BACKGROUND
parkinson’s disease  is a progressive, age-related, neurodegenerative disorder characterized by bradykinesia, resting tremor and gait disturbance.
the major pathological basis of pd is the death of dopaminergic neurons in the substantia nigra pars compacta  and the degeneration of their nerve terminals in striatum  <cit> .
it has been proposed that clinical signs of pd appear at the point when dopaminergic neuronal cell loss exceeds a critical threshold:  <dig> – 80% of striatal nerve terminals and  <dig> – 60% of the snc perikaryons  <cit> .
as a pharmaceutical treatment, ʟ- <dig> -dihydroxyphenylalanine , supplying the precursor of dopamine , is the most commonly applied and alleviates major symptom of pd.
for over 40 years, treatment with ʟ-dopa combined with an inhibitor for peripheral aromatic ʟ-amino acid decarboxylase  such as carbidopa had been established as a gold standard for pd treatment  <cit> .
however, long-term treatment with ʟ-dopa is often complicated by the development of adverse effects such as drug-induced dyskinesia  <cit> .
there have been additional anti-parkinsonian drugs, such as dopamine agonists mostly targeted on d <dig> class receptor, but their pharmaceutical potencies are not drastically better than that of ʟ-dopa.
it is essential to develop novel drugs which can support therapeutic effects of ʟ-dopa with delaying expression of its side effects, or even solely effective against pd symptoms.
serotonergic neurons play an important role in modulating extrapyramidal motor disorders such as pd and drug-induced parkinsonism  <cit> .
some studies showed administration of agonists for 1a subtype of serotonin/5-hydroxytriptamine receptor tertraline and tandospirone) significantly improved various types of extrapyramidal symptoms including antipsychotic-induced bradykinesia and catalepsy, and neurotoxin-induced bradykinesia  <cit> .
therefore, the central serotonergic system is thought to be one of a favorable drug target for the treatment of pd.
it is a well-known fact that serotonergic as well as noradrenergic system is a major target of antidepressants such as selective serotonin-reuptake inhibitor  fluoxetine and fluvoxamine.
recently, a novel antidepressant mirtazapine has been developed and is now approved in many countries for clinical treatment of major depression  <cit> .
mirtazapine is categorized into a noradrenergic and specific serotonergic antidepressant , showing multiple pharmacological actions such as inhibiting presynaptic α <dig> noradrenaline receptor  and selectively activating 5-ht1ar  <cit> .
mirtazapine has higher antidepressant effects than placebo or trazodone, which is equivalent to the effect of tricyclic antidepressant  such as clomipramine and amitriptyline  <cit> .
compared to ssri, mirtazapine showed an earlier onset of antidepressant effects  <cit> .
further, the side effects of mirtazapine are reported to be lower than those of ssri or tca  <cit> .
in  <dig>  nakayama et al.
reported mirtazapine increased da release in the medial prefrontal cortex  of rats with activating 5-ht1ar  <cit> .
they reported that  <dig> or 16 mg/kg of mirtazapine increased da release with dose-dependent manner, and an inhibitor of 5-ht1ar way <dig> significantly decreased the mirtazapine-induced increase of da release.
we hypothesized mirtazapine may be effective on pd if the same mechanism as this 5-ht-dependent increase of da release existed in the nigro-striatal dopaminergic system, too.
in fact with related to pd, some studies reported the clinical efficacy of mirtazapine on parkinsonian tremor in human  <cit> .
however, little is known about the therapeutic effect of mirtazapine for motor dysfunctions other than tremor in pd.
therefore, in this study, we examined the effect of mirtazapine in mice treated with the neurotoxin 1-methyl-4-phenyl- <dig> , <dig> -tetrahydropyridine , one of the typical animal models of pd  <cit> .
RESULTS
effect of mirtazapine on motor dysfunctions induced by mptp
we first assessed effect of mirtazapine on motor dysfunctions induced by mptp using two different behavioral paradigms: the beam-walking and rota-rod tests.
we chose two doses of mirtazapine  in this study based on the previous study by nakayama et al.
in  <dig>  <cit>  in which they showed  <dig>   <dig> and 16 mg/kg of mirtazapine produced a dose-dependent increase in extracellular da levels in mpfc of freely moving rats.
thus we examined minimal  and maximal  doses in mice.
in beam-walking test, mptp-treated mice showed a significantly prolonged duration to traverse a distance of 50 cm than that of the vehicle-treated mice   <dig>  =  <dig> , p <  <dig> , anova).
in contrast, mirtazapine significantly improved the mptp-induced prolongation of the traversal duration when it was treated after mptp with both  <dig> and 16 mg/kg doses , although it did not affect when solely applied compared to the vehicle-treated mice .
