Dopaminergic neuroprotective effects of rotigotine via 5-HT1A receptors: Possibly involvement of metallothionein expression in astrocytes

Astrocytes exert neuroprotective effects through production of antioxidant molecules and neurotrophic factors. A recent study showed that stimulation of astrocyte serotonin 1A (5-HT1A) receptors promotes astrocyte proliferation and upregulation of the antioxidant molecules metallothionein (MT)-1,2, which protect dopaminergic neurons against oxidative stress. Rotigotine, an anti-parkinsonian drug, can bind to dopamine and 5-HT1A receptors. In this study, we examined neuroprotective effects of rotigotine in models of Parkinson's disease and involvement of astrocyte 5-HT1A receptors in neuroprotective effects of rotigotine against dopaminergic neurodegeneration. Rotigotine increased the number of astrocytes and MT-1,2 expression in cultured astrocytes. Pretreatment with conditioned media from rotigotine-treated astrocytes significantly inhibited 6-hydroxydopamine (6-OHDA)-induced dopaminergic neurotoxicity. These effects were completely blocked by a 5-HT1A antagonist or MT-1,2 specific antibody. Subcutaneous administration of rotigotine increased MT-1,2 expression in striatal astrocytes and prevented reduction of dopaminergic neurons in the substantia nigra of a 6-OHDA-lesioned mouse model of Parkinson's disease. These effects were blocked by co-administration with a 5-HT1A antagonist. These results suggest that rotigotine exerts neuroprotective effects through upregulation of MT expression in astrocytes by targeting 5-HT1A receptors. Our findings provide a possible therapeutic application of rotigotine to prevent dopaminergic neurodegeneration in Parkinson's disease.


Introduction
Parkinson's disease (PD) is a progressive neurodegenerative disease. Motor symptoms such as tremor, bradykinesia, and rigidity are induced by degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc), which causes depletion of dopaminergic nerve terminals and dopamine (DA) content in the striatum (Chen et al., 2012;Dauer and Przedborski, 2003;Halliday et al., 1996). PD patients also develop non-motor symptoms including psychological manifestations such as depression, and autonomic disorders such as orthostatic hypotension and constipation (Berg et al., 2013;Grinberg et al., 2010;Singaram et al., 1995). Although pathogenesis in sporadic PD has not been characterized, oxidative stress is thought to be an important neurotoxic factor (Wang and Michaelis, 2010). Major medications used to treat PD patients provide dopaminergic therapy for managing motor disability. Therefore, it is essential to develop neuroprotectants that can prevent or delay progression of dopaminergic neurodegeneration.

Cell culture
Primary cultured neurons and astrocytes were prepared from the mesencephalon and striata of SD rat embryos at 15 days of gestation using a previously described method (Miyazaki et al., 2013). To collect astrocyte conditioned media (ACM) and examine effects of rotigotine on MT expression in astrocytes, astrocyte cultures were prepared using striata of rat embryos. Dissected striata were cut into small pieces with scissors, and then incubated in 0.125% trypsin-EDTA (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 15 min. After centrifugation (1500 g for 3 min), the cell pellet was treated with 0.004% DNase I (Sigma-Aldrich, St. Louis, MO, USA) containing 0.003% trypsin inhibitor (Thermo Fisher) at 37°C for 7 min. Following centrifugation (1500 g for 3 min), cells were plated in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen, San Diego, CA, USA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich) at a density of 2 × 10 5 cells/ cm 2 on poly-D-lysine-coated 6-well plates (Falcon, Corning, NY, USA). Cells were cultured for 4-7 days in the same medium, then subcultured to obtain enriched astrocyte cultures. Subcultured cells were plated at a density of 3.6 × 10 4 cells/cm 2 onto 6-well culture plates (Falcon) for preparation of conditioned media or nuclear protein extraction, or at a density of 2 × 10 4 cells/cm 2 onto four-chambered glass culture slides coated with poly-D-lysine (Falcon) for immunohistochemical analysis. After 7 days of culture, greater than 95% cells expressed astrocyte marker proteins.
To prepare enriched neuronal cultures, the mesencephalon was treated with 0.125% trypsin-EDTA and 0.004% DNase I containing 0.003% trypsin inhibitor as described above. Cells were plated in DMEM containing 10% FBS at a density of 2 × 10 5 cells/cm 2 on poly-Dlysine-coated four-chambered culture slides (Falcon). The medium was replaced within 24 h with fresh medium supplemented with 2 μM cytosine-β-D-arabinofuranoside (Ara-C) (Sigma-Aldrich) to inhibit replication of glial cells, and incubated for 6 days. Ninety-five percent of cells were microtubule-associated protein 2-positive neurons, and 1% of the cells were tyrosine hydroxylase (TH)-positive dopaminergic cells.
Mesencephalic neuronal and striatal astrocyte cocultures were prepared by culturing striatal astrocytes for 4 days, then seeding astrocytes at a density of 4 × 10 4 cells/cm 2 directly onto mesencephalic neurons that had been cultured on four-chambered culture slides for 4 days. All cultures were maintained at 37°C in a 5% CO 2 atmosphere.
After 24 h treatment with ACM, or rotigotine-ACM with or without the antibody, culture media were exchanged for fresh media, and neurons were exposed to 6-OHDA (25 μM) for 24 h. All ACM contained 2 μM Ara-C throughout the neuronal culture treatment period.

