Abstracts

Rationale: A quick and efficient alternative to classical daily amygdala electrical stimulation, a model of temporal lobe epilepsy (TLE) is the fast amygdala kindling (FAK), for example, with 10 daily electrical stimulations in two days. The 21st stimulus is applied at the third day to test, for example, anticonvulsant drugs. Among the main brain areas activated with limbic seizures are the amygdaloid complex, the hippocampal formation and the piriform cortex. Clinical and experimental evidences indicate that olfactory stimulation modulates TLE seizures, either blocking or inducing ictal activity. Our hypothesis is that 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a potent chemical substance derived from fox feces which represents "predator smell", therefore inducing fear, can influence the seizure process. Our main goal was to evaluate the behavioral and electroencephalographic (EEG) effects induced by exposure to olfactory stimulation (TMT) on limbic seizures induced by FAK in Wistar rats. Methods: Wistar male naïve rats (n=18; Ethics Committee of the Ribeirão Preto School of Medicine, Protocol 200/2011), weighing 280-300 g were submitted to FAK, consisting of ten daily electrical stimuli (two days), of bipolar trains of biphasic square waves, 500 μA constant current, 60 Hz, 1 ms pulse, 10 s duration. The gain was adjusted with Quick300 (Axon Instruments) to 2000X, low pass filter 1000 Hz, high pass filter 0,1 Hz and notch filter (60 Hz). EEG signal analysis was made with Acknowledge (Biopac Systems) and Matlab (The Math Works, Inc). After the completion of the FAK protocol, animals were pre-exposed to a saturated chamber of TMT or water (control) at the 21st stimulus. Behavioral responses were measured by either Racine´s (1972) limbic seizure scale or by sequential analysis/neuroethology. Racine’s scale comparisons were done with unpaired Student´s test. Neuroethological analysis used a chi-square statistics with p< 0.05 for statistical significance of pairwise (dyadic) behavioral interactions. Spectral analysis (Fast Fourier Transform, FFT) was done comparing epochs with EEG afterdischarges of TMT and control groups at the 21st stimulus. Mean frequency, peak power, frequency at the peak power and total power in three EEG channels (amygdala, hippocampus and piriform cortex) were measured in the delta, theta, alpha, beta and gamma EEG ranges, considering the frequencies altogether. Results: As expected, the FAK protocol induces a progression of both behavioral and EEG seizure activity. As a completely new finding, exposure of FAK Wistar rats to TMT was able to reduce significantly seizures severity (Racine's scale), corroborated with the behavioral sequential analysis. Furthermore, FFT EEG afterdischarge analysis at the 21st stimulus showed that TMT altered significantly the mean frequency and the peak power in amygdala in all the frequency ranges, except on the gamma one (p < 0,05; Mann-Whitney-U test). The average frequency in the piriform cortex, considering the whole spectrum (not ranges), also presented statistical difference between groups (p < 0,05; Mann-Whitney-U test). Altogether, these data and others from our Lab and the literature are suggestive of the activation of a "fear circuit" by TMT with clear-cut suppression effect on seizure activity evoked by FAK. Conclusions: Both behavioral and EEGraphic evaluations confirm that TMT, a potent molecule with strong biological relevance, in fact, "predator odor", block, with high statistical significance the activity of epileptogenic limbic networks. Ongoing studies are designed to reveal the exact neural substrates in which a "fear related network" can antagonize limbic epileptic activity. Funding: Brazilian Research Foundations: FAPESP, CNPq, PROEX-CAPES and FAEPA Grants.