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Summary: Purpose: Animal studies and anecdotal human case reports have indicated that the subthalamic nucleus (STN) may be a site of anticonvulsant action.
Methods: We tested the hypothesis that continuous electrical stimulation of the STN inhibits seizures acutely. We determined the effects of three stimulation frequencies, 130 Hz, 260 Hz, and 800 Hz, on generalized clonic and tonic–clonic flurothyl seizures. Adult male rats were implanted with concentric bipolar stimulating electrodes in the STN bilaterally. After recovery, rats underwent flurothyl seizures to compare the effects of each stimulation frequency on seizure threshold. Rats were tested 4 times, twice in the stimulated condition, and twice in the unstimulated condition. The order of trials was random, except that stimulation trials alternated with control trials. Flurothyl seizure thresholds under each stimulation condition were compared with control values from the same animal.
Results: Bilateral stimulation of the STN at 130 Hz produced a significant increase in the seizure threshold for clonic flurothyl seizures, whereas stimulation at 260 Hz did not appear to have any effect on seizures. STN stimulation at 800 Hz significantly lowered seizure threshold for tonic–clonic seizures.
Conclusions: We conclude that electrical stimulation of the STN can be anticonvulsant, but the effects appear to depend on the stimulation frequency and the type of seizure.
Generalized convulsive seizures are one of the most debilitating and life-threatening consequences of human epilepsy, particularly in cases of medically intractable epilepsy in which surgical therapy cannot be considered. Indeed, as outlined in the NINDS-CURE benchmarks for epilepsy research (http://www.ninds.nih.gov/about_ninds/epilepsybenchmarks.htm), there is an urgent need for novel therapies that can prevent seizures without causing unacceptable side effects, as frequently develop in patients taking multiple antiepileptic medications (AEDs). The application of focal deep brain stimulation as a potential treatment for convulsive and nonconvulsive seizures has recently gained attention as a result of two factors. First, an emerging understanding of endogenous anticonvulsant subcortical networks has identifed potential targets where focal intervetion may result in widespread changes in seizure susceptibility. Second, the availability of devices for deep brain electrical stimulation offer a means of selectively altering activity in anticonvulsant circuits without lesioning large numbers of neurons.
Over the past two decades, investigators have recognized the importance of subcortical and extrapyramidal motor pathways in the control of seizures. The subthalamic nucleus (STN), a small nucleus receiving major input from cortical and pallidal afferents and projecting to the substantia nigra pars reticulata (SNR) (1–4), has shown anticonvulsant activity in several animal seizure models, including flurothyl convulsions, kindled seizures, and genetic absence epilepsy (5–8). Focal injection of muscimol, a γ-aminobutyric acid subtype A (GABA-A)-receptor agonist, into the STN reduced susceptibility to flurothyl-induced generalized convulsions (5). Bilateral injections of muscimol into the STN decreased by two thirds the number of rats with generalized convulsions produced by amygdala kindling (6), although focal seizures and electrographic discharges were not suppressed. Muscimol injections into the STN of GAERS rats (which experience frequent spontaneous absence-like seizures with synchronous bilateral spike–wave discharges and behavioral arrest) produced a dose-dependent inhibition of electrographic seizures (7). Bilateral excitotoxic lesions in the STN produced a similar but less complete suppression of spontaneous absence seizures (8). Electrical stimulation of the STN bilaterally with 130-Hz pulse trains completely suppressed ongoing absence seizures at the onset of stimulation, but the effect was transient and dissipated after 2 min of continuous stimulation (8). A report that electrical stimulation of the STN is effective against kainic acid–induced convulsions recently appeared in preliminary form (9). Finally, preliminary reports describe the results of STN stimulation in human epilepsy patients. Of a total of eight patients, seven reportedly experienced a 30–100% reduction in seizure frequency (10–12).