6-OHDA-lesioned parkinsonian mice and drug treatment
Male ICR mice weighing 38-42 g (8-week-old) were anesthetized by inhalation of isoflurane (Pfizer, Tokyo, Japan) and placed in a stereotaxic apparatus (Narishige, Tokyo, Japan). All mice received unilateral intrastriatal injections of 6-OHDA (7 μg/site, dissolved in 1 μl of 0.9% saline containing 0.1% ascorbic acid; Sigma-Aldrich) into three sites in the right striatum at the following coordinates: A +1.2 mm, L +2.0 mm, V +3.0 mm; A +0.9 mm, L +1.4 mm, V +3.0 mm; A +0.5 mm, L +2.0 mm, V +3.0 mm from the bregma, according to a mouse brain atlas (Franklin and Paxinos, 1997). The injection rate was 1 μl/2 min. After injection, the syringe was left in the striatum for an additional 3 min before being retracted slowly. Two weeks after 6-OHDA injections, mice that exhibited asymmetric rotation behavior towards the contralateral side (> 30 turns/min) after 0.5 mg/kg apomorphine injection (Sigma-Aldrich) were determined to have developed hemi-parkinsonism. One week after the apomorphine test (3 weeks after 6-OHDA injection), all parkinsonian animals were arbitrarily allocated to groups by the experimenter. Parkinsonian mice were subcutaneously injected with rotigotine (0.125, 0.25 or 0.5 mg/kg/day) or vehicle 2% DMSO in corn oil for 7 days. To examine whether agonism of 5-HT1A by rotigotine was involved in neuroprotective effects, parkinsonian mice were intraperitoneally injected with the 5-HT1A antagonist WAY100635 (0.25, 0.5 mg/kg/day, Sigma-Aldrich) dissolved in saline 1 h prior to rotigotine treatment. The following exclusion criteria were applied: more than 10% severe weight loss, severe behavioral deficits (paralysis, convulsions), or infections. No animal met the exclusion criteria during the study. One day after final injections, mice were perfused transcardially with 4% PFA for immunohistochemical analysis under deep pentobarbital anesthesia (80 mg/kg, i.p.). For Western blot analysis and ELISA, mice were perfused with ice-cold saline under deep anesthesia, and ventral midbrain and striatum tissues were dissected out immediately.
For analysis of proliferative activity induced by rotigotine, striatal astrocyte cultures were counterstained with Hoechst 33342 nuclear stain and counted cell number in 8 fields/well chosen arbitrarily under × 400 magnification. Numbers of MT-and GFAP-immunopositive cells in cell cultures were counted in 8-18 fields/well chosen arbitrarily under × 200 magnification, and expressed as the percentages of MT-immunopositive astrocytes to total cell number. The integrated density of MT was calculated as follows: integrated density (signal density -background density) × area of positive signal. TH-immunopositive cells in cultured cells were counted under a microscope in all areas of each chamber slide.
Cells immunopositive for MT-1,2, GFAP, or S100β in the dorsal striatum of normal ICR mice or parkinsonian mice were counted manually using a microscope at a magnification of × 200. The number of MT-1,2-, GFAP-, or S100β-positive cells and the ratio of MT-1,2-positive cells to GFAP-or S100β-positive cells were evaluated in each section. The number of TH-immunopositive neurons in the SNpc was counted manually under a microscope at × 100 magnification. The boundary between the SNpc and ventral tegmental area was defined by a line extending dorsally from the most medial boundary of the cerebral peduncle. The ratio of lesion area to the intact side was evaluated. No blinding was performed in the counting of immunopositive cell number and measurement of the immunoreactivity. Broken immunostained slices were excluded from the quantitative analyses.
After washing with 20 mM Tris-buffered saline containing 0.1% tween 20 (Wako Pure Chemical Corporation), the blots were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies. Signals were visualized via chemiluminescence using an ELC Western blotting detection system (GE Healthcare). Images were obtained and quantified using a FUJIFILM Luminescent Image Analyzer LAS-3000 (FUJIFILM, Tokyo, Japan) and Multi Gauge (v 3.0) software. For quantitative analysis, the signal ratio of Nrf2 (relative chemiluminescence unit) to that of constitutively expressed Lamin B or TH to α-tubulin protein was calculated to normalize for loading and transfer artifacts. The nuclear fraction for Western blot analysis with low concentration was excluded prior to electrophoresis.

