The anticonvulsant response to valproate in kindled rats is correlated with its effect on neuronal firing in the substantia nigra pars reticulata: a new mechanism of pharmacoresistance

Abstract

Resistance to antiepileptic drugs (AEDs) is a major problem in epilepsy treatment. However, mechanisms of resistance are only incompletely understood. We have recently shown that repeated administration of the AED phenytoin allows selecting resistant and responsive rats from the amygdala kindling model of epilepsy, providing a tool to study mechanisms of AED resistance. We now tested whether individual amygdala-kindled rats also differ in their anticonvulsant response to the major AED valproate (VPA) and which mechanism may underlie the different response to VPA. VPA has been proposed to act, at least in part, by reducing spontaneous activity in the substantia nigra pars reticulata (SNr), a main basal ganglia output structure involved in seizure propagation, seizure control, and epilepsy-induced neuroplasticity. Thus, we evaluated whether poor anticonvulsant response to VPA is correlated with low efficacy of VPA on SNr firing rate and pattern in kindled rats. We found (1) that good and poor VPA responders can be selected in kindled rats by repeatedly determining the effect of VPA on the electrographic seizure threshold, and (2) a significant correlation between the anticonvulsant response to VPA in kindled rats and its effect on SNr firing rate and pattern. The less VPA was able to raise seizure threshold, the lower was the VPA-induced reduction of SNr firing rate and the VPA-induced regularity of SNr firing. The data demonstrate for the first time an involvement of the SNr in pharmacoresistant experimental epilepsy and emphasize the relevance of the basal ganglia as target structures for new treatment options.

Figure 1.

Study design. Adult female Wistar rats were implanted with electrodes for kindling stimulation and recording of afterdischarges. Initially, the stimulation threshold for eliciting afterdischarges (initial ADT, afterdischarge threshold) was determined for each animal followed by daily constant current stimulations. In fully kindled rats, the stimulation threshold for eliciting afterdischarges (post-ADT) was determined for each animal until reproducible postkindling (control) ADTs were observed. For the selection procedure, VPA was administered (200 mg/kg, i.p.) 30 min before ADT determinations. Scorings of side effects induced by VPA included determination of severity of ataxia, hypolocomotion/sedation, abdominal muscle tone, amount of wet dog shake behavior, body temperature, and rotarod performance. VPA trials were repeated at least four times alternating with control trials with injection of saline before stimulation. Within 2 weeks after the last determination of ADT and 24 h after a further kindled seizure was elicited, extracellular single-unit recordings of nondopaminergic, presumably GABAergic nigral neurons were performed in each rat. Finally, rats were deeply anesthetized with chloral hydrate and transcardially perfused to process the brains for histological verification of the kindling site and the recording site.

Figure 2.

Histologically verified kindling sites and single unit recording sites. A, Location of kindling electrodes in the right BLA or the piriform cortex (PC) in 30 rats, drawn on cutouts of coronal sections of the rat brain according to Paxinos and Watson (2007). Filled circles, Animals used for determination of the effect of VPA on kindled ADTs; rhombs, animals used for determination of the effect of VPA on kindled ADTs and for recording of the effect of VPA on SNr neurons (animal names are given within the rhombs to indicate animals that showed a poor or good response to VPA during electrophysiological recordings; compare with Fig. 4); half-filled circle, animal used for determination of the effect of VPA on kindled ADTs and for control recordings with injection of saline during electrophysiological recording; open circles, animals used for control recordings with injection of saline during electrophysiological recording. The distance to bregma in millimeters is given in the left corner of each section. B, Single-unit recording sites in the SNr in animals tested for the efficacy of VPA on SNr firing rates and patterns in kindled rats (rhombs; n = 7; animal names are given within the rhombs to indicate animals that showed a poor or good response to VPA during electrophysiological recordings; compare with Fig. 4) and saline in control rats (half-filled circle and open circles; together n = 4). The distance to bregma in millimeters is given in the right corner of each cutout of coronal sections of the rat brain according to Paxinos and Watson (2007).

