Gabapentin reduces haloperidol-induced vacuous chewing movements in mice.
Ana Paula Chiapinotto Cerettaa, Catiuscia Molz de Freitasb, Larissa Finger Schaffera,
Jeane Binotto Reinheimerc, Mariana Maikéli Dottoc, Elizete de Moraes Reisa, Rahisa
Scusseld, Ricardo Andrez Machado de Ávilad, Roselei Fachinettoa,b
a Programa de Pós-Graduação em Farmacologia, Universidade Federal de Santa
Maria, RS, Brazil
b Programa de Pós-Graduação em Bioquímica Toxicológica, Universidade Federal de
Santa Maria, RS, Brazil
c Curso de Farmácia, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil d Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil
Corresponding author Drª Roselei Fachinetto
Departamento de Fisiologia e Farmacologia, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, 97105-900, Santa Maria RS, Brazil
Tel.: +55-55-3220 8096; fax: +55-55-3220 8241 E-mail address: roseleirf@gmail.com
Abstract
Tardive dyskinesia (TD) is a common adverse effect observed in patients with long- term typical antipsychotic medication and vacuous chewing movements (VCMs) model induced by haloperidol has been used to study these abnormalities in experimental animals. The cause of TD remains unclear, but several lines of evidence suggest that the dopamine receptor supersensitivity and gamma- aminobutyric acid (GABA) insufficiency play an important role in the development of the disorder. This study investigated the effects of treatment with the GABA-mimetic drug gabapentin on the development of haloperidol-induced VCMs. Male mice received vehicle, haloperidol (1.5 mg/kg) or gabapentin (GBP, 100 mg/kg) intraperitoneally during 28 consecutive days. VCMs quantification was evaluated
before the treatment (baseline) and on day 28 as well as open-field activity on 28th
day of the treatment. The administration of gabapentin prevented the manifestation of
haloperidol-induced VCMs. Treatment with haloperidol reduced locomotor activity on open-field test which was prevented by gabapentin. We did not find any difference among the groups nor in tyrosine hydroxylase (TH) or glutamic acid decarboxylase (GAD) immunoreactivity nor monoamines levels in the striatum of mice. These results suggest that the treatment with gabapentin, an analog of GABA, can attenuate the VCMs induced by acutely haloperidol treatment in mice without alterations in dopaminergic (TH) and GABAergic (GAD) markers in striatum.
Keywords: tardive dyskinesia, tyrosine hydroxilase, glutamic acid decarboxylase, dopamine, movements disorders
1. Introduction
Involuntary abnormal orofacial movements are important symptoms presenting a variety of neuropsychiatric conditions including tardive dyskinesia (TD), which appears in ~20-40% of the schizophrenic patients under treatment with typical antipsychotic medication (FAURBYE et al., 1964; ANDREASSEN; JORGENSEN, 2000; SACHDEV, P. S., 2000). The cumulative incidence of this syndrome increases with long-term treatment with antipsychotics and can persists after discontinuation and may be irreversible (HASHIMOTO et al., 1998).
of schizophrenia acting by blockade of D2 dopamine receptors in brain (CREESE;
BURT; SNYDER, 1996). However, the antipsychotic efficacy of haloperidol is limited by the possibility of causing irreversible and disabling involuntary movements as TD in humans (KANE, J. M.; SMITH, 1982). In animals, haloperidol also produces abnormal movements (vacuous chewing movements)(VCMs) which has been used as a valid animal model of orofacial dyskinesia (OD)(BURGER et al., 2005b; BURGER et al., 2005a; COLPO et al., 2007; FACHINETTO et al., 2007; PEROZA et al., 2013; ROPKE et al., 2014).