with rota-rod test, vehicle-treated mice usually remained on the rotating rod for approximately 400–600 sec.
as shown in figure 1b, mptp-treatment significantly decreased the latency to fall from the rod when compared to vehicle-treated mice   <dig>  =  <dig> , p <  <dig> , anova).
on the other hand, administration of 16 mg/kg of mirtazapine after mptp significantly recovered the latency to the level of vehicle-treated group , with no effect when solely applied.
effect of mirtazapine on the striatal da, dopac, hva and turnover rate
using hplc, we quantified the striatal da and its metabolites dopac  and hva  with calculating turnover rate.
as shown in figure  <dig>  administrations of mptp produced marked depletion of da, dopac and hva in striatum   <dig>  =  <dig> , f  <dig>  =  <dig> , f  <dig>  =  <dig> , p <  <dig> , anova), as was reported previously  <cit> .
da turnover, calculated by /da  <cit> , was increased significantly by mptp treatment compared with vehicle .
mirtazapine, when solely applied with 16 mg/kg, showed no significant alterations on them when compared with vehicle-treated group .
furthermore, administrations of mirtazapine after mptp treatment also showed no significant changes on them  both with  <dig> and 16 mg/kg.
however, da turnover was significantly increased by 16 mg/kg of mirtazapine after mptp treatment when compared with vehicle or mptp alone   <dig>  =  <dig> , p <  <dig> , anova), although it did not affect the basal turnover when solely applied with 16 mg/kg .
effect of mirtazapine on the striatal th, dat and vmat <dig> protein expression
with western blot analysis, we examined protein expression of the dopaminergic markers tyrosine hydroxylase , dopamine transporter  and vesicle monoamine transporter  <dig>  in striatum.
treatment with mirtazapine, when solely applied with 16 mg/kg, showed no significant effects on the striatal th, dat and vmat <dig> protein expression of mice when compared to the vehicle-treated group   <dig>  =  <dig> , f  <dig>  =  <dig> , f  <dig>  =  <dig> , p >  <dig> , anova), although vmat <dig> expression showed a slight tendency of decrease with no significance.
mptp significantly decreased th, dat and vmat <dig> protein expressions to  <dig> – 50% of the vehicle treated ones .
mirtazapine did not alter protein expression of th when it was applied after mptp , strongly suggesting it did not affect the process of neurodegeneration of the nigro-striatal dopaminergic neurons triggered by mptp.
in contrast, mirtazapine showed a dose-dependent tendency of recovery of dat protein expression, and 16 mg/kg of mirtazapine showed significant increase of dat when compared with mptp-treated group .
furthermore, both  <dig> and 16 mg/kg of mirtazapine showed significant recovery of vmat <dig> protein expression compared with mptp-treated group .
antagonism of way <dig> on the behavioral effects of mirtazapine
one of the pharmacological effects of mirtazapine is a selective activation of 5-ht1ar with blocking both 5-ht <dig> and 5-ht <dig> receptors.
to examine the involvement of this mechanism in the effects of mirtazapine, we tested way <dig>  a specific antagonist for 5-ht1ar, together with mirtazapine both on beam-walking and rota-rod tests.
in both tests, the therapeutic effects of mirtazapine were almost completely cancelled by pre-treatment with  <dig>  mg/kg of way <dig>   <dig>  =  <dig> , f  <dig>  =  <dig> , p <  <dig> , anova).
we also examined the effects of way <dig> on the basal activities of both tests, and it did not show any significant effects when compared with vehicle-treated group   <dig>  =  <dig> , f  <dig>  =  <dig> , p >  <dig> , anova).
antagonism of prazosin or clonidine on the behavioral effects of mirtazapine
because noradrenergic system regulates both da and 5-ht neurons  <cit>  and α2-nar is one of an inhibitory target of mirtazapine, we tested prazosin, an antagonist for α1-nar, or clonidine, selective agonist for α2-nar, together with mirtazapine both on beam-walking and rota-rod tests.
as shown in figure 6a and b, both of the noradrenergic drugs significantly reduced the effects of mirtazapine   <dig>  =  <dig> , f  <dig>  =  <dig> , p <  <dig> , anova), although their effect in beam-walking test was incomplete when compared to that of way <dig>
as we did in the previous section using way <dig>  we also examined both of the noradrenergic drugs on the basal behavioral activities of beam-walking and rota-rod tests.
in beam-walking test, prazosin did not affect the periods for traversing 50 cm, although clonidine significantly increased it .
in rota-rod test, both prazosin and clonidine significantly shortened the latency to fall from the rotating rod , suggesting some of the effects we have observed contain basal disturbance of these drugs on autonomic system.
antagonism of way <dig>  prazosin and clonidine on the biochemical effects of mirtazapine
we also examined the effects of way <dig>  prazosin and clonidine on the contents of the striatal da and its metabolites with turnover rate of da by hplc both in the vehicle and mptp-treated mice.
as shown in table  <dig>  all of three drugs have no effects on basal da, dopac and hva contents, although prazosin and clonidine decreased basal da turnover significantly when compared with vehicle-treated group .
when these three drugs were administered prior to mirtazapine, all of them significantly reduced the increased da turnover observed in mptp + mirtazapine group   <dig>  =  <dig> , p <  <dig> , anova).
