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Buspirone improves haloperidol-induced Parkinson disease in mice through 5-HT(1A) recaptors.

Background and the purpose of the study The available literatures show that 5-HT1A receptors are widely distributed throughout the basal ganglia, and their activation facilitate dopamine release. Neuroleptic drugs such as haloperidol induce Parkinson-like syndrome through blocking brain D2 receptors. This study aimed to investigate effect of buspirone, a partial agonist of 5HT1A receptor, on motor dysfunctions induced by haloperidol and involvement of 5HT1A receptors in this regard. Methods Study was performed on the male mice weighing 25–30 g. Animals were divided randomly to groups of 10 animals. Motor dysfunction was induced by intraperitoneal (i.p.) injection of haloperidol (1 mg/kg). Catalepsy was assayed by bar-test method 5, 60, 120 and 180 minutes after drug administration and motor imbalance was studied by rotarod test. Results and major conclusion Results showed that buspirone (20 mg/kg, i.p.) decreased significantly haloperidol-induced catalepsy and balance disorder in a dose dependent manner. Furthermore, 8-OH-DPAT (10 mg/kg, i.p.), as an agonist of 5-HT1A receptor, decreased haloperidol-induced catalepsy and balance disorder. The effect of buspirone (20 mg/kg, i.p.) on haloperidol-induced motor disorders was abolished by NAN-190 (10 mg/kg, i.p.), as a 5-HT1A receptor antagonist. From the results it may be concluded that buspirone improves haloperidol-induced catalepsy and balance disorder through activation of 5-HT1A receptors.


INTRODUCTION
Parkinson's Disease (PD) is a progressive neurodegenerative disease occurring in approximately1% of the population of age over 50 years. It's most prominent symptoms are tremor, muscle stiffness and bradykinesia. Some non-motor symptoms such as cognitive behavior disorder and depression are usually observed in patients. The disease is accompanied by preferential loss of dopaminergic neurons of the substanitia nigra pars compacta (SNc) (1) and the influence of other neurotransmitter systems have not been studied extensively. The fact that serotonergic system are also involved in PD has been raised from previous studies (2) and postmortem research has shown reduced levels of basal ganglia serotonin (5-HT) as well as its metabolite 5-hydroxy-indoleatic acid (5HIAA) (3). While there are some reports on the inhibitory effect of 5-HT on striatal dopamine (DA) release (4) , other studies have shown that 5-HT facilitates DA outflow (5). This discrepancy results from differencs in receptor types and subtypes (6). 5-HT 1A receptors are widely distributed throught the basal ganglia (7). They are located on dorsal raphe neurons with efferents to the striatum, and are also localized on cortical neurons sending glutamatergic projections to the basal ganglia (1). Studies have shown that 5-HT 1A receptor stimulation Showed antiparkinsonian effects in 6-hydroxydopamine (6-OHDA)-lesioned rats (8). This effect is most likely caused by the increase in 5-HT 1A receptor activation, resulting in inhibition of serotonin release (1). Although stimulation of 5-HT 1A receptor is associated with an increase in dopamine turnover (9), dopaminergic cell firing (10) and dopamine release (11), suggesting that 5-HT 1A agonists might have potential therapeutic value in the treatment of parkinson's disease, Other studies have reported that administration of the 5-HT 1A agonist did not affect basal DA release in the nucleus accumbens or the striatum (4,12). In animal studies, neurotoxins such as 1-methyl-4phenyl-1,2,3,6-tetrahydroxypyridine (MPTP) and 6-OHDA and neuroleptic drugs (e.g. haloperidol) are used commonly to create experimental model of PD by which certain aspects of the disease such as catalepsy, motor imbalance and slowing of movement can be modeled (1,13). The cataleptic end point of catalepsy was considered the time that both front paws were removed from the bar or if the animal moved its head in an exploratory manner. A cut-off time of 720 seconds was applied (15). The rotarod test was used to assess the ability of the animal to maintain balance on a 1-inch diameter rod revolving at a constant rate of 6 revolutions per minute (16). This test requires a high degree of sensorimotor coordination and is therefore used to test more subtle neurological deficits. The ability of all animals to pass the rotarod test was checked regularly prior and after drug administration.

Expression of data and statistics
Descriptive statistics and comparisons of differences between each data set were calculated by use of InStat software. The data were expressed as mean ± SEM, and were analyzed by one-way ANOVA in each experiment. Statistical significance was accepted at the level of p<0.05. In the case of significant variation (p<0.05), the values were compared by Tukey test.

Effect of 8-OH-DPAT on haloperidol-induced motor impairment
The effect of 8-OH-DPAT (10 mg/kg, i.p.) on haloperidol-induced catalepsy and motor 2 imbalnce was determined. Results showed that 8-OH-DPAT attenuated haloperidol-induced catalepsy (p<0.001) and motor imbalance (p<0.001) in comparision with haloperidol alone. In group of animals treated with 8-OH-DPAT alone, there was not any remarkable alteration in bar test and rotarod elapsed time ( Fig.  2A, 2B).