Although the hypothesis of dopamine receptor supersensitivity has dominated experimental approaches used to study the development of TD this theory does not fully explain some clinical findings and some authors have suggested that TD may in fact be caused by other neurotransmitter disturbances as the gamma-aminobutyric acid (GABA) hypofunction (GUNNE; HAGGSTROM; SJOQUIST, 1984; ANDREASSEN; JORGENSEN, 2000; SACHDEV, 2000). In the GABA hypothesis, TD may be the result of degeneration of striatal GABAergic projection neurons with a decrease in glutamic acid decarboxylase (GAD) activity, a rate-limiting enzyme in the synthesis of GABA, in schizophrenic patients under neuroleptic treatment (GUNNE; HAGGSTROM, 1983; FIBIGER; LLOYD, 1984; GUNNE; HAGGSTROM; SJOQUIST, 1984).
In this context, GABA mimetic drugs like valproic acid or topiramate inhibit the manifestation of reserpine-induced oral dyskinesia (PEIXOTO et al., 2003; ARAUJO et al., 2004) as well as GABAA agonist tetrahydroisoxazolopyridine (THIP) and
GABAB agonist baclofen (PEIXOTO et al., 2005; CASTRO et al., 2006). In this
regard, gabapentin, a new anticonvulsant, has been synthesized as a structural GABA analogue and was expected to cross the blood-brain barrier to exert its effects on GABA receptors (GABAA and GABAB) (TRIBUT et al., 2010). However, little is
known about their actual interaction in these receptors, but it is believed that it can act as GABA agonist by indirect mechanisms. Our group recently demonstrated that gabapentin has a potential antipsychotic-like effect on behavioral parameters in an animal model of schizophrenia (CERETTA et al., 2016). However, there are no study evaluating the gabapentin effects on extrapyramidal symptoms resulting from conventional antipsychotic therapy. So, the aims of the present study were (1) to investigate the effect of repeated treatment with gabapentin on the manifestation of haloperidol-induced VCMs and (2) to verify if the possible protective mechanism of
gabapentin in this model involves neuronal changes on dopaminergic and gabaergic markers.
2. Materials and methods 2.1. Animals
A total of 40 male Albino Swiss mice at 2 months of age (20–30 g) obtained from Central Animal House holding of UFSM were used in this study. The animals were housed in polycarbonate cages (four or five per cage) on a 12-h light-dark cycle (lights on at 7:00 A.M.) in a room with controlled temperature (22 ± 2 ºC) with free access to water and standard laboratory chow (Puro Trato, Santo Augusto, Rio Grande do Sul, Brazil) throughout the experiment and acclimated for at least one week prior behavioral tests. All the animal experimentation was conducted in accordance to the guidelines of the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978), ARRIVE guidelines and the experimental procedure was approved by the Ethic Committee on Animal Use of Federal University of Santa Maria, Brazil under the protocol number CEUA 4562031214/2015 and are in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) (KILKENNY et al., 2010). Every effort was made to minimize the number of animals used and their suffering.
2.2. Drugs
Gabapentin (GBP, from Pharmanostra (Chengdu Hawk Bio-Engineering, China)) and haloperidol (ampoule with 5 mg/mL, from Teuto (Brazil)) were commercially obtained in local pharmacies. All drugs solutions were freshly prepared before administration in physiological saline (0.9% NaCl, vehicle) and administered intraperitoneally (i.p.) for 28 days in a volume of 0.1 ml/10 g body weight. Control groups received an equivalent volume of saline solution.
2.3 Experimental design
The animals were randomly assigned to one of four groups (10 animals per group) in which each animal received two intraperitoneal injections as following: 1) vehicle plus vehicle (control group); 2) GBP plus vehicle; 3) haloperidol plus vehicle; 4) haloperidol plus GBP. These animals received vehicle (0.9% NaCl), GBP (100 mg/kg) or haloperidol (1.5 mg/kg) daily for 28 days. Twenty four hours after the last
injections, animals were observed for quantification of VCMs and open-field behaviors. The doses of GBP and haloperidol were chosen on the basis of previous study (FACHINETTO et al., 2007; CERETTA et al., 2016).