#
##
##
the results are shown as the mean  ± sem of 4– <dig> animals/group.
statistical significance was evaluated by student-newman-keuls test   <dig>  =  <dig> , f  <dig>  =  <dig> , f  <dig>  =  <dig> , f  <dig>  =  <dig> , *p <  <dig> ,**p <  <dig>  compared with mptp-treated group and †p <  <dig>  compared with mptp + mirtazapine group), or by student’s t-test .
rt-pcr detection of mrna for the isoforms of noradrenaline and serotonin receptors
to examine whether the known receptors, which could be affected with mirtazapine directly or for the targets of the inhibitors used in this study, are expressed in striatum, snc and raphe nucleus, we performed rt-pcr.
the specific primers used to detect mrnas for the noradrenaline and 5-ht receptors, α1a, α1b, α1d, α2a, α2b, 5-ht1a, 5-ht2a, 5-ht2b, 5-ht2c and 5-ht <dig> are written in methods.
as shown in figure  <dig>  α1a, α1b, α1d, α2a and α2b noradrenaline receptors were expressed in striatum, snc and raphe.
on the other hand, no 5-ht2br transcript was detected in snc and raphe, while 5-ht1a, 5-ht2a, 5-ht2c and 5-ht <dig> receptors were detected in striatum, snc and raphe .
discussion
in the present study, we found that treatment with mirtazapine in mice significantly improved mptp-induced motor dysfunction.
to our knowledge, this is the first report showing the therapeutic potency of an antidepressant mirtazapine against mptp neurotoxicity in mice.
because mptp mice are one of the most popular models for screening anti-pd agents  <cit> , our results suggest possible use of mirtazapine as a pd therapeutics in clinical patients.
our results of hplc in figure  <dig> and table  <dig> showed mptp increased da turnover in striatum, and mirtazapine further elevated it.
similar results were reported previously by zonisamide, an anti-convulsant drug also effective on pd  <cit> .
increased da turnover with mptp treatment is thought as a compensatory effect exerted by the remained da neurons under the neurotoxic condition  <cit> , although significant behavioral deficits  suggested the compensatory effect was insufficient to keep the normal motor coordination.
further elevation of da turnover with mirtazapine after mptp treatment observed in this study led us to speculate mirtazapine facilitate utilization of da, probably by increasing da release, reuptake, degradation and/or recycling in the da-depleted condition.
supporting this idea, in fact, reduced protein expression of dat by mptp was partially recovered, and that of vmat <dig> was almost completely recovered to the normal level by mirtazapine .
the increase of these transporter proteins would reflect the increased da release from the dopaminergic nerve terminals in striatum by mirtazapine  <cit> .
in contrast, reduction of th expression with mptp was not rescued by mirtazapine , suggesting it did not affect on the neurodegenerative process of mptp.
the effect of mirtazapine in our study was expressed specifically after the treatment of mptp, and the sole treatment with mirtazapine did not alter the behavioral parameters  nor the striatal contents of da and its metabolites with da turnover , whereas the previous report of nakayama et al.
<cit>  showed increased da release by acute and sole treatment of mirtazapine in mpfc of rats.
the discrepancy is probably caused by following differences: schedule of drug administration, method and timing of sampling, method of analysis especially because the lack of real-time measurement of extracellular da levels in our study.
nevertheless of the discrepancy, these results suggest increase of da by mirtazapine in the rodent brain with a short-term administration.
other study, however, reported that two-week administration of mirtazapine completely antagonized the stress-induced increase in dopamine release in the prefrontal cortex  <cit> .
it may be possible that the pharmacological action of miratazapine is different depending on the periods of administration and the amount of da at the targeted synapses.
further examination using microdialysis in striatum measuring extracellular da, na and 5-ht with their metabolites will explore more precise mechanisms of both acute and chronic action of mirtazapine including the hypothesis shown in figure  <dig>
as mentioned in introduction, mirtazapine is categorized into nassa, inhibiting pre-synaptic α2-nar specifically.
mirtazapine also inhibits 5-ht <dig> and 5-ht <dig> receptors which in turn selectively activate 5-ht1r.
it enhances, therefore, the release of noradrenaline and 5-ht1ar-mediated serotonergic transmission  <cit> .