Figure 3.

Mean individual increase of afterdischarge threshold after injection of valproate. Individual control thresholds as well as individual responses to VPA were reproducible in each rat from trial to trial and not just a chance event, indicating that the individual response to VPA was a characteristic feature of the animal. A, Control thresholds and subsequent ADTs after intraperitoneal injection of 200 mg/kg VPA in a poor responder. B, Control thresholds and subsequent ADTs after VPA injection in a good responder. The animal names are given in A and B for comparison with C. C, Individual means of at least four drug values were compared with the individual mean of at least four predrug (control) values for each animal. When all animals were tested (n = 27), relative ADTs determined 30 min after VPA injection were significantly higher compared with predrug values (mean increase ± SEM; 234 ± 31.0% of predrug values; calculated by averaging all individual percentage ADT increases; paired t test, p < 0.0001). Mean group increase of ADTs (234%) is indicated in the figure by the horizontal dotted line. Marked interindividual differences in the relative ADT increase in response to VPA were observed (range, 25.0–640.7% above predrug values), revealing good (above average increase of 234% after VPA) and poor (<234%) responders to VPA. After testing of VPA response in kindled rats, in vivo single-unit recordings were performed and evaluated in seven of these animals (white bars) within 14 d after the last VPA injection and 24 h after a kindled secondarily generalized seizure.

Figure 4.

Individual effect of valproate on spontaneous discharge rates. For evaluation of electrophysiological data, mean 1 min bins were calculated from firing rates relative to the mean individual predrug value (5 min of recording time before drug injection), which was set at 100%. Systemic application of VPA significantly reduced discharge rates of SNr neurons when all animals were considered (n = 7). Importantly, marked individual differences in response to VPA became evident. A, Comparison of maximum reduction of SNr discharge rates within 20 min after injection of VPA reveals marked interindividual differences between animals. The filled rhombs below bars indicate the two animals that were chosen to illustrate representative examples of VPA-induced effects on SNr firing in a poor (B) and a good (C) responder. Relative discharge rates are shown during 5 min before and 20 min after intravenous injection of 100 mg/kg VPA intravenous (dotted line). Representative recordings of the two sample neurons are also shown in B and C. Superimposed spikes (n = 5) of SNr neurons showing biphasic positive-negative waveforms as well as trains of discriminated spikes drawn as raster plots are shown for recordings before (predrug) and after (at the time of maximum reduction of firing rate) injection of VPA, respectively. Superimposed spikes (calibration: 0.5 ms, 25 μV) express biphasic positive-negative waveforms in predrug as well as in postdrug recordings. Raster plots (each with 5 s duration) reflect burst firing pattern independent of the presence of VPA in most of the recordings (refer to Table 4). The mean discharge rates of the shown neurons before or after injection of VPA are given above the trains.

Figure 5.

Correlation between the effect of valproate on afterdischarge thresholds in kindled rats and efficacy of valproate on SNr firing rate and pattern. A, The extent of reduction of discharge rates of SNr neurons by systemic application of VPA was significantly positively correlated with the extent of the anticonvulsant efficacy of VPA in kindled rats (n = 7) (i.e., ADT increase) (Pearson's correlation; r = 0.8654; p = 0.0119). In other words, the more marked VPA raised ADTs, the more marked was the reduction in SNr firing rate by VPA. B, High skewness values reflect irregular firing while a skewness of zero reflects regular firing. The skewness of the ISIH of SNr firing after systemic application of VPA was significantly negatively correlated with the extent of the anticonvulsant efficacy of VPA in kindled rats (i.e., ADT increase) (n = 7; Spearman's correlation; r = −0.8214; p = 0.0341). In other words, the more marked VPA raised ADTs, the more marked was VPA able to induce a regular firing pattern (mirrored as lowered skewness).