TsTx Toxin Isolated from Tityus serrulatus Scorpion Venom‐Induced Spontaneous Recurrent Seizures and Mossy Fiber Sprouting

Abstract

TsTx is a scorpion alpha-type toxin that binds to site 3 of the Na+ channels in a voltage-dependent mode, slowing or blocking the inactivation mechanism of these channels (Possani et al., Eur J Biochem 1999). This binding increases depolarization time of the channel and consequently induces excessive neurotransmitter release. Previously we reported that hippocampal injection of TsTx induces clonic convulsions, electrographic seizures, and hippocampal damage. This investigation was designed to characterize the long-term behavioral, electroencephalographic (EEG), and histopathologic features after a single TsTx injection into the rat hippocampus. Cannulas and electrodes were stereotaxically implanted in the CA1 dorsal hippocampus of rats. Three days after surgery, the animals were injected into the hippocampus with 1 microl of TsTx, 2 microg (n = 9) or 0.1 M phosphate buffer (n = 5). After injection, EEG records and behavioral observations were made during 10 h. For a period of 4 months, the animals were observed through direct visual observation for 8 h/day, 5 days/week for occurrence of convulsive seizures. At the end of the experiment, the animals were processed for histologic analyses. Sections 40- and 20-microm thick were stained according to neo-Timm or Nissl methods, respectively. Cell counts in the cresyl violet-stained sections were performed within the hippocampal pyramidal cell layers (CA1, CA3, and CA4) and granule cell of the dentate gyrus. Fifteen minutes after TsTx injection, facial automatisms, rearing, masticatory jaw movements, sniffing, and wet-dog shakes were observed. In approximately 1 h, limbic convulsions characterized by forelimb clonus, rearing, and falling after generalized clonic convulsion with jumping, wild running, and falling were observed. EEG showed isolated spikes and clusters of spikes that started in the hippocampus and evolved to the cortex, isolated seizures, and epileptic discharges delayed 1-2 min. These recurred repeatedly characterizing the status epilepticus (SE). SE was characterized in 77.5% of animals. Seizures were no longer observed 24 h after the injection. Spontaneous recurrent seizures (SRSs) occurred 31-49 days after TsTx injection. All animals that showed SE in the acute period developed SRSs. Facial myoclonus, generalized clonus, forelimb clonus, rearing, and falling characterized the seizures. The seizure frequency was 1-2 per animal per week. All rats injected with TsTx had significantly fewer cells in CA1, CA3, and CA4 subfield of the hippocampal formation compared with the animals of the control group (p < 0.05, analysis of variance, followed by Tukey test). Neo-Timm-positive granules, normally absent in the supragranular layer, were present in dentate gyrus of rats with TsTx-induced SRSs. Our results suggest that SRSs observed in this study may be a consequence of the TsTx-induced SE. All animals injected with the toxin showed massive neuronal loss in the hippocampal subfields CA1, CA3, and CA4, but only those that had SRSs showed mossy fiber sprouting in the supragranular layer of the dentate gyrus. This shows synaptic reorganization that also is observed in human epileptogenic tissue. These results indicate that TsTx toxin may be a useful tool for studies on neuronal lesions and/or experimental epilepsy. Studies on the mechanisms involved are under investigation.