Prolonged attenuation of amygdala-kindled seizure measures in rats by convection-enhanced delivery of the N-type calcium channel antagonists omega-conotoxin GVIA and omega-conotoxin MVIIA

Abstract

Convection-enhanced delivery (CED) permits the homogeneous distribution of therapeutic agents throughout localized regions of the brain parenchyma without causing tissue damage as occurs with bolus injection. Here, we examined whether CED infusion of the N-type calcium channel antagonists omega-conotoxin GVIA (omega-CTX-G) and omega-conotoxin MVIIA (omega-CTX-M) can attenuate kindling measures in fully amygdala-kindled rats. Rats were implanted with a combination infusion cannula-stimulating electrode assembly into the right basolateral amygdala. Fully kindled animals received infusions of vehicle, omega-CTX-G (0.005, 0.05, and 0.5 nmol), omega-CTX-M (0.05, 0.15, and 0.5 nmol), proteolytically inactivated omega-CTX-M (0.5 nmol), or carbamazepine (500 nmol) into the stimulation site. CED of omega-CTX-G and omega-CTX-M over a 20-min period resulted in a dose-dependent increase in the afterdischarge threshold and a decrease in the afterdischarge duration and behavioral seizure score and duration during a period of 20 min to 1 week after the infusion, indicating an inhibitory effect on the triggering and expression of kindled seizures. The protective effects of omega-conotoxins reached a maximum at 48 h postinfusion, and then they gradually resolved over the next 5 days. In contrast, carbamazepine was active at 20 min but not at 24 h after the infusion, whereas CED of vehicle or inactivated omega-CTX-M had no effect. Except for transient tremor in some rats receiving the highest toxin doses, no adverse effects were observed. These results indicate that local CED of high-molecular-weight presynaptic N-type calcium channel blockers can produce long-lasting inhibition of brain excitability and that they may provide prolonged seizure protection in focal seizure disorders.

Fig. 1

Components of the cannula-bipolar stimulating electrode assembly system used for kindling stimulation and recording, and for CED delivery of solutions into the basolateral amygdala. The combination guide cannula and bipolar stimulating assembly is shown to the right. The guide cannula passes through the threaded left-most pedestal; stimulating electrodes are affixed diametrically opposed to the exterior of the guide cannula below the pedestal. The two wire electrodes pass to the right-most pedestal containing pin connectors to allow electrical connection to the kindling stimulator. The dummy cannula wire-cap assembly is shown to the left. The plastic cap is internally threaded to match the treads of the pedestal of the cannula-electrode assembly. The CED cannula is shown in the center. The plastic stop maintains the tip of the CED cannula 0.5 mm above the tips of the stimulating electrode wires.

Fig. 2

Mean relative AD threshold, AD duration, seizure stage, and duration of behavioral seizures values at intervals from 20 min to 1 week after CED infusion of ω-CTX-G (0.005, 0.05, and 0.5 nmol/infusion) or vehicle. The protocol for AD threshold determination was as described under Materials and Methods. Data are presented as a percentage of change from baseline values calculated as an average of the values obtained in response to at least two stimulation sessions before the infusion. Each data point represents a mean ± S.E.M. of values from 10 rats. *, significantly different from vehicle value at that time point (Tukey’s test) following the detection of a statistically significant main effect by a two-way repeated measures ANOVA. Results of ANOVA and post hoc analysis are presented in Table 1.

Fig. 3

Relative AD threshold, AD duration, seizure stage, and duration of behavioral seizures at intervals from 20 min to 1 week after CED infusion of ω-CTX-M (0.05, 0.15, and 0.5 nmol/infusion) or vehicle. Data are presented as a percentage of change from baseline values calculated as an average of the values obtained in response to at least two stimulation sessions before the infusion. Each data point represents a mean ± S.E.M. of values from 10 to 12 rats. *, significantly different from vehicle value at that time point (Dunnett’s test) following the detection of a statistically significant main effect by a one-way repeated measures ANOVA. Results of ANOVA and post hoc analysis are presented in Table 1.