Despite widespread interest in STN electrical stimulation as a potential new anticonvulsant treatment in humans, few data are available regarding the effects of electrical STN stimulation against convulsive seizure models in animals, and no data have been presented evaluating the effect of different stimulation frequencies on seizure threshold. We hypothesized that electrical stimulation of the STN would decrease susceptibility to generalized convulsions produced by inhalation of flurothyl, a proconvulsant gas, in adult male rats. We tested the effect of bilateral STN stimulation at three stimulation frequencies, 130 Hz, 260 Hz, and 800 Hz by comparing how rapidly seizures developed during exposure to an increasing concentration of flurothyl at each stimulation frequency. Our results indicate that STN stimulation at 130 Hz is anticonvulsant against clonic, but not tonic–clonic seizures, and that stimulation at 800 Hz is proconvulsant for tonic–clonic seizures. Stimulation at 260 Hz did not change seizure susceptibility, in contrast to anticonvulsant and proconvulsant effects seen at 130 and 800 Hz, respectively.
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Electrical stimulation of the STN did not produce any lesion in the STN visible under light microscopy (Fig. 1B, top). It should be noted, however, that some evidence of mechanical electrode trauma from implantation and repeated convulsions was present in most animals, and may have masked evidence of subtle damage caused by electrical stimulation. The average stimulation currents per STN electrode that were used at each stimulation frequency are summarized in Table 1. The difference in the maximal tolerated stimulation current was significantly higher at 800 Hz than at either 130 or 260 Hz, indicating that greater stimulation intensities was better tolerated (i.e., did not produce motor effects) at the highest stimulation frequency. Comparison of the control exposure trials showed that the flurothyl threshold declined significantly for clonic seizures (from 177 ± 13 μl to 143 ± 9 μl; p < 0.05; n = 15), and nearly significantly for tonic seizures (281 ± 19 μl to 238 ± 23 μl; p = 0.062; n = 15), consistent with a kindling effect from repeated seizures after flurothyl exposure.
Table 1. Maximum tolerated stimulation intensity at each of the stimulation frequencies testeda
|Stimulation frequency||Number of trials in each condition (N)||Stimulus intensity (mean ± SEM)||Range of stimulation intensity|
|130 Hz||17||342 ± 20 μA||200–500 μA|
|260 Hz||5||320 ± 25 μA||250–400 μA|
|800 Hz||12||418 ± 22 μAa||300–500 μA|
The effect of STN electrical stimulation depends on stimulation frequency
Electrical stimulation of the STN at 130 Hz increased the clonic seizure threshold by 28% (n = 17; p < 0.05), indicating an anticonvulsant effect (Fig. 2A). Stimulation at 260 Hz did not change the threshold for clonic convulsions (Fig. 2B). Stimulation at 800 Hz slightly lowered the clonic seizure theshold, but the change was not significant (Fig. 2C). In contrast to the effect on clonic seizures, electrical stimulation of the STN at 130 Hz (n = 17) was associated with only a modest increase in the threshold for tonic–clonic seizures that was not significant (Fig. 3A). STN stimulation at 260 Hz also did not affect the tonic–clonic seizure threshold (Fig. 3B). Conversely, stimulation at 800 Hz significantly lowered threshold for tonic–clonic seizures by 16% (n = 12; p < 0.05), a proconvulsant effect (Fig. 3C). Comparison of the difference in seizure threshold for each clonic and tonic–clonic seizures shows that increasing stimulation frequency results in a diminishing anticonvulsant effect that becomes a proconvulsant effect at the highest stimulation frequency tested, 800 Hz (Fig. 4).
Figure 2. Frequency-dependent effects of stimulation on clonic seizure threshold. Stimulation at the lowest frequency tested, 130 Hz, significantly raised the seizure threshold for clonic flurothyl-induced seizures, whereas stimulation at higher frequencies did not alter seizure threshold. Statistical comparisons were performed between the stimulated and unstimulated conditions in the same animal (*p < 0.05).
