the Master's thesis tamate levels also had behavioral signs related to convulsions. presented by Luciene Toshie Takeishi Ossanai to the Post-Graduation This phenomenon – the effect happens in a very strong manner or it Program in Laboratory Research in Public Health of the Disease Control does not happen – is a feature of scorpion venoms and correlates with Coordinating Agency of São Paulo State Public Health Ministry. Thanks clinical cases of envenoming. It was observed that scorpion bites in are due to the Venom Commission of the Butantan Institute for pro- human patients, in addition to pain, usually cause only minor effects viding the venom. Dr. A. Leyva provided the English editing of the such as small changes in heart rate and increased secretions, but some- manuscript. times a large number of serious effects develop, including convulsions and death. A similar effect of the venom was also observed in other experimental conditions. In a study demonstrating the central effects References of T. serrulatus and T. bahiensis scorpion venom, it was shown that some of the animals injected with venom directly into the hippocampus Andrade MV, Caramez MP, Abreu EM, Dolnikoff M, Omar ED, Velasco IT, et al. Lung – had a great increase in HVA concentration, whereas others showed no compliance, plasma electrolyte levels and acid base balance are affected by scorpion envenomation in anesthetized rats under mechanical ventilation. Comp Biochem alteration (Nencioni et al., 2009). In pregnant rats, changes in reproduc- Physiol C Toxicol Pharmacol 2004;138:97-104. tive performance and an incomplete ossification of skull bones were Arantes EC, Prado WA, Sampaio SV, Giglio JR. A simplified procedure for the fraction of observed only in some offspring of dams treated with T. serrulatus scor- Tityus serrulatus venom: isolation and partial characterization of TsTx-IV, a new neurotoxin. Toxicon 1989;27:907–16. pion venom (Barão et al., 2008; Cruttenden et al., 2008). Barão AAS, Nencioni ALA, Dorce VAC. Embryotoxic effects of maternal exposure to Tityu The same situation was observed in the histological analysis. Tb serrulatus scorpion venom. J Venomous Anim Toxins Incl Trop Dis 2008;14: V-4 caused an intense lesion with many pyknotic neurons in several 322–37. Becerril B, Corona M, Coronas FIV, Zamudio F, Calderon-Aranda ES, Fletcher PL, et al. animals, whereas others had only a mild or no lesion. Neuronal loss Toxic peptides and genes encoding toxin γ of the Brazilian scorpions Tityus is generally associated with an increase in glutamate levels and bahiensis and Tityus serrulatus. Biochem J 1996;313:753–60. cytosolic calcium concentration. Rapid accumulation of extracellular Becerril B, Marangoni S, Possani LD. Toxins and genes isolated from scorpions of the genus Tityus. Toxicon 1997;35:821–35. glutamate following injury results in the excessive activation of gluta- Bertazzi DT, Assis-Pandochi AI, Azzolini AE, Talhaferro VL, Lazzarini M, Arantes EC. 2+ mate receptors, massive influx of Ca into cells, excitotoxic neuronal Effect of Tityus serrulatus scorpion venom and its major toxin, TsTX-I, on the com- cell dysfunction, and cell death (Choi, 1988; Gardoni and Di Luca, plement system in vivo. Toxicon 2003;41:501–8. 2006). Scorpion toxins act on sodium channels, increasing the time Carvalho FF, Nencioni ALA, Lebrun I, Sandoval MRL, Dorce VAC. Behavioral, electroencephalographic, and histopathologic effects of a neuropeptide isolated from Tityus of depolarization. Consequently, there is an increase in neurotrans- serrulatus scorpion venom in rats. Pharmacol Biochem Behav 1998;60:7-14. mitter release, mainly glutamate, which binds to its receptors, there- Carvalho FF, Nencioni ALA, Lebrun I, Dorce VAC, Sandoval MRL. Convulsive effects of by opening ion channels. A high quantity of glutamate leads to the some isolated fractions of Tityus