2.4 Behavioral assessment tools
Behavioral tests were conducted during the light phase of the cycle (between 9:00 A.M. and 3:00 P.M.) using independent experimental groups of mice and these animals were housed and acclimated to the experimental room for one hour before testing. The animal’s behavior was analyzed by an observer who was blind of the pharmacological treatments.
2.4.1 Quantification of VCMs
To quantify the occurrence of VCMs, mice were placed individually in observation cages (20 cm x 20 cm x 19 cm) containing mirrors under the floor to allow behavioral quantification when the animal was faced away from the observer. VCMs are operationally defined as single mouth openings in the vertical plane not directed towards physical material, the numbers were counted. The incidence (in numbers) of tongue protrusions (visible extension of the tongue outside of the mouth and not direct at anything) and the duration (in seconds) of jaw tremor (high frequency fasciculation of the mouth or jaw) also were considered. All the movements occurring during a period of grooming were not taken into account. The behavioral parameters were measured continuously for 6 min after a period of 6 min of adaptation (BUSANELLO et al., 2011) and analyzed before the treatment (baseline)
and on 28th day of experimental protocol.
2.4.2 Open field
Spontaneous locomotor and exploratory activity of the animals were assessed in an open field test (ARCHER, 1973) performed in a sound attenuated room and with low light. All animals were placed individually in the center quadrant of an open field arena (white acrylic walls and floor, 44 cm X 44 cm X 44 cm) divided into 16 equal areas and the number of crossing (total number of the black lines crossed with four paws, parameter related to locomotor activity), and rearing responses (total number of stand up responses on two paws, parameter related to exploration activity)
were measured in a 5 minutes session with no habituation. This experiment was performed immediately after VCMs quantification.
2.5 Tissue preparation and Western blotting assay
After the last behavioral test, on day 28, mice were euthanized by cervical dislocation and the brains were rapidly dissected and put on ice. The striatum was separated and immediately frozen on powdered dry ice and thereafter stored at −80 °C. Tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD) immunoreactivity were carried out as described by De Freitas et al. (2016). Briefly, the striatum was homogenized in 400 μL of lysis buffer (4 % sodium dodecyl sulfate (SDS), 2 mM EDTA, 50 mM Tris, 0.5 mM Na2VO4, 2 μg/mL aprotinin, 0.1 mM
benzamidine, 0.1 mM PMSF), boiled for 6 min and centrifuged at 8000 rpm at 4°C for 10 min. The supernatant was used to determine protein concentration (LOWRY et al., 1951). Then, the samples (30 μg) were mixed with 10% glycerol and 8% 2- mercaptoethanol and resolved by 10% SDS-PAGE and transferred into nitrocellulose membrane (Millipore, USA). Proteins on the membrane were stained with a Ponceau solution (0.5% Ponceau plus 5% glacial acetic acid in water), as a loading control (ROMERO-CALVO et al., 2010). After staining, the membranes were dried and scanned for quantification. The membranes were blocked with 1 % bovine serum albumin and incubated with primary antibodies against TH (1:10000; Millipore; AB152) or GAD67 (1:7500, Millipore; MAB5406) overnight at 4°C. After, the membranes were incubated with alkaline phosphatase-coupled secondary antibody (1:10 000; Millipore). The reaction was determined by a colorimetric assay using nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) as a substrate (TREVISAN et al., 2013). The membranes were dried, scanned, and quantified. Finally, all values were normalized using Ponceau quantification.
