version ISSN 0104-7930
J. Venom. Anim. Toxins vol.8 no.1 Botucatu 2002
EFFECTS OF SCORPION Tityus serrulatus VENOM TOXIN TS-8F ON RAT LEARNING AND MEMORY
1 Laboratory of Pharmacology, School of Veterinary Medicine - São Paulo University, 2 Laboratory of Pharmacology and 3 Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo, SP, 05503 900, Brazil.
ABSTRACT: Scorpion venom neurotoxins are responsible for toxicity and pharmacological effects. They are active in sodium and potassium channels leading to an increase in the release of neurotransmitters, such as glutamate. Glutamate is found in large quantities in the hippocampus (HPC) and is involved in the long-term potentiation (LTP) induction. The HPC is known to be related to certain kinds of memory. The aim of this study is to evaluate the effects of Tityus serrulatus TS-8F toxin on rat behavior with emphasis on learning and memory. We analyzed the effects of different doses of TS-8F on rat behavior in home cages, open-field (habituation), inhibitory avoidance, T-maze, and hippocampus morphology. In the first two experiments, 0.05µg/animal dose of TS-8F did not cause convulsion but led to a decrease in locomotion (LO) frequency in the open-field first session. During the second session, rats receiving 0.03µg/animal TS-8F showed a decrease in LO and rearing frequency (RE); controls only showed decreased LO; and those receiving 0.05µg/animal showed no significant changes. In inhibitory avoidance, T-maze, and HPC morphology experiments no significant differences were observed. It is concluded that TS-8F may exert some influence in rat learning and memory and seems to be useful as a pharmacological tool. Further research is required to elucidate all possible uses of this toxin.
KEY WORDS: learning and memory, scorpion neurotoxin, Tityus serrulatus, T-maze, open-field, passive avoidance, TS-8F toxin.
Tityus serrulatus is the most dangerous scorpion in Brazil, causing severe envenoming and even death. Toxins in the venom are responsible for toxicity and pharmacological effects. These toxins, acting through binding to two distinct external sites of sodium channels (15,23), induce an increase in the release of noradrenaline (17), dopamine (27), gamma-aminobutyric acid (GABA), aspartate, and glutamate (8) in the peripheral and /or central nervous system (CNS).
Scorpion venoms have been shown to be useful as neurobiological tools, facilitating research on ionic channels. Some toxins from these venoms are also useful in CNS studies. A previous work reports that TS-8F induces convulsions and HPC cell damage (6). These seem to be as a consequence of excessive releasing of excitatory amino acids, such as glutamate. This hypothesis is consistent with in vitro studies in brain cortex slices where tityustoxin was able to release glutamate, aspartate, and GABA (8).
We have recently studied the convulsant effects of TS-8F (6). TS-8F microinjections into rat dorsal hippocampus (HPC) induced convulsions. This effect seems to be involved in glutamate release, which acts on HPC metabotropic and ionotropic receptors, causing excessive postsynaptic calcium increase, leading to convulsions and neuronal death (25). In this work to evaluate this hypothesis, we administered subconvulsant doses of TS-8F directly in rat HPC. We expected the toxin to cause an increase in glutamate release sufficient to induce long-term potentiation (LTP), a long-lasting enhancement of synaptic transmission that may correspond to one of the mechanisms underlying certain forms of memory (21). The aim of this study is to evaluate the effects of subconvulsant doses of TS-8F on rat behavior in home cage, open-field, inhibitory avoidance, T-maze, and HPC morphology.
MATERIALS AND METHODS
Animals, surgery, TS-8F administration, and histological analysis
Seventy-nine male Wistar rats (210-250 g) were housed in groups of 5 or 6 per cage and kept under normal laboratory conditions at 21 ± 2°C and 12/12 h light/dark cycle, starting at 07:00 h. Food and water were provided ad libitum. Each animal was randomly assigned to one of the experimental groups receiving 1µl of phosphate buffer, pH 7.4, or subconvulsant doses of TS-8F administered in the dorsal HPC CA1 region. For implantation of cannulae, each animal was anesthetized (3.0 ml/Kg, IP) with pentobarbitone (1.0 g) and chloralhydrate (4.0 g) in 100 ml of 0.9% NaCl and placed in a stereotaxic frame. The skin was carefully removed and the skull exposed. A hole was drilled into the skull for unilateral insertion of a guide cannula into HPC CA1 region. Coordinates were caudally, 4.8; laterally, 3.5; depth, 3.0 mm (19). The guide cannula was then lowered and cemented to the head with dental acrylic. After surgery, the animals were housed individually and allowed to recover for 5 to 7 days.