based on these pharmacological properties of mirtazapine and from our results of the experiments using nar- and 5-htr-related reagents, we illustrated a hypothetical mechanism of mirtazapine action on pd-related nigro-striatal dopaminergic system with serotonergic and noradrenergic systems .
way <dig>  a selective inhibitor for 5-ht1ar, clearly cancelled the therapeutic effects of mirtazapine on mptp-induced neurotoxicity without affecting the basal behavioral or the biochemical parameters , strongly suggest the involvement of this receptor for the effects of mirtazapine.
from the dorsal raphe nuclei, 5-ht neurons innervate directly to the nigral da neurons to inhibit the firing of them with 5-ht2ar-dependent manner  <cit> .
inhibition of this 5-ht2ar by mirtazapine results in increase of da release.
further, the recurrent innervation of 5-ht neurons via 5-ht1ar in raphe negatively controls cell firing and release of 5-ht  <cit> .
thus, activation of 5-ht1ar by mirtazapine can reduce 5-ht release, and it also results in the increased da release.
in fact, our rt-pcr results showed expression of both 5-ht2ar mrna in snc and 5-ht1ar in raphe .
recently, it has been reported that 5-ht2ar antagonists m <dig> improved motor impairments in the mptp-induced mouse model of pd  <cit> .
other studies also revealed that stimulation of 5-ht1ar recovered the motor disorders caused by lesions of da neurons or da depletion  <cit> .
thus, both 5-ht1ar and 5-ht2ar are attractive targets as novel pd therapeutics.
α2-nar is another inhibitory target of mirtazapine.
in fact, clonidine, a selective agonist for this receptor reduced the effects of mirtazapine, although it also affected basal activity of mice and striatal da turnover , probably of its inhibitory effect on peripheral sympathetic system.
noradrenaline inhibits the neurotransmitter release via presynaptic α2-nar, and mirtazapine increases neurotransmitter release by inhibiting this receptor  <cit> .
although precise mechanism is still unknown, we speculate this receptor could also function as presynaptic hetero receptor on da neuron to regulate da release negatively, the same as presynaptic autoreceptor on noradrenergic nerve terminal .
actually we detected α2a and α2b mrna isoforms both in striatum and snc .
prazosin, an antagonist for α1-nar, also showed similar effects as clonidine.
we suppose noradrenergic input on da would activate da neuron via α1-nar in snc as illustrated in figure  <dig>  since similar stimulatory mechanism was reported in 5-ht and other neurons  <cit> .
however, we could not clearly discriminate peripheral and central effects of the noradrenergic drugs in this study.
it will be difficult to use clonidine  and prazosin  to address the mechanism for mirtazapine effect in this mouse model with a systemic administration.
precise mechanism of the effect of mirtazapine through noradrenergic system should be examined, with applying more specific inhibitors directly into specific brain regions, for example.
it is a well known fact that long-term treatment of ʟ-dopa in pd patients causes various adverse side effects such as wearing-off, dyskinesia, psychiatric symptoms, and so on  <cit> .
a recent study has demonstrated that treatment with a ssri fluoxetine significantly suppressed ʟ-dopa-induced rotational behavior in 6-hydroxydopamine -treated rats with 5-ht1ar-dependent manner  <cit> .
furthermore, a selective α2-nar antagonist fipamezole reduced ʟ-dopa-induced dyskinesias in mptp-treated monkeys  <cit> .
these observations suggest that nassa mirtazapine could be a possible novel therapeutic drug for pd, particularly in regard to avoiding the adverse side effects of ʟ-dopa.
depression in pd patients is the most common psychiatric disturbance  <cit> , and ssris are now often used for the treatment  <cit> .
recently, nassa is also used and shown to be effective in the treatment of depression in pd patients as well as ssri  <cit> .
however, the effect of nassa on motor dysfunction in pd patients is still unknown.
together with these reports and our results in this study, it is highly expected that mirtazapine has dual therapeutic effects both on depression and pd in humans.
our study here and further detailed examinations will open the next door of clinical trial to examine mirtazapine on pd.
CONCLUSIONS
our present study provides the first evidence that mirtazapine has a therapeutic potency against mptp neurotoxicity in mice.
because pd patients sometimes show depression together, it is highly expected that mirtazapine has dual therapeutic effects both on depression and pd in humans.