2.6 Monoamines estimation
Quantification of dopamine (DA), 5-hydroxytryptamine (5-HT) and norepinephrine (NE) tissue levels in the striatum were estimated by HPLC (DE BENEDETTO et al., 2014). The samples were kept frozen at−80 °C until analysis. After homogenization in 200 µl of 0.2 M perchloric acid containing 3 mM cysteine and subsequent centrifugation, the supernatant was used for high pressure liquid chromatography (HPLC) followed by fluorimetric detection. The system comprised a
Ascentis® C18 chromatography column (250 mm x 2.1 mm 5 μm; Supelco®, USA), LC-20AT pump coupled to a SIL-20AHT autosampler and an RF-20A fluorescence detector, and LC Solution Shimadzu software (Kyoto, Japan). The column temperature was maintained at 35 °C and the flow rate was 0.3 mL/min. The mobile phase was acetate buffer (pH 3.5, 12 mM acetic acid, 0.26 mM EDTA)–methanol (86:14, v/v). The fluorescence was monitored at excitation and emission wave lengths of 279 nm and 320 nm, respectively. Peaks were identified by comparing their retention time in the sample (tissue extracts) solution with that of standard solution. The samples had an injection volume of 20 μL.
2.6. Statistical analysis
Data are expressed as mean ±standard error of the mean (SEM). Statistical analysis of data was carried out by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Statistical significance was assumed at p<0.05 or less. 3. Results
3.1. Effects of gabapentin on haloperidol-induced VCMs in mice
Fig.1 shows the effect of gabapentin administration on the frequency of VCMs of mice treated with 1.5 mg/kg of haloperidol. Haloperidol treatment increased the number of VCMs compared to the vehicle group (F(3,36)=43.30; p<0.0001). Co- treatment with GBP (100 mg/kg) attenuated haloperidol-induced VCM.
Table 1 shows the effect of GBP or haloperidol on the frequency of tongue protrusions (quantified in numbers) and facial twitching (duration in seconds). One- way analysis of variance revealed no statistical differences among the groups (p>0.05).
3.2. Effects of gabapentin on open-field behaviors
Fig.2 shows the number of locomotion (A) and rearing (B) observed in an open-field test immediately after quantification of VCMs. Haloperidol decreased the number of crossing after 28 days of treatment (F3,36)=3.602; p<0.05). The co- treatment with GBP could avoid this effect. Analysis of variance revealed no statistical differences among the groups when rearing responses were evaluated (p>0.05).
3.3. Effects of GBP and/or haloperidol in TH and GAD levels in striatum of mice
Due to the beneficial effect obtained with the treatment of 100 mg/kg of GBP in preventing haloperidol-induced VCMs, we also investigated if this effect might be involved with changes in levels of important marker of the dopaminergic system, TH and gabaergic pathway as GAD in the striatum. The analysis of western blotting revealed no change in TH (Fig. 3A) or GAD (Fig. 3B) levels in any experimental group (p>0.05).
3.4 Effects of GBP and/or haloperidol on dopamine (DA), norepinephrine (NE) and serotonin (5-HT) in striatum of mice
Any statistical difference was found to DA, 5-HT and NE levels in the striatum of mice under GBP or haloperidol treatment (Table 2).
4. Discussion
Our results demonstrated that the co-administration of the anticonvulsant GBP was effective in reducing the manifestation of VCMs induced by haloperidol in mice. In addition, haloperidol treatment caused a decrease in locomotor activity observed in the open field test which was prevented by gabapentin. However, any treatment altered markers of dopaminergic and gabaergic pathways when analyzed TH and GAD striatal immunoreactivity or monoamines levels. To our knowledge, this is the first study reporting the effects of gabapentin in an animal model of TD.
TD has been attributed to a dopaminergic supersensitivity caused by a long- term administration of antipsychotics as haloperidol by blockade of presynaptic dopamine D2 receptors leading to a compensatory increase in the synthesis and
release of dopamine. Moreover, there is a blockade of its reuptake with development of hypersensitivity postsynaptic striatal dopaminergic receptors (KLAWANS, H. L., JR., 1973; LERNER, P. et al., 1977; SULZER et al., 1995; JONES et al., 1998; SACHDEV, P. S., 2000). In rodents, a long-term administration of haloperidol causes an increase orofacial movements serving as animal model of TD. Previous studies corroborate this idea (KELLEY et al., 1989; ABI-DARGHAM, 2004; BURGER et al., 2005a; COLPO et al., 2007; FACHINETTO et al., 2007; SALAMONE et al., 2008; PEROZA et al., 2013). Some studies also related that chronic administration of
haloperidol leads a glutamate excitotoxicity, oxidative stress and GABAergic hypofunction (THAKUR et al., 2015; DATTA et al., 2016).