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Figure 3. Frequency-dependent effects of stimulation on tonic–clonic seizure threshold. Stimulation at the lowest two frequencies tested, 130 and 260 Hz, did not significantly change the seizure threshold for tonic–clonic flurothyl-induced seizures, whereas stimulation at 800 Hz did lower seizure threshold. Statistical comparisons were performed between the stimulated and unstimulated conditions in the same animal (*p < 0.05).
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Figure 4. Summary of difference in seizure threshold as a function of stimulation frequency. For both clonic and tonic seizures, the frequency of stimulation determines whether the effect on seizures will be anti- or proconvulsant. The greatest anticonvulsant effects are seen on clonic seizures with 130-Hz stimulation.
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The anticonvulsant effect of 130 Hz electrical stimulation was specific to STN stimulation, rather than arising from the lesion produced by electrode placement. The threshold for clonic (155 ± 10 μl [mean ± sterr]; n = 17) and tonic–clonic seizures (267 ± 11 μl; n = 17) in animals with implanted STN electrodes did not differ significantly from the clonic (154 ± 11 μl; n = 10) and tonic–clonic (263 ± 11 μl; n = 10) seizure thresholds obtained in naïve unimplanted animals (n = 10). In animals with both electrodes outside the STN (n = 4), clonic seizure threshold during electrical stimulation at 130 Hz (168 ± 46 μl) was similar to the unstimulated state (154 ± 29 μl). “Misses” were all in the region of the posterior STN either mediodorsal or ventrolateral to the STN (Fig. 1A).
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The results indicate that electrical stimulation of the STN can be an effective way of increasing seizure threshold; however, the effects depend on the frequency of stimulation. In adult male rats, 130-Hz electrical stimulation of the STN was effective against flurothyl-induced generalized clonic convulsive seizures. Stimulation at 260 Hz did not produce any anticonvulsant benefit, whereas stimulation at 800 Hz actually increased susceptibility to tonic–clonic flurothyl-induced seizures. The anticonvulsant action of stimulation at 130 Hz appears to result from stimulation, rather than from lesional effects after electrode insertion. Moreover, analysis of “misses” shows that the effect of stimulation appears to be specific to the STN and its immediate vecinity (15).
The stimulation frequencies tested were chosen for several reasons. There is evidence that 130-Hz stimulation in the STN is anticonvulsant in a rat model of absence epilepsy (7) and possibility in kainic acid seizures (9), and that STN stimulation reduces action-potential activity within in the SNR (16), part of an intrinsic subcortical anticonvulsant circuit. A stimulation rate of 800 Hz was chosen to exceed the maximum in vitro firing rate (300–500 Hz) of STN neurons (17,18), to increase the likelihood of stimulation-induced depolarization blockade. The 800-Hz stimulation also was tested for anticonvulsant efficacy because STN stimulation applied to a primate model of tremor determined that the greatest benefits and fewest undesired effects resulted from the highest stimulation frequencies of ≤1,000 Hz (19). The stimulation rate of 260 Hz was selected to be intermediate between the the maximal firing rate of STN neurons and 130-Hz stimulation. Frequencies <100 Hz were not tested as in vitro data indicates that stimulation in this frequency range excited (rather than suppressed) neuronal activity in the substantia nigra pars reticulata (16).
In this experiment, we observed that the threshold for undesired motor effects was increased with higher stimulation frequencies (Table 1). The increase in the threshold for undesired effects, however, did not translate into increased efficacy against seizures, because the effect of stimulation changed from anti- to proconvulsant as frequency increased. An unexpected observation was that the seizure thresholds for clonic and tonic–clonic seizures in unstimulated animals (the “control” condition) varied between the groups tested at each stimulation frequency (Figs. 2–4).