 parkinson’s disease is a progressive, age-related, neurodegenerative disorder characterized by bradykinesia, resting tremor and gait disturbance. the major pathological basis of pd is the death of dopaminergic neurons in the substantia nigra pars compacta and the degeneration of their nerve terminals in striatum. it has been proposed that clinical signs of pd appear at the point when dopaminergic neuronal cell loss exceeds a critical threshold: – 80% of striatal nerve terminals and – 60% of the snc perikaryons. as a pharmaceutical treatment, ʟ- -dihydroxyphenylalanine, supplying the precursor of dopamine, is the most commonly applied and alleviates major symptom of pd. for over 40 years, treatment with ʟ-dopa combined with an inhibitor for peripheral aromatic ʟ-amino acid decarboxylase such as carbidopa had been established as a gold standard for pd treatment. however, long-term treatment with ʟ-dopa is often complicated by the development of adverse effects such as drug-induced dyskinesia. there have been additional anti-parkinsonian drugs, such as dopamine agonists mostly targeted on d class receptor, but their pharmaceutical potencies are not drastically better than that of ʟ-dopa. it is essential to develop novel drugs which can support therapeutic effects of ʟ-dopa with delaying expression of its side effects, or even solely effective against pd symptoms. serotonergic neurons play an important role in modulating extrapyramidal motor disorders such as pd and drug-induced parkinsonism. some studies showed administration of agonists for 1a subtype of serotonin/5-hydroxytriptamine receptor tertraline and tandospirone) significantly improved various types of extrapyramidal symptoms including antipsychotic-induced bradykinesia and catalepsy, and neurotoxin-induced bradykinesia. therefore, the central serotonergic system is thought to be one of a favorable drug target for the treatment of pd. it is a well-known fact that serotonergic as well as noradrenergic system is a major target of antidepressants such as selective serotonin-reuptake inhibitor fluoxetine and fluvoxamine. recently, a novel antidepressant mirtazapine has been developed and is now approved in many countries for clinical treatment of major depression. mirtazapine is categorized into a noradrenergic and specific serotonergic antidepressant, showing multiple pharmacological actions such as inhibiting presynaptic α noradrenaline receptor and selectively activating 5-ht1ar. mirtazapine has higher antidepressant effects than placebo or trazodone, which is equivalent to the effect of tricyclic antidepressant such as clomipramine and amitriptyline. compared to ssri, mirtazapine showed an earlier onset of antidepressant effects. further, the side effects of mirtazapine are reported to be lower than those of ssri or tca. in nakayama et al. reported mirtazapine increased da release in the medial prefrontal cortex of rats with activating 5-ht1ar. they reported that or 16 mg/kg of mirtazapine increased da release with dose-dependent manner, and an inhibitor of 5-ht1ar way significantly decreased the mirtazapine-induced increase of da release. we hypothesized mirtazapine may be effective on pd if the same mechanism as this 5-ht-dependent increase of da release existed in the nigro-striatal dopaminergic system, too. in fact with related to pd, some studies reported the clinical efficacy of mirtazapine on parkinsonian tremor in human. however, little is known about the therapeutic effect of mirtazapine for motor dysfunctions other than tremor in pd. therefore, in this study, we examined the effect of mirtazapine in mice treated with the neurotoxin 1-methyl-4-phenyl-, -tetrahydropyridine, one of the typical animal models of pd.  effect of mirtazapine on motor dysfunctions induced by mptp we first assessed effect of mirtazapine on motor dysfunctions induced by mptp using two different behavioral paradigms: the beam-walking and rota-rod tests. we chose two doses of mirtazapine in this study based on the previous study by nakayama et al. in in which they showed and 16 mg/kg of mirtazapine produced a dose-dependent increase in extracellular da levels in mpfc of freely moving rats. thus we examined minimal and maximal doses in mice. in beam-walking test, mptp-treated mice showed a significantly prolonged duration to traverse a distance of 50 cm than that of the vehicle-treated mice  = , p < , anova). in contrast, mirtazapine significantly improved the mptp-induced prolongation of the traversal duration when it was treated after mptp with both and 16 mg/kg doses, although it did not affect when solely applied compared to the vehicle-treated mice. with rota-rod test, vehicle-treated mice usually remained on the rotating rod for approximately 400–600 sec. as shown in figure 1b, mptp-treatment significantly decreased the latency to fall from the rod when compared to vehicle-treated mice  = , p < , anova). on the other hand, administration of 16 mg/kg of mirtazapine after mptp significantly recovered the latency to the level of vehicle-treated group, with no effect when solely applied. effect of mirtazapine on the striatal da, dopac, hva and turnover rate using hplc, we quantified the striatal da and its metabolites dopac and hva with calculating turnover rate. as shown in figure  administrations of mptp produced marked depletion of da, dopac and hva in striatum  = , f  = , f  =, p < , anova), as was reported previously. da