serrulatus scorpion venom: behavioral, electroencephalographic and neuropathological aspects. J Venomous Anim Toxins 2000;6: stimulation of many receptors, allowing enhanced calcium entrance 238–60. into the cell. The calcium mobilization caused by Tb V-4 was corrob- Cestèle S, Catterall WA. Molecular mechanisms of neurotoxin action on voltage-gated orated by our experiments with confocal microscopy. sodium channels. Biochimie 2000;82:883–92. Choi DW. Glutamate neurotoxicity and disease of the nervous system. Neuron 1988;1: Despite that the toxin caused a marked increase in calcium mobi- 623–34. lization, again, it was only seen in some slices. It should be noted that Cologna CT, Marcussi S, Giglio JR, Soares AM, Arantes EC. Tityus serrulatus scorpion this characteristic of the toxin, i.e., an all or none effect, has been gen- venom and toxins: an overview. Protein Pept Lett 2009;16:920–32. Conceição IM, Jurkiewicz A, Fonseca DR, Opperman AR, Freitas TA, Lebrun I, et al. Selec- erally observed only with the whole venom and never before with tive release of ATP from sympathetic nerves of rat vas deferens by the toxin TsTX-I isolated toxins. from Brazilian scorpion Tityus serrulatus. J Pharmacol 2005;144:519–27. Pharmacological studies involving scorpion neurotoxins provide Couraud F, Jover E. Mechanism of action of scorpion toxins. In: Tu AT, editor. Handbook of natural toxins: insect poisons, allergens and other invertebrate venoms. New important information about the toxic aspects of envenoming. More- York: Marcel Dekker; 1984. p. 659–78. over, animal neurotoxins may be useful for identifying the sequence Coutinho-Netto J, Abdul-Ghani AS, Norris PJ, Thomas AJ, Bradford HF. The effects of of events underlying neurotransmission and, thereby, represent a scorpion venom toxin on the release of amino acid neurotransmitters from cere- – potential tool for the study of ion channels and epilepsy models. bral cortex in vivo and in vitro. J Neurochem 1980;35:558 65. Cruttenden K, Nencioni AL, Bernardi MM, Dorce VAC. Reproductive toxic effects of Tityus serrulatus scorpion venom in rats. Reprod Toxicol 2008;25. 497-03. Conclusions Cunningham MD, Ferkany JW, Enna SJ. Amino acid receptors: a gallery of new targets for pharmacological intervention. Life Sci 1994;54:135–48. Dorce VAC, Sandoval MRL. Effects of Tityus serrulatus crude venom on the GABAergic The present results suggest that Tb V-4 toxin may be responsible, and dopaminergic systems of the rat brain. Toxicon 1994;32:1641–7. at least in part, for the epileptic and behavioral effects observed with Fisher RS. Glutamate and epilepsy. In: Fisher RS, Coyle JT, editors. Neurotransmitters and epilepsy. New York: Wiley-Liss; 1991. p. 131–45. the crude venom in experimental animals. Fletcher PL, Fletcher M, Fainter LK, Terrian DM. Action of new world scorpion venom We suggest that the convulsive and degenerative effects induced and its neurotoxins in secretion. Toxicon 1996;34:1399–411. by the toxin could be due to the enhanced release of excitatory Freire-Maia L, Campos JA. Pathophysiology and treatment of scorpion poisoning. In: — amino acids that are involved in the most important pathways of Ownby CL, Odell GV, editors. Natural toxins characterization, pharmacology and therapeutics. New York: Pergamon Press; 1989. p. 139–59. the hippocampus. Fukuhara YD, Dellalibera-Joviliano R, Cunha FQ, Reis ML, Donadi EA. The kinin system Although no significant differences were observed for the group, in the envenomation caused by the Tityus serrulatus scorpion sting. Toxicol Appl – some animals had high levels of glutamate and neuronal loss after Pharmacol 2004;196:390 5. Gardoni F, Di Luca M. New targets for pharmacological intervention in the gluta- toxin injection. matergic synapse. Eur J Pharmacol 2006;545:2-10. This is the first work demonstrating that a toxin isolated from