Data from humans and rodents studies support the evidence of GABAergic dysfunction in pathophysiology of TD after antipsychotics treatment, these changes are related to a reduction of GABA release or even loss of GABAergic terminals in basal ganglia with consequent reduction of the rate-limiting enzyme in the synthesis of GABA, GAD. This would contribute to striatal dopaminergic hyperexcitability culminating in the appearance of orofacial dyskinetic movements (GUNNE; HAGGSTROM; SJOQUIST, 1984; GERLACH; CASEY, 1988; ANDREASSEN; JORGENSEN, 2000; SACHDEV, P. S., 2000). Also, it was reported a reduction in the levels of GAD and GABA in patients with TD, supporting the "GABA hypothesis" (THAKER et al., 1987; ANDERSSON et al., 1989). Supporting this idea, the administration of GABA-mimetic drugs such as tiagabine and progabide were effective in reducing the development of VCMs induced by neuroleptics in rats (KANEDA et al., 1992; GAO et al., 1994).
Our group has demonstrated that gabapentin prevent the hyperlocomotion, memory deficit and social isolation induced by amphetamine in an animal model of schizophrenia (CERETTA et al., 2016). In the present study, GBP was also able to decrease haloperidol-induced orofacial movements as well restored locomotor activity in mice. These data suggest that GBP could promote an improvement in psychotic-like symptoms without causing OD in experimental animals.
Considering the previously described involvement of gabaergic and dopaminergic system, we also examined if the protective effect of gabapentin on haloperidol-induced VCMs in mice could involve alterations in TH and monoamines levels and/or GAD immunoreactivity. However, we did not find significant differences in any of the groups tested.
Haloperidol may exhibit biochemical changes in the dopaminergic system depending on the duration of drug treatment. At first, a short-term treatment causes an increase in dopamine synthesis characterized by TH activation. However, tolerance to repeated doses of haloperidol may happen, with return to the baseline state (LERNER, P. et al., 1977).
Studies have suggested indirect pharmacological actions on glutamate and dopamine by increasing GABA synthesis, reducing the dopaminergic activity and decreasing the excitability of glutamatergic system (RANDRUP; MUNKVAD, 1969;
WASSEF et al., 1999; TUOMINEN; TIIHONEN; WAHLBECK, 2006; LANDMARK, 2007). We suggest that as GPB was synthesized as a structural GABA analogue (inhibitory neurotransmitter), it can have pharmacological actions that lead to changes in the interneuron transmission which reinforce the inhibitory signaling pathways, may be acting indirectly in neurotransmitter levels through the release of synaptic vesicles per share in voltage-gated calcium channels (GEE et al., 1996; TAYLOR et al., 1998; GABRIEL, 2010). Thus, GBP could reduce synaptic release of neurotransmitters as dopamine and glutamate with postsynaptic decrease of calcium influx resulting in modulatory effects on excitability of the central nervous system (TAYLOR et al., 1998; STEFAN; FEUERSTEIN, 2007). GBP also increase cellular GABA by inhibiting degradation restoring the inhibitory tone of dopaminergic and glutamatergic pathways (ERRANTE; PETROFF, 2003). Our findings suggest a lack of biochemical changes in striatal dopaminergic and gabaergic metabolism. We can not rule out the possibility of an effect on different markers or neurotransmitter systems as well as testing another treatment schedule and evaluating other brain structures besides the striatum.