The frequency-specific effects of STN stimulation may be related to the structures involved in the control of clonic and tonic seizures. The substantia nigra pars reticulata (SNR) has been implicated in the control of clonic seizures (20), whereas brainstem structures may be involved in the control of tonic seizures (21). The 130-Hz stimulation of the STN decreases the firing rate of SNR neurons and increases GABA levels in the SNR (16–22). Indeed, application of either muscimol or electrical stimulation directly in the anterior SNR is anticonvulsant, and the anterior SNR may mediate the anticonvulsant effects of STN manipulations (23,24). The mechanisms through which 130-Hz electrical stimulation of the STN may decrease the spontaneous firing rate in the SNR includes (a) depletion of glutamate in the presynaptic terminal of STN projections, (b) retrograde activation of pallidal neurons resulting in increased inhibition from the pallidum on the substantia nigra pars reticulata (SNR) and the entopeduncular nucleus, and (c) firing blockade at the STN neuron (16,25,26).
Our data indicate that firing blockade is not the anticonvulsant mechanism of STN stimulation. Stimulation frequencies of 260 and 800 Hz, which should produce the most robust blockade of STN firing, did not produce an anticonvulsant effect. Stimulation at 130 Hz, on the other hand, a frequency within the dynamic range of STN neurons and with intermediate effects on STN firing in vitro, produced the greatest anticonvulsant benefit. Our findings and published reports from others (18) support the conclusion that STN stimulation in vivo does not functionally inactivate the STN.
Because stimulation likely affects not only the somata of STN neurons, but also presynaptic terminals in the STN and fibers of passage through the STN, the net effect of STN stimulation most likely reflects the frequency-dependent contribution from multiple elements (15). Instead, stimulation of the STN may provoke neuronal firing, orthodromically fire efferent axonal projections and fibers of passage, and antidromically propagate activity to the pallidum. As the STN and basal ganglia form a network with rhythmic and pacemaker properties (27), the net effect of STN stimulation–induced changes in excitation and inhibition in the extrapyramidal system may be to alter the rhythmic properties of these circuits (28). In vitro recordings show that STN neurons fire rhythmically, and that the pattern of STN firing changes significantly in response to changes in GABAergic input arriving in the STN (29–31). Changes in circuit resonance produced by focal stimulation at the STN could reduce the susceptibility of extrapyramidal circuit elements to entrainment by seizures, and thus could retard the evolution and generalization seizures.
Our data also indicate that changing stimulation frequency not only affects the anti- or proconvulsant effects of stimulation, but that stimulation at different frequencies may selectively affect different types of seizures. There is evidence that generalized clonic seizures originate in forebrain structures, whereas generalized tonic seizures originate in the brainstem (32,33). The efficacy of continuous STN stimlation against clonic seizures indicates that STN stimulation at 130 Hz can disrupt forebrain networks mediating clonic convulsions but appears less effective at disrupting brainstem seizure networks responsible for tonic–clonic convulsions. The finding that STN stimulation at 800 Hz produces proconvulsant effects against tonic–clonic seizures but not clonic seizures raises the possibility that STN stimulation selectively activates or suppresses specific seizure-mediating networks in the forebrain or in the brainstem, depending on the frequency of stimulation.
The frequency-dependent effects of electrical stimulation on the STN are complex, and it seems unlikely that the effects of stimulation seizures can be understood solely in terms of the direct effect of stimulation on STN neurons. Additional studies examining the effects of stimulation in vivo on STN firing and on the activity in other extrapyramidal nuclei will add to the understanding of the mechanism of anticonvulsant action of STN stimulation and may give additional insight into the most effective parameters for anticonvulsant stimulation.
Acknowledgment: This study was supported by NIH grants NS41340 (F.A.L.), NS41366 (L.V.), and NS20253 (S.L.M.), and a CURE Foundation grant (L.V.). Dr. Moshé also is the recipient of a Martin A. and Emily L. Fisher fellowship in Neurology and Pediatrics. The authors are grateful to Jana Velíšková, M.D. Ph.D., for assistance and advice regarding preparation and testing of animals for this study.