TS-8F toxin was purified as previously described (6), added to phosphate buffer, pH 7.4, at the moment of administration, and a total volume of 1 µl was injected within 5 min. Doses used were 0.03, 0.05, 0.1, 0.3, 0.6, and 1 µg/animal. Phosphate buffer or TS-8F was injected immediately before home cage behavior or 15 min prior to open-field first exposure, inhibitory avoidance train, and T-maze tests. After experimental procedures, the animals were anesthetized with ethylic ether and perfused through the heart (left ventricle) with phosphate buffer followed by 10% formalin solution. The extracted brains were stored in 10% formalin for a minimum of 1 week. The brains were then dehydrated with alcohol and fixed in paraffin. Paraffin blocks containing the brains were sectioned at 10 µm in the coronal plane using a microtome. The sections were mounted on slides and stained with cresyl violet for the observation of cannula location and HPC damage. Only the animals that had the cannula inserted into the appropriate location were chosen for further data analysis. For the evaluation of HPC damage, a cell count was made of three sections per brain, using a reticulum of 100 µm2. The intact cells of the HPC CA1, CA3, CA4, and dentate gyrus (DG) were counted.
Behavior tests were performed between 08:00 and 12:00 h. All experiments were in strict compliance with the National Institute Health Guide for Care and Use of Laboratory Animals (1985).
Determination of subconvulsant doses
Following administration of 0.03, 0.05, 0.1, 0.3, 0.6, or 1.0µg/animal TS-8F, convulsant effects were analyzed in home cages. Observation period was 1 hour. Alterations related to convulsion, such as facial and limb myoclonus, wet dog shakes, limb, clonus, and tonic-clonic seizures were observed. This experiment was carried out in order to determine the subconvulsant doses used in the other experiments.
Habituation in the open-field
Rat novelty-induced activities were measured in an open-field (4). All measurements were made in a quiet room in the morning. The animals received 0.03 or 0.05 µg/animal TS-8F or phosphate buffer 15 minutes prior to testing. The test started by placing the rat in the center of the arena. Each test lasted 10 min; the following frequencies were recorded: locomotion (the number of sectors penetrated by the animal), rearing (raising of the forepaws), immobility duration, and the number of fecal bolli deposited (defecations). One week later, they were exposed to the apparatus again. The same parameters were again registered, and an evaluation of animal habituation to the open-field was made, by comparing the parameters in the first and second sessions. Decreases in defecation, rearing, and locomotion frequencies and increase in immobility duration were considered indicative of habituation.
Step-through passive avoidance
The passive avoidance chamber consisted of two compartments each with a metal grid floor separated by a motor driven guillotine door. On the training day 15 minutes after 0.03 µg/animal TS-8F or 1.0 µl of phosphate buffer administration, the animal was placed in the light compartment with the guillotine door open. Latency to enter the dark compartment was measured in seconds, and this behavior was punished with a 0.9 mA foot shock for 2s via the grid floor. The animal was then returned to its home cage and holding room for 24 h. One day (Test) and one week (Retest) after the training session, each animal was placed in the light compartment for one trial per day, and latency to enter the dark compartment was measured. No additional foot shocks were administered during these trials.
Procedure in the T-maze
The T-maze had a start arm and left and right arms (50x13x35 cm). At the extremity of each left and right arm a 2-cm diameter, 1.5-cm deep food cup was placed on the floor. The T-maze was located in a quiet room. The animals were familiarized with the maze, food, and food containers on two consecutive days before the beginning of the experiments. On these two days, two trials per rat were carried out. The animals were deprived of food for 24 h; they then received 0.03 or 0.05 µg/ animal TS-8F or phosphate buffer and were returned to their home cages. After 15 minutes, the experimental phase (test and retest 24h) began. At the beginning of the experimental session, 15 trials per rat were performed each day. Only one arm of the T-maze was baited with food. The correct choice was the left arm for half rats and the right arm for the other half. Each rat was put in the start arm, and upon reaching the end of the right or the left arm was removed from the maze and put in a separate waiting box for 10 s and then returned to the maze as before. A correct trial ended with the rat eating the food. An incorrect trial (error) ended with the rat reaching the empty food cup. The time taken for each trial was recorded using a stopwatch. On the experimental days, the number of errors was recorded.
Data were analyzed using INSTAT version 2. The mean and mean standard error were calculated for all parameters. Comparisons between the different groups were made using the one-way analysis of variance. Further comparisons were performed using Tukey-Kramers test, with 5% significance level. Latency in the T-maze was analyzed by Kruskall-Wallis non-parametric test.
