Four weeks after SE, prior to FLX treatment, 16 animals were randomly divided into two groups (n = 8 per group), and underwent seizure monitoring for 1 week. In each of the groups, seizures were observed in five animals. The minimal/maximal/median seizure count over 1 week, was 0/5/1.5 in group 1 and 0/4/1.0 in group 2 (P > 0.05 between the groups). After vehicle (group 1) and FLX (group 2) treatment (8 weeks after SE), seizure incidence and frequency were within the same statistical range as during the first monitoring session (B), although there was a trend toward a decrease in seizure frequency in the FLX-treated animals. In the vehicle treated group, seven animals exhibited seizures, but after FLX, seizures were recorded in all eight rats. The minimal/maximal/median seizure frequency was 0/15/5 in vehicle-treated rats and 1/5/2 after FLX administration (P > 0.05). During the two observation periods combined, each of the animals exhibited at least one seizure.
Fig. 3 Spontaneous seizures after SE and the effects of FLX. (A) An example of spontaneous electrographic seizure 4 weeks after SE. Arrows show the onset and the end of the seizure. (B) Spontaneous seizure count during 1-week recording periods before and after (more ...)
During the first test prior to FLX treatment, ADT was significantly lower and ADD—significantly longer in post-SE animals (two groups, n = 8 each), as compared to controls (two groups, n = 7 each; A–D). Furthermore, in contrast to naive rats, post-SE animals developed behavioural seizures in response to threshold stimulation (D).
Fig. 4 Changes in afterdischarge properties after SE and the effects of FLX treatment. (A and B) Examples of afterdischarge in naive (A) and post-SE (B) animals. Horizontal bars and numbers on the top indicate test train and the applied current. Arrows indicate (more ...)
In naive rats, treatment with FLX significantly elevated ADT (from 1.1 ± 0.14 mA to 1.8 ± 0.12 mA, n = 7, P < 0.05); ADD was not affected by FLX treatment (C). Administration of FLX to post-SE animals (n = 8) reversed the increase of excitability which had been induced by SE. Thus, ADT increased from 0.53 ± 0.1 mA before treatment to 1.6 ± 0.14 mA after FLX; ADD shortened from 64 ± 14.1 s to 24.1 ± 5.6 s (P < 0.05 for both parameters, D). Further, the severity of convulsions in response to threshold stimulation decreased from 2.5 ± 0.6 to 1.2 ± 0.4 (P < 0.05, D).
During the first test, prior to FLX treatment, the naive animals exhibited a strong preference towards saccharin solution over tap water (two groups, n = 7 each; A). In contrast to controls, post-SE animals consumed statistically equal amounts of regular water and saccharin solution (two groups, n = 8 each; A). The introduction of saccharin did not alter total volume of consumed fluid (saccharin + regular water) in both control and post-SE rats (data not shown). The administration of FLX did not modify saccharin consumption in both the naive and post-SE animals (A) nor change the total volume of consumed fluid (data not shown).
Fig. 5 Changes in behaviour after SE and the effects of FLX treatment. (A) Saccharin consumption. Taste preference is expressed as percent of volume of saccharin solution of a total fluid (water + saccharin) consumed over 24 h. ‡P < 0.05 versus (more ...)
Prior to FLX (n = 7) or vehicle (n = 7) treatment, total immobility time in the naive rats was between 75 s and 110 s. In post- SE animals (two groups, n = 8 each) immobility time was between 90 s and 210 s. The increase in immobility time after SE was statistically significant (P < 0.05, B). Administration of FLX to naive rats resulted in significant shortening of immobility time under conditions of FST (from 80 ± 4.6 s to 48 ± 3.5 s P < 0.05, B). However, similar treatment with FLX, did not improve forced swim behaviour in post-SE animals: in this group immobility time was 148 ± 26 s before, and 147 ± 22 s after FLX administration (B).
5-HT release from the hippocampus
Pre-treatment with PCA (n = 4) led to 7-fold reduction of 5-HT release from the rat hippocampus, as compared to naive rats (A and B), thus confirming the validity of the used technique for detecting 5-HT release in vivo. In the hippocampus of vehicle-treated post-SE animals (n = 6) the release of 5-HT in response to raphe stimulation was significantly diminished as compared to vehicle-treated controls (n = 7). Chronic treatment with FLX did not affect 5-HT release in both control (n = 7) and post-SE (n = 7) animals (A and B).
Fig. 6 5-HT release from the hippocampus in response to raphe stimulation and the effects of FLX treatment. (A) Mean ± SEM Faradaic currents under CA1 and CA3 carbon fibre electrodes in response to dorsal raphe stimulation. *P < 0.05 versus respective (more ...)
5-HT concentration and turnover in the hippocampus
In hippocampal tissue of vehicle-treated post-SE animals (n = 6), both the concentration of 5-HT and its turnover were significantly lower as compared to control rats (n = 7; ). Chronic treatment with FLX did not alter 5-HT concentration in hippocampi of both the naive (n = 7) and post-SE (n = 7) rats. At the same time, FLX administration to controls significantly inhibited 5-HT turnover. Treatment of post-SE animals with FLX resulted in a further decrease of the 5-HIAA/5-HT ratio, which was lower than both the FLX-free post-SE group and the FLX-treated control subjects ().
Fig. 7 5-HT concentration and turnover in the hippocampus, and the effects of FLX treatment. Data are shown as mean ± SEM for control and post-SE groups, both vehicle and FLX-treated. 5-HT concentration is plotted on the left, and turnover—on (more ...)
In the absence of FLX treatment, no correlation was found between the frequency of seizures and any of other parameters (). At the same time, animals with more profound changes in afterdischarge properties exhibited longer immobility time in the FST and lower preference towards saccharin (). Furthermore, longer immobility time during FST was observed in animals with slower 5-HT turnover in, and release from the hippocampus. However, parameters of serotonergic transmission were not associated with changes in taste preference. Likewise, no correlation was found between the extent of behavioural changes in forced swim and taste preference tests. Finally, no statistical association was revealed between the changes in either of the two of afterdischarge indices and the extent of serotonergic deficits ().
Correlation analysis between spontaneous seizures, brain excitability, behavioural and biochemical impairments after status epilepticus
After FLX treatment, no correlation was observed between both seizure frequency and afterdischarge properties on the one hand and behavioural indices on the other hand (−0.06 > r > +0.065, P >0.05 for all pairs of variables). Correlation between 5-HT turnover and behavioural alterations in FST also disappeared after FLX administration (P >0.05); at the same time, correlation between 5-HT release from the hippocampus and the extent of forced swim behavioural deficits was statistically significant as it had been without FLX treatment (r = −0.81, P < 0.05).