Parkinson’s disease (PD) is characterized by degeneration of nigrostriatal dopamine (DA) neurons. The primary drug used to treat PD symptoms is L-DOPA, but side effects such as dyskinesias limit its use. Previous findings show that L-DOPA treatment induces extracellular signal-regulated kinase (ERK1/2), a MAP-kinase protein. γ-aminobutyric acid (GABA) is intimately involved in basal ganglia function. Our previous study using a unilaterally lesioned rat model of PD indicated that elevating GABA levels by GABA transaminase inhibitor, aminooxyacetic acid significantly attenuated L-DOPA-induced ERK phosphorylation in the striatum and substantia nigra (SN). The aim of the present study was to assess the role of GABA-A and GABA-B receptor by using a selective agonists, muscimol and baclofen respectively, on L-DOPA-induced ERK phosphorylation in the striatum and SN. Unilaterally 6-OHDA-lesioned rats were prescreened by apomorphine induced rotation test for the extent of DA loss. Lesioned rats were treated with L-DOPA alone or after muscimol or baclofen pretreatment. Appropriate control groups were used. Phospho-ERK levels, tyrosine hydroxylase (to ascertain DA loss) and substance P (an indirect marker for DA loss) levels were assessed by immunohistochemistry using coronal slices at the level of striatum and SN. L-DOPA administration induced a robust increase (>300%) in phospho-ERK1/2 levels in the striatum and SN. Muscimol as well as baclofen pretreatment attenuated the L-DOPA-induced increase in phospho-ERK1/2 levels by >60% in the striatum and SN. Muscimol and baclofen pretreatment also greatly reduced the number of L-DOPA induced phospho-ERK1/2 stained cells in the striatum as well as the contralateral rotational behavior. The present data taken together with our previous study indicate that the L-DOPA induced increase in ERK1/2 is attenuated by GABA via a GABA-A and GABA-B receptor linked mechanism. The study provides further insight into a dopamine-GABA-ERK interaction in the therapeutic and/or side effects of L-DOPA in the basal ganglia.
Dopaminergic neurons in the nigrostriatal pathway of the basal ganglia degenerate in Parkinson’s disease (PD). An imbalance in DA function leads to motor complications associated with the disease [1-3]. D1 receptors receive diminishing amounts of DA compensation by becoming supersensitive to the neurotransmitter. It is well established that L-DOPA and other DA agonists ameliorate certain motor deficits [4-6]. Long-term administration of L-DOPA, however, often leads to side effects. These include dyskinesias, characterized by loss of voluntary motor control as well as the emergence of involuntary motor control [7,8].
Numerous reports have stated that activation of supersensitive D1 receptors in the DA-depleted striatum leads to the phosphorylation of ERK1/2, which belongs to a class of mitogen-activated protein kinases (MAPKs) and is implicated in transcriptional and translational efficiency [5,9,10]. Reports involving both hemiparkinsonian rodents [10-12] and non-human primates [5,8,13]
have attributed this phenomenon to the development of motor side effects. The involvement of supersensitive D1 receptor stimulation and ERK1/2 signaling in the striatum and SN was further supported by more recent studies. D1 agonist, SKF-38393 increased phospho-ERK1/2 levels in the striatum and in the substantia nigra (SN), and both responses were blocked by D1 antagonist SCH- 23390 [
GABAergic medium-sized spiny neurons (MSNs) comprise about 95% of the striatum [
GABA receptors are of two major subtypes: ionotropic (GABA-A) and metabotropic G-protein-coupled (GABAB) receptors [
Animals were kept as previously described [
Lesions of the nigrostriatal DA pathway were made in the right median forebrain bundle (MFB) as previously described [
The extent of the unilateral 6-OHDA lesion was assessed by apomorphine-induced rotation test as previously described [
The following groups of 6-OHDA-lesioned animals were used: a) vehicle + vehicle (control); b) vehicle + LDOPA; c) muscimol + vehicle; d) muscimol + L-DOPA; e) vehicle + baclofen; f) L-DOPA + baclofen; the numbers of animals in each group were 3, 6, 3, 6, 3,6 respectively. In the present study we administered muscimol or baclofen 30 min prior to L-DOPA and perfused the animals transcardially 30 minutes after L-DOPA administration. RO-4-4602 (50 mg/kg, i.p., Hoffmann-La Roche, Inc.) was given 30 minutes prior to L-DOPA administration (94.5 mg/kg, i.p., Sigma-Aldrich, Inc.). Vehicle + vehicle served as the independent control group. The experimental design allowed the comparison of not only the relative changes from the lesioned versus intact side in the same animal, but also each drug treatment group versus the independent control group. Ten minutes after L-DOPA injection, rats were observed for 5 minutes to observe the rotational response. An overdose of sodium pentobarbital (100 mg/kg, i.p., Sigma-Aldrich, Inc.) was used to perfuse animals.
The protocol for immunohistochemistry is as previously described [
Quantification of Immunostaining. The immunostaining intensities for TH, SP and phospho-ERK1/2 in the striatum and SN of the intact and lesioned sides were determined as previously described [
Quantification of Striatal Phospho-ERK1/2 Labeled Cell Counts. Counts of phospho-ERK1/2 immunostained cells were performed as previously described [
Stastical Analysis. All values are depicted as mean ± SEM. Group means were compared using SigmaStat (Systat Software, Inc.) to apply one-way analysis of variance followed by Newman-Keuls multiple range test. P < 0.05 was considered significant.