turnover, calculated by /da, was increased significantly by mptp treatment compared with vehicle. mirtazapine, when solely applied with 16 mg/kg, showed no significant alterations on them when compared with vehicle-treated group. furthermore, administrations of mirtazapine after mptp treatment also showed no significant changes on them both with and 16 mg/kg. however, da turnover was significantly increased by 16 mg/kg of mirtazapine after mptp treatment when compared with vehicle or mptp alone  = , p < , anova), although it did not affect the basal turnover when solely applied with 16 mg/kg. effect of mirtazapine on the striatal th, dat and vmat protein expression with western blot analysis, we examined protein expression of the dopaminergic markers tyrosine hydroxylase, dopamine transporter and vesicle monoamine transporter in striatum. treatment with mirtazapine, when solely applied with 16 mg/kg, showed no significant effects on the striatal th, dat and vmat protein expression of mice when compared to the vehicle-treated group  = , f  = , f  = , p > , anova), although vmat expression showed a slight tendency of decrease with no significance. mptp significantly decreased th, dat and vmat protein expressions to – 50% of the vehicle treated ones. mirtazapine did not alter protein expression of th when it was applied after mptp, strongly suggesting it did not affect the process of neurodegeneration of the nigro-striatal dopaminergic neurons triggered by mptp. in contrast, mirtazapine showed a dose-dependent tendency of recovery of dat protein expression, and 16 mg/kg of mirtazapine showed significant increase of dat when compared with mptp-treated group. furthermore, both and 16 mg/kg of mirtazapine showed significant recovery of vmat protein expression compared with mptp-treated group. antagonism of way on the behavioral effects of mirtazapine one of the pharmacological effects of mirtazapine is a selective activation of 5-ht1ar with blocking both 5-ht and 5-ht receptors. to examine the involvement of this mechanism in the effects of mirtazapine, we tested way a specific antagonist for 5-ht1ar, together with mirtazapine both on beam-walking and rota-rod tests. in both tests, the therapeutic effects of mirtazapine were almost completely cancelled by pre-treatment with  mg/kg of way  =, f  = , p < , anova). we also examined the effects of way on the basal activities of both tests, and it did not show any significant effects when compared with vehicle-treated group  = , f  = , p > , anova). antagonism of prazosin or clonidine on the behavioral effects of mirtazapine because noradrenergic system regulates both da and 5-ht neurons and α2-nar is one of an inhibitory target of mirtazapine, we tested prazosin, an antagonist for α1-nar, or clonidine, selective agonist for α2-nar, together with mirtazapine both on beam-walking and rota-rod tests. as shown in figure 6a and b, both of the noradrenergic drugs significantly reduced the effects of mirtazapine  = , f  = , p < , anova), although their effect in beam-walking test was incomplete when compared to that of way as we did in the previous section using way we also examined both of the noradrenergic drugs on the basal behavioral activities of beam-walking and rota-rod tests. in beam-walking test, prazosin did not affect the periods for traversing 50 cm, although clonidine significantly increased it. in rota-rod test, both prazosin and clonidine significantly shortened the latency to fall from the rotating rod, suggesting some of the effects we have observed contain basal disturbance of these drugs on autonomic system. antagonism of way prazosin and clonidine on the biochemical effects of mirtazapine we also examined the effects of way prazosin and clonidine on the contents of the striatal da and its metabolites with turnover rate of da by hplc both in the vehicle and mptp-treated mice. as shown in table  all of three drugs have no effects on basal da, dopac and hva contents, although prazosin and clonidine decreased basal da turnover significantly when compared with vehicle-treated group. when these three drugs were administered prior to mirtazapine, all of them significantly reduced the increased da turnover observed in mptp + mirtazapine group  =, p < , anova). # ## ## the results are shown as the mean  ± sem of 4– animals/group. statistical significance was evaluated by student-newman-keuls test  = , f  = , f  = , f  = , *p < ,**p <  compared with mptp-treated group and †p <  compared with mptp + mirtazapine group), or by student’s t-test. rt-pcr detection of mrna for the isoforms of noradrenaline and serotonin receptors to examine whether the known receptors, which could be affected with mirtazapine directly or for the targets of the inhibitors used in this study, are expressed in striatum, snc and raphe nucleus, we performed rt-pcr. the specific primers used to detect mrnas for the noradrenaline and 5-ht receptors, α1a, α1b, α1d, α2a, α2b, 5-ht1a, 5-ht2a, 5-ht2b, 5-ht2c and 5-ht are written in methods. as shown in figure  α1a, α1b, α1d, α2a and α2b noradrenaline receptors were expressed in striatum, snc and raphe. on the other hand, no 5-ht2br transcript was detected in snc and raphe, while 5-ht1a, 5-ht2a, 5-ht2c and 5-ht receptors were detected in striatum, snc and raphe. discussion in the present study, we found that treatment with mirtazapine in mice significantly improved mptp-induced motor dysfunction. to our