Enzyme-linked immunosorbent assay (ELISA)
Levels of MT-1 in cultured astrocytes and striatal tissue of parkinsonian mice were measured by enzyme-linked immunosorbent assay (ELISA) with mouse MT-1 ELISA kit (SEB199Mu; Cloud-Clone Corp., Katy, TX, USA) according to the manufacturer's protocol. To extract total cell lysate from cultured astrocytes or striata of parkinsonian mice for ELISA, cells or tissues were homogenized using RIPA buffer containing PMSF. After centrifugation to remove cellular debris, the supernatants were stored at −80°C until analyses. Protein concentration were determined using the Lowry-based Bio-Rad DC protein assay kit (Bio-Rad) with bovine serum albumin as a standard.

Statistical analysis
Values were expressed as means ± SEM. Differences between groups were examined for statistical significance using one-way analysis of variance (ANOVA) followed by post-hoc Fisher's PLSD test. A p value less than 0.05 was considered statistically significant. The sample size in our study was determined based on our previous reports (Miyazaki et al., 2013). Data are expressed as means ± SEM (n = 3). *p < 0.05, **p < 0.01 vs. control group.
N. Isooka, et al. Neurochemistry International 132 (2020) 104608 channel sites (Forster et al., 1995). Simultaneous treatment with WAY100635 almost completely blocked rotigotine-induced MT-1,2 expression as shown in the results of MT-1,2-and GFAP-double immunostaining ( Fig. 2A-C). In this study, we measured MT-1,2 levels using total cell lysates from cultured astrocytes by ELISA. Rotigotine significantly upregulated MT levels in astrocytes, but the MT upregulation was not significantly inhibited by WAY100635. (Fig. 2D). MT-1,2 gene expression is regulated by the transcriptional factor Nrf2 (Shih et al., 2003). In the present study, we examined Nrf2 levels in nuclei of astrocytes after 6 h treatment with rotigotine (1 μM) and WAY100635 (10 nM) by Westernblot analysis. Nrf2 levels were significantly increased by rotigotine treatment, and nuclear translocation of Nrf2 was completely blocked by WAY100635 (Fig. 2E). These results suggested that rotigotine-induced Nrf2 nuclear translocation and MT upregulation occurred through astrocyte 5-HT1A receptor signaling.

