Male Wistar rats (220 - 270 g) were treated according to guidelines approved by the European Ethics Commitee (86/609/EEC) and the ethical guidelines of the Committee of Health Guide for the Care and Use of Laboratory Animals of School of Veterinary Medicine of Buenos Aires University. The experimental protocol was approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) of School of Medicine of Buenos Aires University. Furthermore, efforts were made to minimize the number of animals used in the study and their suffering. The animals were housed individually in cages and kept under environmentally controlled conditions (23˚C - 25˚C, 12 h normal light/ dark cycles (lights on from 6:00-18:00 h)). Animals were provided with water and food ad libitum. They were acclimated for 7 - 10 days before surgery. Experiments were carried out each day between 10:00-17:00 h.

The surgery was carried out according to Toibaro et al. [28]. The rats were deeply anaesthetized with intraperitoneal injections of a ketamine/xylazine mixture at a concentration of 85.0 mg/ml ketamine and 15.0 mg/ml xylazine. The rats were fixed in a stereotaxic frame according to the method of Paxinos and Watson 1998 [32] and the skull was exposed. Five holes were drilled, two of them for anchor screws, two for placement of a stainless steel wire used as ground and reference and one for placement of the electrode. A bipolar recording and stimulating electrode (0.2 mm in diameter) was chronically implanted in the right ventral hippocampus, using the following coordinates in mm from bregma and skull surface: anteroposterior −5.3; lateral 5.2; depth 7.5 [32]. The electrodes consisted in two twisted stainless steel Teflon-coated wires (tip distance 0.5 - 1.0 mm) insulated except for 0.5 mm at the tip. Electrode was connected to a miniature receptacle, which was embedded in the skull with dental cement. At least 7 - 10 days were allowed for recovery from surgery before starting the experiment. Following completion of the experimental protocol, all animals were anaesthetized by an intraperitoneal injection of ketamine (85 mg/ml) and xylazine (15 mg/ml) and then transcardially perfused with 4% paraformaldehyde. Brains were frozen and coronally sectioned into 40 μm slices with a microtome and slide mounted, allowing for confirmation of electrode placements. The data of the animals with false placement of their electrode position or existence of any abnormality, such as lesion, were not included in the results.

All the recordings were performed after the rat had been transferred from the home cage to a recording box (30 × 30 × 30 cm). The head-stage of the rat was connected to a flexible, shielded cable. Evoked responses were recorded in the freely moving rat while the animal was awake with its eyes open.

On day 0, seven to ten days post surgical recovery, the afterdischarge threshold (ADT) for each animal was determined (Figure 1). The ADT is the minimum intensity that evokes an afterdischarge (AD) of 5 s or longer, defined as a two-fold increase in the EEG baseline. An initial current of 50 μA (peak-to-base; 2 s trains of biphasic square wave pulses, 20 Hz) was applied and then increased in steps of 50 μA (to a maximum of 400 μA), with 5 minutes intervals between current delivery. The animals that did not elicit AD with a current intensity of

400 μA were excluded from the experiment.

On day 1, the animals were assigned to two groups: the control group (hRK; n = 7) and the group treated with HFS (HFS + hRK; n = 9). All animals received the rapid kindling stimulus. The hRK stimulus consisted in 12 daily stimulations during three consecutive days. Each stimulus was carried out at ADT intensity with 10 s trains of biphasic square wave pulses, at a frequency of 20 Hz with intervals of 30 minutes between each of them. The HFS (biphasic square wave pulses 50 μA, 130 Hz for 30 seconds) was applied to the treated group immediately before each one of the 12 RK stimulations on three stimulation days. HFS parameters were determined according to Goodman et al. 2005 [19] and our preliminary experiments [28].

Electroencephalogram (EEG) during each AD was recorded through the hippocampal electrodes. All of the recording were obtained and reviewed by using a digital system (Stellate-Bioscience system, Bioscience S.R.L, Buenos Aires, Argentina). The signal was amplified 1000 times, filtered 0.5 - 40 Hz (3 dB/octave), and digitized at a sampling rate of 200 Hz. The behavioral manifestation was classified following an adjusted version of the scale of Racine [33: Racine et al., 1972]: stage I: immobility, facial clonus, wet dog shakes; stage II: head nodding, chewing, automa-tisms; stage III: clonus of one forelimb; stage IV: rearing, bilateral forelimb clonus; stage V: rearing, bilateral fore-limb clonus, loss of balance and falling [34-36]. Stages I - III were considered as focal seizures, while stages IV and V were considered as generalized seizures (GS) [22]. When the animals exhibited three consecutive stage 5 seizures, they were regarded as fully kindled. An investigator who had no information about the animal group and the number of stimulations scored the behavioral responses. After the completion of the experiment, the incidence of full kindling, the mean of the number of stimulations necessaries to reach the stage IV and V (generalization of seizures), the behavioral progression of kindling (stages I - V; according to Racine’s scores), and the cumulative daily AD duration (ADD) were monitored to evaluate the effect of the HFS during the kindling acquisition. Values for ADD were calculated by adding the duration of ADs recorded after each of the daily twelve stimulations.

A two-way analysis of variance (ANOVA) followed by a post hoc Bonferroni’s test was done to compare the ADD and the number of stimulations required to achieve different stages between hRK (control) and HFS+hRK groups. The behavioral progression of kindling (stages I - V) was analyzed by using Mann Whitney U test. In the case of comparing generalized seizure incidence, chisquare test was used. Data are expressed as mean and standard error of the mean (SEM). Statistical analysis was carried out by GraphPad Prism 4.0 for Windows. Statistical comparisons between HFS and LFS data were performed using one way ANOVA followed by Student-Newman-Keuls post test. For all analysis, the tests were performed two-sided and a p < 0.05 was considered significant.

All the animals used in this experiment fulfilled the above mentioned criteria, 7 rats were assigned to the control group (hRK) and 9 rats to the treated group (HFS + hRK). The following parameters were analyzed: incidence of full kindling, seizure stage achieved per day, number of stimulations needed to reach the different stages and daily afterdischarge duration (ADD).

At the end of the experimental protocol, all control rats were fully kindled, while in HFS group only 2 of 9 rats (22.2%) became fully kindled (p < 0.01; Chi-square test; Figure 1(A)). The seizure stage achieved by the HFS + hRK group was significantly lower (p < 0.01; Mann Whitney U test) than the control group (hRK) on the third day, as shown in Figure 1(B).

HFS group required a higher number of stimulations to reach stage III (12.4 ± 2.6; p < 0.05; two-way ANOVA followed by post hoc Bonferroni’s test) compared to control animals (5.0 ± 1.6). This effect was also observed to achieve stage IV-V, but without reaching significance (HFS: 17.4 ± 2.9; hRK: 13.7 ± 1.9; p > 0.05) (Figure 2).

Regarding ADD, HFS group showed a non significant decreased duration from second and third day in relation to control group.

The HFS group showed a lower incidence of full kin-

dling when compared to LFS group, but did not reach significance (Figure 3).

We did not observe any differences in relation to the daily achieved seizure stage, between HFS and LFS treatments (LFS data previously published by our group [28]; Figure 1(b) for HFS).

ADD has not been significantly modified by HFS neither LFS. Despite both HFS and LFS show a decreased ADD.

The animals of HFS group (n = 9) have achieved stage IV - V after 17.4 ± 2.9 stimulations, while animals of LFS group (n = 7) have needed more stimulations (26.1 ± 3.9) to reach the same seizure stages (p < 0.01) (LFS data previously published by our group [28]; Figure 2 for HFS).