Determination of subconvulsant doses
TS-8F dose of 1.0 µg/animal caused forelimbs clonus (n=4). Doses of 0.6 (n=2) and 0.3 (n=2) induced wet dog shakes and facial myoclonus, while 0.1 µg/animal (n=5) produced forelimb myoclonus in two animals and wet dog shakes in another two. Neither phosphate buffer (n=3) nor TS-8F 0.05 µg/animal (n=3) induced behavioral changes. TS-8F doses of 0.03 and 0.05 µg/animal were used in the other experiments.
Behavior activities of TS-8F and control rats in response to a novel environment were compared in open-field, 15 minutes and 1 week after administration. Analysis of our data revealed an impairment of explorative behavior after TS-8F injection of 0.05 µg/animal, manifesting as a significant decrease of horizontal locomotor activity (ambulation), F (2.21)=7.125, p<0.05 (Figure 1) 15 minutes after injection. During the second trial, controls showed habituation to open-field revealed by decreased rearing frequency, F(2.21)=2.005, p<0.01 (Figure 2); a similar effect was recorded in the 0.03 µg/animal TS-8F group F(2.21)=4.067, p<0.05 (Figure 2). In parallel with changes in vertical movements, ambulation of animals which received 0.03 µg/animal TS-8F also decreased, F(2.21)=3.699, p<0.01 (Figure 1). No other significant changes were observed in relation to immobility duration and number of fecal bolli.
|Figure 1. Intensity of ambulation (A) and rearing (B) of control and TS-8F animals in open-field in the first and second trials. Eight control and eight TS-8F rats were used in each trial point to determine behavior activities in open-field. * p<0.05 vs. the control group; **p<0.05 vs. first trial (Tukey-Kramer test). Data are expressed as mean and mean standard error.|
|Figure 2. Mean latencies (sec) to enter dark chamber of control and TS-8F- animals in inhibitory avoidance during train, test, and retest days. Eight control and nine TS-8F rats were used to determine learning and memory in inhibitory avoidance. ANOVA, p>0.05.|
Learning and memory in passive avoidance
In this experiment, 0.05 µg/animal TS-8F was not used as it had been shown to decrease locomotion and could interfere with the results. Only two groups were evaluated: the group receiving phosphate buffer and the other receiving 0.03 µg/animal TS-8F. Figure 2 shows the mean latencies to enter the dark compartment by members of each group during training, test, and retest. During training, test, and retest, no significant changes were observed (p>0.05).
Learning and memory in T-maze
Performance of rats receiving phosphate buffer, 0.03, or 0.05 µg/animal TS-8F on the first and second days in the T-maze test is shown in Table 1 . One-way ANOVA demonstrated no significant differences between groups in relation to number of errors (p>0.05). The Kruskall-Wallis non-parametric test did not detect significant differences between groups on the time taken for each trial (p>0.05).
|Table 1. Performance of the rats given different doses of TS-8F and controls in T-maze in the first and second days (Mean ± SEM).|
Intact cell counting of the HPC CA1, CA3, CA4, and dentate gyrus analyzed by ANOVA did not show significant changes between phosphate buffer and TS-8F groups (p>0.05).
T. serrulatus venom is known to act on sodium channels inducing the release of neurotransmitters, such as glutamate (8) that exerts an action through the binding to metabotropic and ionotropic receptors, leading to a postsynaptic calcium increase (21). This seems to play a physiological role in plasticity phenomena, such as long-term potentiation (LTP) and pathological states that involve excessive or inappropriate glutamatergic neuronal transmission, such as seizure and excitotoxicity (25). Microinjection of 1.0, 2.0, 3.0, or 5.0 µg of T. serrulatus venom into the HPC induced immobility, wet dog shakes, orofacial automatism, and tonic-clonic seizure (24). Microinjection of 1.0 or 2.0µg TS-8F into rat HPC also leads to orofacial automatism, wet dog shakes, and myoclonus (6). These actions might be caused by an excessive increase in glutamate release in the HPC.
In this work, the effects of different TS-8F doses were first studied to determine doses that would not cause convulsant behavior or HPC damage; these doses were then used in learning and memory experiments. TS-8F doses were administered from 1.0 µg, known to cause convulsant effects, and progressively down to 0.05 µg/animal, which did not lead to convulsion.
If higher doses of TS-8F cause seizure, it would be reasonable to think that lower doses would lead to LTP, a long-lasting enhancement of synaptic transmission that may correspond to one of the mechanisms underlying certain forms of memory (21), and thus enhancing learning and memory.
