Effects of zonisamide on c-Fos expression under conditions of tacrine-induced tremulous jaw movements in rats: A potential mechanism underlying its anti-parkinsonian tremor effect

1. Introduction 

There is growing interest in the therapeutic potential of zonisamide, an antiepileptic drug, on motor symptoms of Parkinson's disease (PD) [1], [2], [3]. A recent, randomized, placebo-controlled study revealed that zonisamide could effectively reduce the duration of “off” time in PD patients treated with l-DOPA [4]. In addition, zonisamide may be effective for the treatment of tremor disorders that include not only l-DOPA-resistant parkinsonian tremor [5], but also essential tremors [6], [7]. Thus, zonisamide is thought to have clinical potential as an anti-tremor drug [3]. However, the pharmacological mechanisms of zonisamide, particularly with respect to its beneficial effects on PD, are unknown. Experimentally, we have previously reported that zonisamide effectively suppressed tacrine-induced tremulous jaw movements (TJMs) [8], a proposed pharmacological model of parkinsonian tremor [9], [10], [11], [12], [13]. Symptomatologically, there is an unavoidable gap between tacrine-induced jaw movements in rats and parkinsonian tremor. However, the application of rodent models to the analysis of the mechanism of tremor is possible if appropriate pathophysiological translation is undertaken [13]. Thus, in the present study, we examined the potential mechanisms of this inhibitory effect of zonisamide on tacrine-induced TJMs by determining the expression of c-Fos, a transcription factor and a protein product of an immediate early gene, c-fos, in the various tremor-related anatomical sites of the brain [13], [14]. Since c-Fos expression markedly increases when a neuron is activated by external or internal stimuli [15], the analysis of c-Fos expression has been used as a tool for mapping activated neurons at a cellular resolution [16], [17]. Determining potential mechanisms involved in the inhibitory effect of zonisamide on the tacrine-induced c-Fos expression will help to further understand the pharmacological mechanisms underlying the anti-parkinsonian tremor effect of zonisamide.

3. Results 

3.3. Cortex 

There was a significant and extensive increase in c-Fos expression in the cerebral cortex following tacrine administration (Fig. 1). The increase in c-Fos expression was present in all the cortical areas including the frontal, parietal, temporal, and occipital cortices. Zonisamide significantly suppressed the tacrine-induced increase in c-Fos expression in the cortex in a dose-dependent manner (Fig. 1).

View Large Image Download to PowerPoint

Fig. 1 (A) c-Fos immunostaining of the frontal cortex in rats treated with vehicle or zonisamide (ZNS) following administration of tacrine. Nissl staining is also demonstrated on the right. Each Roman numeral denotes the corresponding cortical layer. Scale bar: 200μm. (B) Corresponding optical densities of the cortex are shown. Tacrine-induced increase of c-Fos immunoreactivity was significantly suppressed by zonisamide (50mg/kg). Asterisk indicates P<0.05.

3.4. Basal ganglia and diencephalon 

There was a significant increase in c-Fos expression in the dorsal and medial striatum but not in the ventrolateral striatum following tacrine administration (Fig. 2). In the dorsal striatum, administration of zonisamide prior to tacrine treatment significantly decreased the number of c-Fos-positive nuclei, although there was no difference between the 5 and 50mg/kg zonisamide doses. In contrast, in the ventrolateral part of the striatum, which is regarded as a primary site of action of tacrine-induced TJMs, there was no significant alteration in the number of c-Fos-positive nuclei following pretreatment with zonisamide (Fig. 2).

View Large Image Download to PowerPoint

Fig. 2 (A) Schematic of the striatum. Areas surrounded by three squares indicate dorsal, medial, and ventrolateral parts of the striatum, respectively. (B) Number of c-Fos-positive nuclei in the dorsal, medial, and ventrolateral parts of the striatum in rats treated with vehicle or zonisamide (ZNS) following administration of tacrine. Data are expressed as means±SE (N=4–5). Following administration of tacrine, in the medial and dorsal striatum, the number of c-Fos-positive nuclei increased significantly compared to control (saline alone; indicated as #) (P<0.05). Only in the dorsal striatum was the tacrine-induced increase of c-Fos-positive nuclei significantly suppressed by ZNS. In the ventrolateral and medial parts of the striatum, there was no significant effect of zonisamide on the number of c-Fos-positive nuclei. Asterisk indicates P<0.05.

In the nucleus accumbens, c-Fos expression was markedly induced in the shell, but not in the core, following tacrine administration. This increase was suppressed in a dose-dependent manner by zonisamide pretreatment (Fig. 3). In the other basal ganglia structures such as the globus pallidus, entopeduncular nucleus, subthalamic nucleus, and the substantia nigra, c-Fos expression levels following tacrine administration were generally too low to be analyzed (data not shown). In the parasubthalamic nucleus, located just medially to the subthalamic nucleus, there was a significant increase in c-Fos expression following administration of tacrine, which was dose-dependently suppressed by zonisamide pretreatment (Fig. 4). In the ventrolateral nucleus of thalamus, c-Fos expression levels induced by tacrine were generally low, and insufficient to evaluate the suppressive effects of zonisamide pretreatment.