Gerfin-Moser A, Grogg F, Rietschin L, Thompson SM, Streit P. Alterations in glutamate T. bahiensis scorpion venom has an effect on the central nervous system. but not GABAA receptor subunit expression as a consequence of epileptiform activity in vitro. Neuroscience 1995;67:849–65. Lourenço GA, Lebrun I, Dorce VAC. Neurotoxic effects of fractions isolated from Tityus Conflict of interest statement bahiensis scorpion venom (Perty, 1984). Toxicon 2002;40:149–57. Massensini AR, Moraes-Santos T, Gomez MV, Romano-Silva MA. Alpha- and The authors declare that there are no conflicts of interest. beta-scorpion toxins evoke glutamate release from rat cortical synaptosomes with different effects on [Na+]i and [Ca2+]i. Neuropharmacology 1998;37:289–97. Nencioni ALA, Carvalho FF, Lebrun I, Dorce VAC, Sandoval MRL. Neurotoxic effects of Acknowledgments three fractions isolated from Tityus serrulatus scorpion venom. Pharmacol Toxicol 2000;86:149–55. Nencioni ALA, Lebrun I, Dorce VAC. A microdialysis study of glutamate concentration in This work was supported by FAPESP (Fundação de Amparo à Pes- the hippocampus of rats after TsTX toxin injection and blockade of toxin effects by quisa do Estado de São Paulo; grant 03/13502-2), CNPq (INCTTox) glutamate receptor antagonists. Pharmacol Biochem Behav 2003;74:455–63. 236 L.T. Ossanai et al. / Life Sciences 91 (2012) 230–236

Nencioni AL, Lourenço GA, Lebrun I, Florio JC, Dorce VAC. Central effects of Tityus Sampaio SV, Coutinho-Neto J, Arantes EC, Toyama MH, Novello JC, Giglio JR. TsTX-VII, a serrulatus and Tityus bahiensis scorpion venoms after intraperitoneal injection in new toxin from Tityus serrulatus scorpion venom able to induce the release of neu- rats. Neurosci Lett 2009;463:234–8. rotransmitters from brain synaptosomes not blocked by tetrodotoxin. Biochem Nishikawa AK, Caricati CP, Lima ML, Dos Santos MC, Kipnis TL, Eickstedt VR, et al. Mol Biol Int 1997;41:1255–63. Antigenic cross-reactivity among the venoms from several species of Brazilian Severino DN, Pereira RL, Knysak I, Cândido DM, Kwasniewski FH. Edematogenic activity of scorpions. Toxicon 1994;32:989–98. scorpion venoms from the Buthidae family and the role of platelet-activating factor Paxinos G, Watson C. The rat brain in stereotaxic coordinates. San Diego: Academic and nitric oxide in paw edema induced by Tityus venoms. Inflammation 2009;32:57–64. Press; 1998. Tian C, Yuan Y, Zhu S. Positively selected sites of scorpion depressant toxins: possible Pimenta AMC, Martin-Euclaire M-F, Rochat H, Figueiredo SG, Kalapothakis E, Afonso roles in toxin functional divergence. Toxicon 2008;51:555–62. LCC, et al. Purification, amino-acid sequence and partial characterization of two Trequattrini C, Zamudio FZ, Petris A, Prestipino G, Possani LD, Franciolini F. Tityus toxins with anti-insect activity from the venom of the South American scorpion bahiensis toxin IV-5b selectively affects Na channel inactivation in chick dorsal Tityus bahiensis (Buthidae). Toxicon 2001;39:1009–19. root ganglion neurons. Comp Biochem Physiol 1995;112:21–8. Possani LD, Martin BM, Fletcher M, Fletcher PL. Discharge effects on pancreatic Umeda Y, Amano M, Suemaru K, Yamaguchi T, Kitamura Y, Gomita Y, et al. The influ- exocrine secretion produced by toxins purified from Tityus serrulatus scorpion ence of hyperactivity of the hypothalamic pituitary–adrenal axis and hyperglyce- venom. J Biol Chem 1991;266:3178–85. mia on the 5HT2A receptor-mediated wet dog shake responses in rats. Acta Med Revelo MP, Bambirra EA, Ferreira AP, Diniz CR, Chávez-Olórtegui C. Body distribution of Okayama 2007;61:311–7. Tityus serrulatus scorpion venom in mice and effects of scorpion antivenom. Vornov JJ. Mechanisms of neurotransmitter receptor actions. In: Fisher RS, Coyle JT, ed- Toxicon 1996;34:1119–25. itors. Neurotransmitters and epilepsy. New York: Wiley-Liss; 1991. p. 17–31.