In control, muscimol or baclofen-treated animals, no rotations were observed. In animals treated with vehicle + L-DOPA, an average of 13.3 ± 1.6 rotations per minute was observed. Finally, in animals pretreated with muscimol or baclofen followed by L-DOPA, averages of only 0.9 ± 0.6 and 0.9 ± 0.5 rotations, respectively, were observed, indicating that muscimol or baclofen pretreatment greatly attenuated L-DOPA-induced rotations (
Immunoreactive Intensity of TH, SP, and PhosphoERK1/2 in the Striatum (Figures 2 and 3). A greater than 90% decrease in TH immunoreactivity in the lesioned striatum was observed for control, muscimol, baclofen, L-DOPA, muscimol + L-DOPA and baclofen + L-DOPA groups. The lesioned striatum of animals in all aforementioned groups demonstrated a moderate loss of SP immunoreactivity. Muscimol, baclofen, L-DOPA, musicmol + L-DOPA or baclofen + L-DOPA treatments did not alter the basal loss of TH or SP immunoreactivity in the lesioned striatum. In the unlesioned side (intact striatum), none of the treatment groups showed phosphoERK1/2 immunoreactivity. In control, muscimol or baclofen treated rats, phospho-ERK1/2 immunoreactivity was not apparent in the lesioned or intact striatum. LDOPA treatment resulted in a robust activation of phospho-ERK1/2 in the striatum in the lesioned side as compared to the intact side. Pretreatment of muscimol or baclofen to stimulate GABA-A or GABA-B receptors followed by L-DOPA administration resulted in significantly lower levels of phospho-ERK1/2 in the striatum as compared to phospho-ERK1/2 in the L-DOPA group.
Cells Labeled with Phospho-ERK1/2 in the Striatum (
cimol + L-DOPA and baclofen + L-DOPA treatments significantly decreased the amount of phospho-ERK1/2 labeled cells as compared to the rats treated with LDOPA alone.
Results similar to those seen in the striatum were observed within the SN with regard to changes in TH, SP, and phospho-ERK1/2 immunoreactivity. L-DOPA treatment resulted in the robust activation of phospho-ERK1/ 2 in the SN. Administration of muscimol or baclofen to stimulate GABA-A or GABA-B receptors followed by L-DOPA treatment resulted in significantly lower levels of phospho-ERK1/2 in the SN as compared to phospho-ERK1/2 in the L-DOPA group (Figures 2 and 3).
In the present study, we reported a new finding that pretreatment with GABA-A agonist, muscimol, or GABA-B agonist, baclofen attenuated the L-DOPA-induced rotational response as well as increase in phospho-ERK1/2 levels both in the striatum and SN. These data together with our previous study [
Basal ganglia activity depends on the direct (striatonigral) and indirect (striatopallidal) pathway, subserved by D1 and D2 receptors, respectively [
GABAergic medium-sized spiny neurons (MSNs) are morphologically the main neuron type in the striatum [15,16] and the SN pars reticulata (SNr) [15,16]. DA regulates GABA-releasing MSN activity by activating DA receptors [8,32,33]. It has been reported that LDOPA administration induces alterations in GABA transmission in the striatum and SN [17,18,20]. Restored DA activity via D1 receptor activation by L-DOPA administration in unilaterally 6-OHDA lesioned rats results in enhanced GABA transmission in the basal ganglia [
The D1 receptor-mediated enhancement of GABA transmission has been linked to GABA-A receptor activation [17,35]. GABA-A receptor activation resulted in increased striatal and nigral DARPP-32 phosphorylation [
Ionotropic GABA-A receptors are generally located on symmetrical synapses, whereas metabotropic GABA-B receptors are found in all regions of the plasma membrane. Because of this localization, it has been concluded that GABA-A receptors in the basal ganglia mediate fast GABA transmission, and GABA-B receptors are involved in subtle and more complex GABAergic effects [
Other studies have also suggested that GABA may have a beneficial rather than detrimental effect in treating PD. Local GABA-A receptor activation in the striatum or SNr offers beneficial effects in experimental Parkinsonism. GABA-A receptor agonist muscimol, injected into the SNr of parkinsonian monkeys, has been reported to ameliorate motor symptoms [
GABA is also implicated in the effects of adenosine A2A receptor antagonists that appear to be useful in the treatment of PD. Adenosine A2A receptors are densely localized in the basal ganglia, concentrated on GABAergic MSNs [
In summary, we report for the first time that stimulation of GABA-A or GABA-B receptors attenuates the L-DOPA-induced phospho-ERK1/2 levels in the striatum and SN and also L-DOPA induced rotations. The results of this study further support the importance of GABA systems in L-DOPA induced effects, suggesting the possibility of GABAergic drugs to enhance therapeutic benefits and/or reduce motor side effects of L-DOPA therapy.
This work was supported in part by a Research Enhancement Award from IUPUI, a summer research internship award from IUSM-NW, and an award from IUN Research Fund. The generous supply of SP antiserum used in this study from Dr. J.-S. Hong, NIEHS, Research Triangle Park is greatly appreciated.