Rotigotine required striatal astrocytes to protect dopaminergic neurons
To examine the role of astrocytes on neuroprotective effects of rotigotine against dopaminergic neurodegeneration, enriched mesencephalic neuronal cultures or mesencephalic neuronal and striatal astrocyte cocultures were treated with rotigotine following by oxidative stress induced by 6-OHDA exposure (Fig. 3A). In enriched neuronal cultures, rotigotine failed to prevent reduction of TH-positive dopaminergic neurons following 6-OHDA exposure ( Fig. 3B and C and Suppl. Fig. 1). Conversely, in neuronal and astrocyte cocultures, rotigotine exerted protective effects against 6-OHDA-induced damage of neuritis and reduction of dopaminergic neurons (Fig. 3B, D and Suppl. Fig. 1). These results suggested that neuroprotective effects of rotigotine are mediated by astrocytes.
N. Isooka, et al. Neurochemistry International 132 (2020) 104608 signals were colocalized with GFAP-positive astrocytes ( Fig. 9A and B). Rotigotine (0.25 mg/kg/day) treatment significantly increased MT-1,2 expression in reactive astrocytes, and WAY100635 (0.25, 0.5 mg/kg/ day) completely blocked this effect ( Fig. 9C and D). Repeated rotigotine (0.5 mg/kg/day) administration did not induce MT-1,2 expression in the striatum of parkinsonian mice, and WAY100635 (0.5 mg/kg/day) had no effect on MT expression (Suppl. Fig. 6A-C). Furthermore, we measured MT-1 content by ELISA using striatal total cell lysate from tissues of parkinsonian mice treated with rotigotine (0.25 mg/kg) and/ or WAY100635 (0.25, 0.5 mg/kg). There was a tendency to increase in MT content on the lesioned side of parkinsonian mice, but rotigotine failed to induce further increase in MT expression (Suppl. Fig. 7). To examine effects of rotigotine administration on astrocytic MT-1,2 expression in the SNpc, we performed double immunostaining of MT-1,2 and S100β or GFAP using slices of SNpc of parkinsonian mice administered with rotigotine (0.25 mg/kg) and WAY100635 (0.25 or 0.5 mg/kg). There was no change in MT-1,2 expression in the astrocytes of SNpc in any groups (Suppl. Figs. 8,9). These results suggested that rotigotine induced MT-1,2 expression in striatal astrocytes, and protected dopaminergic neurons via 5-HT1A receptor signaling in parkinsonian mice.