Effect of buspirone and NAN-190 co-injection on haloperidol-induced motor impairment
As it has been shown in figure 3A, NAN-190 (10 mg.kg, i.p.) as a 5-HT 1A receptor antagonist, abolished (p<0.001, 0.05) anticataleptic effect of buspirone (20 mg/kg, i.p.). This drug also decreased (p<0.001, 0.01) immobility induced in rodents by typical neuroleptics (e.g., haloperidol, chlorpromazine, fluphenazine) is a behavioral method to study nigrostriatal function and its modulation by other neurotransmitters (14). Drugs which attenuate haloperidol-induced motor disorders might reduce the extrapyramidal signs of PD (15). Thus investigation of the effects of buspirone on extrapyramidal side effects of haloperidol appeared of interest. The present study was designed to determine effect of buspirone on catalepsy and motor imbalance induced by haloperidol as well as possible involvement of 5-HT 1A receptors by using 8-OH-DPAT and NAN-190 as agonist and antagonist of 5-HT 1A recepotrs respectively.

Chemicals
All chemicals were obtained from Sigma Chemical Company (USA) except for busprione and haloperidol that were purchased from Heumann Company (Germany) and Daru-Pakhsh Company (Iran) respectively. Solutions were prepared fresh on the days of experimentation by dissolving drugs in physiological saline (0.9% NaCl). The drugs were injected intraperitonealy (i.p.) and movement disorders were assessed by bar test and rotarod of 5, 60, 120 and 180 min after drugs administration.

Animals
The experiments were carried out on male Swiss albino mice weighing 25-30 g. Animals were housed in standard polypropylene cages, ten per cage, under a 12:12 h light/dark schedule at an ambient temperature of 23±2 °C and had access to food and water freely. All experiments were carried out under the ethical guidelines of the Tabriz University of Medical Sciences, for the care and use of laboratory animals (National Institutes of Health Publication No 85-23, revised 1985).

Motor impairment study
Motor impairment was induced with haloperidol (1 mg/ kg, ip) and measured by bar test and rotarod methods at 5, 60, 120 and 180 minute after drug administration. In each tests animals were used only once. The dose of the haloperidol was chosen to produce a moderate degree of catalepsy and motor imbalance, so that either attenuation or potentiation of the both phenomenon could be detected (15). The drugs (and saline for the controls) were injected ip with a 29-G needle, 15 min before haloperidol. All observations were made between 9:00 AM and 16:00 PM in a quiet room by an observer who was blind to treatments. Catalepsy was measured by means of a standard bar test, as the time that mouse maintained an imposed position with both front limbs extended and resting on a 3-cm high wood bar (0.9 cm in diameter). The the effect of buspirone (20 mg/kg, i.p.) on haloperidolinduced motor imbalance (Fig. 3B).

DISCUSSION
Parkinson's disease is caused by degeneration of dopaminergic neurons of substantia nigra, pars compacta. The most important characteristic of this disease is reduction of dopamine release from the end of striatal dopaminergic nerves. This disease is accompanied with movement disorders e.g. tremor, muscle rigidity and slow movement (1). Some neurotoxins such as MPTP, 6-OHDA, or neuroleptic drugs (e.g. haloperidol) are used frequently to create experimental models of this disease (1, 13). In this study effect of buspirone and possible involvement of 5-HT 1A receptors on haloperidol-induced catalepsy and motor imbalance was investigated. Bar test (15) and rotarod (17) are standard tests that are usually used to evaluate catalepsy and motor imbalance disoreders, respectively. Results of this study showed that acute administration of haloperidole induced catalpsy and imbalance in the rotating rod (rotarod) which is in accordance with results of other studies (13) where haloperidol (D2 antagonist) was used to create an empirical model of Parkinson disease.   Buspirone, as a partial agonist of 5-HT 1A receptors, improved prominently haloperidol-induced catalepsy and motor imbalance. This finding substantiates studies reporting that 5-HT 1A receptor agonists attenuate movement disorders induced by dopamine neurotoxins which was assessed by other experimental methods (18). Haloperidol is a potent typical neuroleptic drug which is used for treatment of some neuropsychiatric diseases. Parkinson-like syndromes and extrapyramidal symptoms are the major problems resulting from the use of this drug in psychotic patients (19). Therefore, special attention are paied to adjuvant therapy to reduce the severity of motor complications induced by haloperidol or any other neuroleptic drugs. Apart from affecting 5-HT 1A receptors, buspirone has also D 2 and α 2 -adrenoceptor blocking effects (20, 21). Therefore, it is possibile that its effects on improvement of haloperidol induced motor deficiency may be due to its effects on these receptors. In this study it was shown that 8-OH-DPAT could reduce haloperidol-induced movement disorders considerably and NAN-190 (5-HT 1A antagonist) abolished improving effect of buspirone on motor disorders induced by haloperidol. It might be due to effects of buspirone on and as a result involvement of á 2 or D 2 receptors may be neglected.
It has been shown that the 5-HT 1A receptor is present on dorsal raphe neurons with efferents to the striatum, and on cortical neurons sending glutamatergic projections to the basal ganglia (1). Stimulation of 5-HT 1A receptors in these regions leads to decrease and increase of serotonin and dopamine release respectively (9) by the inhibition of adenyl cyclase and opening of potassium channels (22). Therefore, it may be suggested that the effect of buspirone on haloperidol-induced motor disorders is due to decrease of inhibitory effects of serotonin on the release of dopamine. Anxiety is a co-morbid problem that is observed commonly in patients suffering from psychosis (23). Therefore, adjuvant therapy with buspirone in addition of reducing symptoms of anxiety, might be decrease in movement complications induced by haloperidol or other neuroleptic drugs. On the basis of results of this study further investigation on a possible usefulness of buspirone in decreasing motor side effects of neuroleptic drugs is suggested.