In clinical studies, GBP has demonstrated efficacious in the treatment of patients with bipolar disorder and schizoaffective disorder (BENNETT; GOLDMAN; SUPPES, 1997; CABRAS et al., 1999). It also had a beneficial effect on antipsychotic-induced akathisia in patients unresponsive to conventional therapy (USISKIN et al., 2000; PFEFFER; CHOUINARD; MARGOLESE, 2005), treatment of TD, the antipsychotic-induced blepharospasm, oromandibular in involuntary movements (HARDOY et al., 2003) and prophylaxis of seizures induced by clozapine (USISKIN et al., 2000). The administration of gabapentin with other antipsychotics showed an improvement in patients with treatment-resistant schizophrenia and was able to stabilize the aggressive behavior. However, with the administration of gabapentin suspension, the behavior was restored .
In conclusion, gabapentin has shown neuroprotective effect in haloperidol- induced OD and bring a new perspective that modulation in GABAergic system might be involved in the protection against antipsychotic-induced TD. Furthermore, the mechanism by which gabapentin acts seems not to involve alterations in TH and GAD markers in striatum. From clinical approaches, our findings indicate that gabapentin might have potential therapeutic use for the treatment of TD. Additional
studies should be performed to investigate the mechanisms which are involved in gabapentin effects.
Acknowledgement: This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS). We also acknowledge fellowships from CNPq, CAPES and FAPERGS All authors declare there are no potential financial, personal, or otherwise conflicts of interest.
References
Abi-Dargham, A., 2004. Do we still believe in the dopamine hypothesis? New data bring new evidence. Int J Neuropsychopharmacol 7 Suppl 1, S1-5.
Andersson, U., Haggstrom, J.E., Levin, E.D., Bondesson, U., Valverius, M., Gunne, L.M., 1989. Reduced glutamate decarboxylase activity in the subthalamic nucleus in patients with tardive dyskinesia. Mov Disord 4(1), 37-46.
Andreassen, O.A., Jorgensen, H.A., 2000. Neurotoxicity associated with neuroleptic- induced oral dyskinesias in rats. Implications for tardive dyskinesia? Prog Neurobiol 61(5), 525-541.
Araujo, N.P., Abilio, V.C., Silva, R.H., Pereira, R.C., Carvalho, R.C., Gonzalez, C., Bellot, R.G., Castro, J.P., Fukushiro, D.F., Rodrigues, M.S., Chinen, C.C., Frussa- Filho, R., 2004. Effects of topiramate on oral dyskinesia induced by reserpine. Brain Res Bull 64(4), 331-337.
Archer, J., 1973. Tests for emotionality in rats and mice: a review. Anim Behav 21(2), 205-235.
Bennett, J., Goldman, W.T., Suppes, T., 1997. Gabapentin for treatment of bipolar and schizoaffective disorders. J Clin Psychopharmacol 17(2), 141-142.
Burger, M.E., Fachineto, R., Alves, A., Callegari, L., Rocha, J.B., 2005a. Acute reserpine and subchronic haloperidol treatments change synaptosomal brain glutamate uptake and elicit orofacial dyskinesia in rats. Brain Res 1031(2), 202-210. Burger, M.E., Fachinetto, R., Zeni, G., Rocha, J.B., 2005b. Ebselen attenuates haloperidol-induced orofacial dyskinesia and oxidative stress in rat brain. Pharmacol Biochem Behav 81(3), 608-615.
Busanello, A., Barbosa, N.B., Peroza, L.R., Farias, L.E., Burger, M.E., Barreto, K.P., Fachinetto, R., 2011. Resveratrol protects against a model of vacuous chewing movements induced by reserpine in mice. Behav Pharmacol 22(1), 71-75.
Cabras, P.L., Hardoy, M.J., Hardoy, M.C., Carta, M.G., 1999. Clinical experience with gabapentin in patients with bipolar or schizoaffective disorder: results of an open-