Discussion
In the present study, we demonstrated dopaminergic neuroprotective effect of rotigotine via 5-HT1A receptor in parkinsonian models. Rotigotine administration prevented dopaminergic neurodegeneration in parkinsonian mice, which were assessed by counting TH-positive cells. However, the results of TH Western blot analysis using tissue samples showed neuroprotective tendency, but there was no significance. By immunohistochemistry, TH-positive signal in each neuron in the lesioned side of 6-OHDA-injected mice was stronger than that in the control side. These observations suggest compensatory increase of TH expression in the lesioned-side SN of parkinsonian mice. Western blot analysis reflects expression of TH protein but not neuronal survival. Therefore, we suppose it is difficult to reproduce the results of TH immunostaining by Western blot analysis.
We showed that upregulation of MT-1,2 in astrocytes could be involved in rotigotine-mediated neuroprotection in this study. MT is a cysteine-rich protein, which has strong antioxidative, anti-apoptotic, and anti-inflammatory properties (Giralt et al., 2002;Murakami et al., 2014;Pan et al., 2013;Penkowa, 2006;Ruttkay-Nedecky et al., 2013). In the brain, two major isoforms, MT-1 and -2, are expressed mainly in astrocytes and secreted in response to oxidative stress (Chung et al., 2004). We previously demonstrated that astrocytes produced MT-1,2 in response to excess DA-induced oxidative stress, and extracellular MT-1,2 protected dopaminergic neurons against DA-induced neurotoxicity (Miyazaki et al., 2011). In addition, MT-1 binds DA quinone, a dopaminergic neuron-specific oxidative stress product formed by DA oxidation, and can protect dopaminergic neurons against DA quinone neurotoxicity (Asanuma et al., 2003;Graham, 1978;Hastings et al., 1996;Miyazaki et al., 2007;Rabinovic and Hastings, 1998). It is well known that 6-OHDA induces dopaminergic neurotoxicity via reactive oxygen species. Previous studies reported that 6-OHDA also produce pquinone by auto-oxidation, played a pivotal role in 6-OHDA-induced neurotoxicity, and glutathione (GSH) conjugated with p-quinone and provided neuroprotection (Izumi et al., 2005). Taken together with these previous reports, it is suggested that up-regulation of MT-1,2 expression induced by rotigotine can provide neuroprotection against oxidative stress-induced dopaminergic neurodegeneration. It is also supposed by a previous report, which demonstrated that rotigotine decreased reactive oxygen species in rotenone-treated primary cultured cells from midbrain (Radad et al., 2014). The involvement of up-regulation of anti-oxidative property in neuroprotective effects of rotigotine would be examined in the future study. In this study. In this study, we showed that MT expression was increased after rotigotine treatment via 5-HT1A receptor, which were assessed by counting the number of MT-1,2-positive astrocytes. Unlike the results of immunohistochemistry Fig. 6. Neuroprotective effects of rotigotine administration against dopaminergic neurodegeneration in parkinsonian mice. (A) Flow chart of the study design. 6-OHDA was administered to the right striatum of ICR mice (male, 8 weeks old) to prepare parkinsonian mice. Fourteen days after 6-OHDA injection, apomorphine rotation test was performed to confirm development of parkinsonism. Rotigotine (0.125, 0.25, 0.5 mg/kg/day) or vehicle (2% DMSO in corn oil) was administered subcutaneously to the parkinsonian mice for 7 days. Parkinsonian mice were perfused with a fixative one day after the final administration. (B) Representative photomicrographs of TH immunostaining in the SNpc of parkinsonian mice after repeated administration of rotigotine for 7 days. Scale bar: 200 μm. (C) Ratio of nigral TH-immunopositive cells in the lesioned side to the control side of parkinsonian mice after treatment with rotigotine (0.125, 0.25, 0.5 mg/kg/day, s.c.) or vehicle (2% DMSO in corn oil) for 7 days. Each value is presented as the mean ± SEM (n = 5). *p < 0.05, **p < 0.01 vs. the control side of each group, # p < 0.05 vs. the vehicletreated group. N. Isooka, et al. Neurochemistry International 132 (2020) 104608 using brain slices, ELISA showed increasing tendency of MT content in the lesioned-side striatum of parkinsonian mice, and rotigotine (0.25 mg/kg) failed to induce further increase in MT expression. As mentioned above, MT-1,2 are expressed mainly in astrocytes and protein secreted from astrocytes are consumed by neurons to reduce oxidative stress. Therefore, we suppose that it is difficult to detect changes in MT expression using tissue homogenates of parkinsonian mice. As shown in this study, MT-1,2 were induced specifically in GFAP-positive reactive astrocyte. Because astrocytes were activated dramatically in the lesioned-side striatum of 6-OHDA-injected mice, we could observe MT upregulated at least in the lesioned side. We demonstrated that rotigotine promoted Nrf2 nuclear translocation via astrocyte 5-HT1A receptor, resulting in increased MT-1,2 expression. Nrf2 is a master transcription factor, which responds to oxidative stress and induces various antioxidative molecules including MT-1,2 (Miyazaki et al., 2011;Suzuki et al., 2013aSuzuki et al., , 2013b. GSH is also synthesized in astrocytes regulated by Nrf2 and secreted into the extracellular space to protect neurons against oxidative stress. Previous studies showed that GSH levels are lower in the SN of PD patients compared to those in control subjects (Jenner et al., 1992;Riederer et al., 1989;Sian et al., 1994;Sofic et al., 1992). Therefore, we examined GSH content in conditioned media from roigotine-treated astrocytes. However, GSH levels in rotigotine-ACM were not different from levels in control-ACM (data not shown). N. Isooka, et al. Neurochemistry International 132 (2020) 104608 Previously, we reported that treatment with 5-HT1A full agonist promoted astrocyte proliferation via secretion of S100β in vitro and in vivo (Miyazaki et al., 2013). S100β is a calcium binding protein and expressed primarily in astrocytes. Stimulation of 5-HT1A receptor on astrocytes promotes S100β secretion, and extracellular S100β exerts autocrine effects that promote astrocyte proliferation. To assess effect of rotigotine on astrocyte proliferation, we counted astrocytes in cultured cells or normal or parkinsonian mice. In the present study, we used anti-GFAP and anti-S100β antibodies to detect astrocytes by immunohistochemistry. In cell cultures, all astrocytes express GFAP, so we counted GFAP-positive cells. On the other hands, an anti-GFAP antibody detected mainly fibrous activated astrocytes in the brain of mice. To assess all types of astrocytes, including protoplasmic astrocytes in vivo, we used anti-S100β antibody. Treatment with rotigotine (0.1, 1 μM) increased the number of GFAP-positive cultured astrocytes. Rotigotine also showed the increasing tendency of S100β-positive astrocyte number in normal mice and control side of parkinsonian mice. However, the number of GFAP-positive activated astrocytes was not changed by rotigotine. These results suggested that rotigotine has weak proliferation effect of astrocyte, but not astrocyte activation.
In this study, we used two doses of rotigotine (0.25 or 0.5 mg/kg). In contrast to rotigotine (0.25 mg/kg)-treated group, rotigotine (0.5 mg/kg) did not upregulate MT-1,2 expression, but the drug could inhibit the reduction of nigral dopaminergic neurons in parkinsonian mice. These data suggest that rotigotine may activate several neuroprotective pathways including not only Nrf2-MT pathway but also other pathways, which are not regulated by Nrf2, via 5-HT1A receptor. In addition, rotigotine (0.25 mg/kg) induced MT expression rather than rotigotine (0.5 mg/kg). In the previous study, we reported that effects of 5-HT1A full agonist on astrocytes are not exhibited in a dose-dependent manner (Miyazaki et al., 2013). There is an optimal dose of 5-HT1A agonist to activate astrocytic antioxidative property. Taken together, we suppose rotigotine (0.5 mg/kg) may affect other neuroprotective pathways rather than MT upregulation. WAY100635 (0.25 mg/ kg) administration could annul rotigotine (0.25 mg/kg)-induced MT upregulation in astrocytes, but not dopaminergic neuroprotective effect. As mentioned above, we demonstrated that rotigotine induced Nrf2 translocation to nuclei via 5-HT1A receptor. Taken together with these results from in vivo and in vitro, it is suggested that rotigotine exerts neuroprotective effects by not only MT upregulation but also other mechanisms induced by 5-HT1A receptor stimulation. We supposed that WAY100635 (0.25 mg/kg) is not sufficient to block all neuroprotective mechanisms. Fig. 8. Rotigotine upregulated MT-1,2 expression in S100β-positive striatal astrocytes in parkinsonian mice via 5-HT1A receptors. Effects of administration of rotigotine (0.25 mg/kg/day, s.c.) and/or WAY100635 (0.25, 0.5 mg/kg/day, i.p.) for 7 days on MT-1,2 expression in striatal astrocytes in parkinsonian mice. We performed double-immunostaining of S100β and MT-1,2. (A) Images of confocal laser microscope. Scale bar: 100 μm. (B) Representative photomicrographs of S100β and MT-1,2 double immunostaining. Scale bar: 50 μm. (C) Number of S100β-and MT-1,2-potive cells in the striatum of parkinsonian mice. (D) Ratio of MT-1,2-positive cells to S100β-positive cells. Data are presented as means ± SEM (n = 5). *p < 0.05 vs. the same side of the vehicle-treated group, # p < 0.05, ## p < 0.01 vs. the same side of the rotigotine-treated group.