knowledge, this is the first report showing the therapeutic potency of an antidepressant mirtazapine against mptp neurotoxicity in mice. because mptp mice are one of the most popular models for screening anti-pd agents, our results suggest possible use of mirtazapine as a pd therapeutics in clinical patients. our results of hplc in figure  and table  showed mptp increased da turnover in striatum, and mirtazapine further elevated it. similar results were reported previously by zonisamide, an anti-convulsant drug also effective on pd. increased da turnover with mptp treatment is thought as a compensatory effect exerted by the remained da neurons under the neurotoxic condition, although significant behavioral deficits suggested the compensatory effect was insufficient to keep the normal motor coordination. further elevation of da turnover with mirtazapine after mptp treatment observed in this study led us to speculate mirtazapine facilitate utilization of da, probably by increasing da release, reuptake, degradation and/or recycling in the da-depleted condition. supporting this idea, in fact, reduced protein expression of dat by mptp was partially recovered, and that of vmat was almost completely recovered to the normal level by mirtazapine. the increase of these transporter proteins would reflect the increased da release from the dopaminergic nerve terminals in striatum by mirtazapine. in contrast, reduction of th expression with mptp was not rescued by mirtazapine, suggesting it did not affect on the neurodegenerative process of mptp. the effect of mirtazapine in our study was expressed specifically after the treatment of mptp, and the sole treatment with mirtazapine did not alter the behavioral parameters nor the striatal contents of da and its metabolites with da turnover, whereas the previous report of nakayama et al. showed increased da release by acute and sole treatment of mirtazapine in mpfc of rats. the discrepancy is probably caused by following differences: schedule of drug administration, method and timing of sampling, method of analysis especially because the lack of real-time measurement of extracellular da levels in our study. nevertheless of the discrepancy, these results suggest increase of da by mirtazapine in the rodent brain with a short-term administration. other study, however, reported that two-week administration of mirtazapine completely antagonized the stress-induced increase in dopamine release in the prefrontal cortex. it may be possible that the pharmacological action of miratazapine is different depending on the periods of administration and the amount of da at the targeted synapses. further examination using microdialysis in striatum measuring extracellular da, na and 5-ht with their metabolites will explore more precise mechanisms of both acute and chronic action of mirtazapine including the hypothesis shown in figure  as mentioned in introduction, mirtazapine is categorized into nassa, inhibiting pre-synaptic α2-nar specifically. mirtazapine also inhibits 5-ht and 5-ht receptors which in turn selectively activate 5-ht1r. it enhances, therefore, the release of noradrenaline and 5-ht1ar-mediated serotonergic transmission. based on these pharmacological properties of mirtazapine and from our results of the experiments using nar- and 5-htr-related reagents, we illustrated a hypothetical mechanism of mirtazapine action on pd-related nigro-striatal dopaminergic system with serotonergic and noradrenergic systems. way a selective inhibitor for 5-ht1ar, clearly cancelled the therapeutic effects of mirtazapine on mptp-induced neurotoxicity without affecting the basal behavioral or the biochemical parameters, strongly suggest the involvement of this receptor for the effects of mirtazapine. from the dorsal raphe nuclei, 5-ht neurons innervate directly to the nigral da neurons to inhibit the firing of them with 5-ht2ar-dependent manner. inhibition of this 5-ht2ar by mirtazapine results in increase of da release. further, the recurrent innervation of 5-ht neurons via 5-ht1ar in raphe negatively controls cell firing and release of 5-ht. thus, activation of 5-ht1ar by mirtazapine can reduce 5-ht release, and it also results in the increased da release. in fact, our rt-pcr results showed expression of both 5-ht2ar mrna in snc and 5-ht1ar in raphe. recently, it has been reported that 5-ht2ar antagonists m improved motor impairments in the mptp-induced mouse model of pd. other studies also revealed that stimulation of 5-ht1ar recovered the motor disorders caused by lesions of da neurons or da depletion. thus, both 5-ht1ar and 5-ht2ar are attractive targets as novel pd therapeutics. α2-nar is another inhibitory target of mirtazapine. in fact, clonidine, a selective agonist for this receptor reduced the effects of mirtazapine, although it also affected basal activity of mice and striatal da turnover, probably of its inhibitory effect on peripheral sympathetic system. noradrenaline inhibits the neurotransmitter release via presynaptic α2-nar, and mirtazapine increases neurotransmitter release by inhibiting this receptor. although precise mechanism is still unknown, we speculate this receptor could also function as presynaptic hetero receptor on da neuron to regulate da release negatively, the same as presynaptic autoreceptor on noradrenergic nerve terminal. actually we detected α2a and α2b mrna isoforms both in striatum and snc. prazosin, an antagonist for