View Large Image Download to PowerPoint

Fig. 3 (A) c-Fos immunostaining of the nucleus accumbens in rats treated with vehicle or zonisamide (ZNS) following administration of tacrine. Scale bar: 100μm. (B) Number of c-Fos-positive nuclei in the nucleus accumbens in rats treated with vehicle or ZNS following administration of tacrine. Number of c-Fos immunoreactive nuclei was significantly increased following the administration of tacrine. There was a significant suppressive effect of ZNS on the tacrine-induced increase of c-Fos in a dose-dependent manner. Asterisk indicates P<0.05.

View Large Image Download to PowerPoint

Fig. 4 (A) Nissl staining of the subthalamic area in rats. CP: cerebral peduncle; OT: optic tract; PLH: posterior lateral hypothalamus; PSTN: parasubthalamic nucleus; STN: subthalamic nucleus; VM: ventromedial thalamus. (B) c-Fos immunostaining of the parasubthalamic nucleus in rats treated with vehicle or zonisamide (ZNS) following administration of tacrine. Scale bar: 100μm. (C) Number of c-Fos-positive nuclei in the parasubthalamic nucleus in rats treated with vehicle or zonisamide (ZNS) following administration of tacrine. Number of c-Fos immunoreactive nuclei significantly increased following administration of tacrine. Administration of ZNS significantly suppressed the tacrine-induced increase of c-Fos. There was no significant difference in the suppressive effect of ZNS between 5 and 50mg/kg. Asterisk indicates P<0.05.

4. Discussion 

The present study demonstrated that administration of tacrine induced abundant c-Fos expression in various anatomical sites in the brain. Further, zonisamide pretreatment could largely suppress these increases, particularly in the cortex and dorsal striatum. To date, there has been no prior report examining the effects of an acetylcholinesterase inhibitor on c-Fos expression. However, c-Fos expression has been shown to be highly inducible in many forebrain structures following cholinergic receptor stimulation by pilocarpine, a muscarinic agonist, including the cerebral cortex, nucleus accumbens, amygdala, neocortex, and the hypothalamus [18], [19]. Similarly, it is likely that administration of tacrine could widely enhance cholinergic neurotransmission in the brain, thereby resulting in massive induction of c-Fos expression.

In the present study, the tacrine-induced c-Fos expression, particularly that in tremor-related structures, was analyzed to determine the anti-TJM effect of zonisamide. Unfortunately, the primary tremor-generator of TJMs remains to be fully determined, although it has been proposed that M4 receptors in the ventrolateral striatum play a primary role in the generation of TJMs induced by cholinomimetics in rats [12], [20]. However, tacrine-induced c-Fos expression levels were not significantly altered by zonisamide pretreatment in the ventrolateral striatum, suggesting that zonisamide may not directly influence neuronal activities in the primary sites responsible for generating TJMs. It should be noted that neuronal activation is not always associated with an increase of c-Fos expression [21]. Thus, another possibility, that the physiological mechanisms underlying c-Fos expression in the ventrolateral striatum may differ from those in the dorsal or medial striatum, should not be excluded. Indeed, c-Fos expression levels were not increased following administration of tacrine. On the other hand, in the dorsal striatum as well as in the nucleus accumbens, tacrine-induced c-Fos expression levels significantly decreased following administration of zonisamide. Since zonisamide has been shown to have no direct action on cholinergic receptors [2], [3], the reduction in c-Fos expression levels in these structures by zonisamide may be indirectly induced. Currently, the pathophysiological significance of the tacrine-induced c-Fos expression is unclear; however, when considering the anti-tremor as well anti-parkinsonian effects of zonisamide, it is of interest that zonisamide can alter c-Fos expression in the dorsal striatum or the nucleus accumbens.

It is not surprising that zonisamide significantly suppressed the tacrine-induced expression of c-Fos in the cortex, since cholinergic agents produce experimental convulsions [18], [19] and zonisamide is an antiepileptic drug. Although it remains uncertain whether the reduction of cortical c-Fos expression by zonisamide is directly related to its anti-TJM mechanism, cortical suppression is likely to be related to the sedative actions of zonisamide, possibly contributing to the tremor reduction.

The general absence of an increase in c-Fos expression in the trigeminal motor nucleus following administration of tacrine was unexpected, unless it was determined that repetitive muscle activities in temporal muscles differentially play a prominent role in the generation of TJMs [20]. Zonisamide suppressed the tacrine-induced increase of c-Fos expressions in the parasubthalamic nucleus; anatomically this nucleus has massive projections to trigeminal motor nucleus [22], [23]. Thus, it is interesting to note that zonisamide could affect c-Fos expression in those anatomical sites, such as the nucleus accumbens [24] or parasubthalamic nucleus [22], that are related to ingestive behaviors when considering the anti-obesity action of zonisamide.

5. Conclusion 

Zonisamide did not affect the tacrine-induced c-Fos expression in the ventrolateral striatum, the primary tremor-generating site responsible for tacrine-induced tremors [10], [12]. However, zonisamide suppressed the tacrine-induced c-Fos expression in the dorsal striatum and the cortex, both of which are architectures in the cortico-basal ganglia-thalamocortical circuits. Speculatively, zonisamide may contribute to down-regulation of the transmission efficiency or suppression of the tremor-related repetitive firings in such cortico-basal ganglia circuits, resulting in lowered tremor amplitude. There are methodological limitations in this type of experimental study that uses analysis of c-Fos expression. Thus care should be taken when translating these observations to the pharmacological mechanisms underlying clinical effects of zonisamide. However, we hope that these data will further contribute to our understanding of potential pharmacological actions underlying the anti-parkinsonian effects, particularly those on tremor, of zonisamide.