Conclusion
The present study demonstrated that the anti-parkinsonian agent, rotigotine, increased expression of antioxidant MTs in astrocytes and protected dopaminergic neurons against oxidative stress via 5-HT1A receptors. Furthermore, administration of rotigotine prevented dopaminergic neurodegeneration in parkinsonian mice. These results suggested that rotigotine upregulates antioxidant molecules by targeting 5-HT1A receptors on astrocytes, resulting in neuroprotection. Our findings suggested that rotigotine may be effective as a disease-modifying treatment for PD.

Funding and conflict of interest
This work was supported by Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan. N.I., I.M., and M.A. received research and travel expenses from Otsuka Pharmaceutical Co., Ltd. Other authors have no conflicts of interest other than funding support for this study provided by Otsuka Pharmaceutical Co., Ltd. The funding organization had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
All experiments were conducted in compliance with the ARRIVE guidelines.

Author contribution statement
Nami Isooka: performed experiments and analyzed the data, writing paper. Ikuko Miyazaki: designed the research, performed experiments and analyzed the data, writing paper. Ryo Kikuoka: performed experiments and analyzed the data. Kouichi Wada: performed experiments and analyzed the data. Erika Nakayama: performed experiments and analyzed the data. Kotaro Shin: performed experiments and analyzed the data. Daichi Yamamoto: performed experiments and analyzed the data. Yoshihisa Kitamura: assisted with some of the in vivo experiments. Masato Asanuma: designed the research, organized the project, writing paper. Effects of administration of rotigotine (0.25 mg/kg/ day, s.c.) and/or WAY100635 (0.25, 0.5 mg/kg/ day, i.p.) for 7 days on MT-1,2 expression in striatal astrocytes in parkinsonian mice. We performed double immunostaining of GFAP and MT-1,2. (A) Images of confocal laser microscope. Scale bar: 100 μm. (B) Representative photomicrographs of GFAP and MT-1,2 double immunostaining. Scale bar: 50 μm. (C) Number of GFAP-and MT-1,2-positive cells in the striatum of parkinsonian mice. (D) Ratio of MT-1,2-positive cells to GFAP-positive cells on the lesioned side. Data are presented as means ± SEM (n = 5). *p < 0.05 vs. the same side of the vehicle-treated group, # p < 0.05, ## p < 0.01 vs. the same side of the rotigotinetreated group.