Fig. 4

Area-under-the-curve analysis of the data from Figs. 2 and 3. The area under the curve of the percentage of change values for the period 20 min to 1 week following infusion for every time course data set in each animal was determined using the trapezoidal method. The data points represent the mean ± S.E.M. of the values (in percent-days) for all animals receiving a specific toxin and dose. Open squares (□) and circles (○) indicate control values for the group of animals tested with ω-CTX-G (■) and ω-CTX-M (●), respectively. *, significantly different from corresponding vehicle value at that dose (Tukey’s test) following the detection of a statistically significant main effect by a two-way repeated measures ANOVA.

Fig. 5

Baseline values for AD threshold, AD duration, seizure stage, and duration of behavioral seizures before each of four sequential CED infusions (test sessions I–IV) of ω-CTX-G and ω-CTX-M at various doses and vehicle in the experiments presented in Figs. 2 and 3. The values for test session I represent the kindling measures for 22 animals before any infusion (true baseline). The treatments in later test session (II–IV) were presented in random order. The baseline values were calculated by averaging seizure measures in response to at least three electrical stimulations before an infusion. Each bar represents the mean ± S.E.M. There were no significant differences between the mean values of any of the seizure measures among the four test sessions (two-way repeated measures ANOVA), indicating that the baseline before CED infusion was stable during repeated CED infusion and testing.

Fig. 6

CED causes minimal damage in the amygdala and surrounding brain structures in contrast to bolus infusion, which causes cavitation. A, brain obtained from an animal that had been fully kindled and that had received three CED infusions of ω-CTX-G and an additional vehicle infusion. The animal had experienced multiple stimulation-induced kindled seizures. Top, cresyl violet-stained coronal section through the track of the cannula-electrode assembly and right amygdala. Arrow indicates location of track. There is minimal tissue disruption along the track and no damage apparent in the amygdala. Bottom, the adjacent silver-stained section shows some staining in the right striatum, thalamus, and internal capsule, indicating neuronal disintegration. There is no damage apparent in the amygdala. B, brain obtained from a rat infused with 0.05 nmol of ω-CTX-G at a rate of 2.5 μl/min (10-fold greater than for CED). Tracks of the stimulating electrode wires are seen to extend into the right basolateral amygdala and a cavity is present above the nucleus. A second cavity is present in the alveus due to damage from the guide cannula and tracking of the infused solution up the cannula. Cavitation was observed in seven of eight rats treated in a similar manner.

Fig. 7

Assessment of the influence of repeated kindling stimulation on recovery from the toxin effects. AD threshold, AD duration, seizure stage, and duration of behavioral seizures before the infusion (baseline control values) and 7 and 8 days after CED infusion of vehicle, ω-CTX-G (0.05 nmol), and ω-CTX-M (0.5 nmol). After the infusions, the animals were not electrically stimulated until 7 days after the infusion. Bars represent the mean ± S.E.M. of the values from six rats. Baseline values for each animal are averages from at least three electrical stimulations before the infusion.

Fig. 8

Lack of effect of CED infusion of proteolyzed ω-CTX-M (0.5 nmol) on kindling measures. Mean relative AD, AD duration, seizure stage, and duration of behavioral seizures values at intervals from 20 min to 72 h after CED infusion of proteolyzed ω-CTX-M (eight rats) or vehicle (eight rats). Each data point represents a mean ± S.E.M. There were no significant differences between the values for the proteolyzed toxin and vehicle by a two-way repeated measures ANOVA.

Fig. 9

Mean relative AD threshold, AD duration, seizure stage, and duration of behavioral seizures values at intervals from 20 min to 1 week after CED infusion of 500 nmol of carbamazepine (eight rats) or vehicle (six rats). *, significantly different from vehicle value (Tukey’s test) following the detection of a statistically significant main effect by a two-way repeated measures ANOVA.

Fig. 10

Effects of CED infusion of ω-CTX-G (0.05 nmol), ω-CTX-M (0.5 nmol), or vehicle on locomotor activity at intervals from 20 min to 96 h after the infusion presented as the percentage of change from baseline (right). Each data point represents the mean ± S.E.M. of experiments with six rats. The dotted lines represent no change from the baseline values. The 18 animals were habituated to the locomotor-activity chamber in five daily sessions before the infusion (left). Baseline values were the averages of the activity counts during the last two habituation sessions.