α1-nar, also showed similar effects as clonidine. we suppose noradrenergic input on da would activate da neuron via α1-nar in snc as illustrated in figure  since similar stimulatory mechanism was reported in 5-ht and other neurons. however, we could not clearly discriminate peripheral and central effects of the noradrenergic drugs in this study. it will be difficult to use clonidine and prazosin to address the mechanism for mirtazapine effect in this mouse model with a systemic administration. precise mechanism of the effect of mirtazapine through noradrenergic system should be examined, with applying more specific inhibitors directly into specific brain regions, for example. it is a well known fact that long-term treatment of ʟ-dopa in pd patients causes various adverse side effects such as wearing-off, dyskinesia, psychiatric symptoms, and so on. a recent study has demonstrated that treatment with a ssri fluoxetine significantly suppressed ʟ-dopa-induced rotational behavior in 6-hydroxydopamine -treated rats with 5-ht1ar-dependent manner. furthermore, a selective α2-nar antagonist fipamezole reduced ʟ-dopa-induced dyskinesias in mptp-treated monkeys. these observations suggest that nassa mirtazapine could be a possible novel therapeutic drug for pd, particularly in regard to avoiding the adverse side effects of ʟ-dopa. depression in pd patients is the most common psychiatric disturbance, and ssris are now often used for the treatment. recently, nassa is also used and shown to be effective in the treatment of depression in pd patients as well as ssri. however, the effect of nassa on motor dysfunction in pd patients is still unknown. together with these reports and our results in this study, it is highly expected that mirtazapine has dual therapeutic effects both on depression and pd in humans. our study here and further detailed examinations will open the next door of clinical trial to examine mirtazapine on pd.  our present study provides the first evidence that mirtazapine has a therapeutic potency against mptp neurotoxicity in mice. because pd patients sometimes show depression together, it is highly expected that mirtazapine has dual therapeutic effects both on depression and pd in humans. 
mirtazapine, a noradrenergic and specific serotonergic antidepressant, shows multiple pharmacological actions such as inhibiting presynaptic α noradrenaline receptor and selectively activating 5-hydroxytriptamine 1a receptor. 1-methyl-4-phenyl-, -tetrahydropyridine parkinson’s diseasemirtazapinenoradrenergic and specific serotonergic antidepressant serotonin dopamine biochemical examinations by high performance liquid chromatography and western blot analysis suggested mirtazapine facilitated utilization of dopamine by increasing turnover and protein expression of transporters, without affecting on neurodegenerative process by mptp. mirtazapine was also reported to increase dopamine release in the cortical neurons with 5-ht dependent manner. because pd patients sometimes show depression together, it will be a useful drug for a future pd treatment. to examine whether mirtazapine has a therapeutic potency in parkinson’s disease, we examined this compound in 1-methyl-4-phenyl-, -tetrahydropyridine -treated mice model of pd. our results showed mirtazapine had a therapeutic potency against pd in a mouse model. mptp-induced motor dysfunction, assessed by beam-walking and rota-rod tests, was significantly improved by administration of mirtazapine. these therapeutic effects of mirtazapine were reduced by administration of way an inhibitor for 5ht1ar, or of clonidine, a selective agonist for α2-nar, or of prazosin, an inhibitor for α1-nar, respectively. male c57bl/ mice were subjected to mptp treatment to establish a pd model. mirtazapine was administered once a day for 3 days after mptp treatment.
 mirtazapine, a noradrenergic and specific serotonergic antidepressant, shows multiple pharmacological actions such as inhibiting presynaptic α noradrenaline receptor and selectively activating 5-hydroxytriptamine 1a receptor. mirtazapine was also reported to increase dopamine release in the cortical neurons with 5-ht dependent manner. to examine whether mirtazapine has a therapeutic potency in parkinson’s disease, we examined this compound in 1-methyl-4-phenyl-, -tetrahydropyridine -treated mice model of pd.  male c57bl/ mice were subjected to mptp treatment to establish a pd model. mirtazapine was administered once a day for 3 days after mptp treatment. mptp-induced motor dysfunction, assessed by beam-walking and rota-rod tests, was significantly improved by administration of mirtazapine. biochemical examinations by high performance liquid chromatography and western blot analysis suggested mirtazapine facilitated utilization of dopamine by increasing turnover and protein expression of transporters, without affecting on neurodegenerative process by mptp. these therapeutic effects of mirtazapine were reduced by administration of way an inhibitor for 5ht1ar, or of clonidine, a selective agonist for α2-nar, or of prazosin, an inhibitor for α1-nar, respectively.  our results showed mirtazapine had a therapeutic potency against pd in a mouse model. because pd patients sometimes show depression together, it will be a useful drug for a future pd treatment. 1-methyl-4-phenyl-, -tetrahydropyridine parkinson’s diseasemirtazapinenoradrenergic and specific serotonergic antidepressant serotonin dopamine 