The next step was to evaluate behavior in the open-field. The administration of 0.05 µg TS-8F significantly decreased ambulation. This is consistent with previous results where it was observed that Mesobuthus tamulus venom administered to rat brain ventricles showed a reduction of exploratory activity in the open-field (2). The hypothesis of a functional antagonism between dopaminergic and glutamatergic systems has been described in literature (13). This could explain ambulation decrease caused by the administration of 0.05 µg TS-8F, which could increase glutamate release, antagonizing the dopaminergic system and decreasing locomotion frequency. However, ambulation decrease could indicate a motor impairment, suggesting a decreased dose because it might interfere with rat performance in inhibitory avoidance. Decreasing TS-8F dose to 0.03 µg/animal did not affect the inhibitory avoidance parameters observed in open-field. In relation to open-field, changes were not observed in the number of fecal bolli, usually related to rat emotionality. However, during microinjection, an increased defecation was seen, probably caused by stimulation of the autonomic nervous system provoked by the manipulation.
Evaluation of habituation in open-field second trial showed a decrease in rearing frequencies in the controls and 0.03 µg TS-8F groups, indicating that these animals habituated to the open-field. Furthermore, rats receiving 0.03 µg TS-8F habituated strongly, showing a decrease in ambulation. Rats receiving 0.05 µg TS-8F showed no differences between the second and first trials. However, as ambulation was decreased in the first trial in relation to controls, these animals might have also habituated to the open-field. If they did not habituate, we would expect an increase in ambulation and rearing during the second trial, similar to levels of controls in the first trial, indicating that they did not remember the open field.
During inhibitory avoidance training, both groups behaved similarly reacting to light as an adverse stimulus, running to the dark chamber, and receiving shock. In the test trial, the latency to enter in the dark chamber of experimental group increased approximately twice. Although not significant, it shows a tendency of the toxin to enhance learning and memory.
In T-maze, during familiarization, all groups behaved similarly showing homogeneity. On test and retest days, no significant differences in the number of errors were observed between groups. Despite ambulation decrease in open-field first session in 0.05 µg/animal TS-8F group, no differences between groups were observed in the time taken for each trial, indicating that the toxin might have interfered with motivation because when a stimulus is given, food deprivation in this case, animals reacted similarly to controls.
Cell counting of HPC CA1, CA3, CA4, and dentate gyrus areas showed no damage in either hemisphere, which was expected since the animals did not suffer convulsions and no learning and memory impairment was observed.
Glutamate is considered one of the major transmitters in the CNS (11) and there is considerable evidence that activation of glutamate receptors is crucial for memory processing. Drugs that facilitate glutamate receptor activation have been found to improve learning (10,20), while antagonists impair a wide range of tasks in several species (5,10,18). Furthermore, a number of neurological disorders displaying memory pathology, including Alzheimers disease, show abnormalities in glutamatergic neurons and binding to glutamate receptors (12,16). Glutamate receptors have also been found to have a central role in HPC long-term potentiation (LTP), a widely accepted neuronal model of memory formation, whereby a high frequency stimulation of presynaptic fibers induces a long-lasting enhancement of synaptic transmission (3). LTP induction, at least in HPC CA1 and dentate gyrus regions, depends on the activation of NMDA class of glutamate receptor (7), while the AMPA/kainate glutamate receptors are responsible for expressing potentiation (9). There is also evidence that the metabotropic class of the glutamate receptor may be essential for LTP because antagonists to this type of receptor prevent LTP induction as well as some features of LTP maintenance (1,14,22,26).
The present findings provide evidence that TS-8F may exert some influence in learning and memory. This effect may be due to toxin glutamate release. However, its use as a pharmacological tool to study cognition requires further experiments, such as the use of this toxin in other memory tasks, under different administration periods. Also, other less convulsant toxins present in Tityus serrulatus venom must be evaluated to enlarge the range of useful doses, since TS-8F doses that causes convulsant effects is too close to the one that enhances memory.
The authors would like to thank Dr. Deborah Cory-Selechta for her suggestions concerning learning and memory protocols. This paper is part of the MS dissertation presented by Viviane M. Mauro to the Department of Pathology - School of Veterinary Medicine/USP with a fellowship from CNPq 157.505.628-32.
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Received May 8, 2001
Accepted July 30, 2001
V. A. C. Dorce - Laboratório de Farmacologia - Instituto Butantan, Av. Dr. Vital Brasil, 1500, 05503-900, São Paulo, SP, Brazil. Phone: 55 11 37267222 - Extension 2133 - FAX: 55 11 3726 1505