DRD3 is selectively expressed in astrocytes but not in microglial cells

We previously appreciated a selective expression of the drd3 transcript in primary astrocytes, which was not detected in primary microglial cells [31]. To evaluate whether this differential expression is actually observed at the protein level, we performed immunofluorescence analyses in which we compared the expression of DRD3 in primary cultures of microglia and astrocytes obtained from wild-type (WT) C57BL/6 mice. In these analyses, the PC12 cell line, which has been shown to express DRD3 [38], was used as a positive control. The results show significant immunoreactivity associated to the anti-DRD3 antibody in comparison to the isotype matched control in both primary astrocytes and PC12 cells, but not in primary microglial cells (Fig. 1). Thus, these results suggest that, in agreement with our previous analyses at the level of drd3 transcript, DRD3 is selectively expressed in astrocytes but not in microglial cells.

Fig. 1 DRD3 is selectively expressed in astrocytes but not in microglial cells. Astrocyte (top panels) and microglial (middle panels) cultures were obtained from wild-type mice as described in materials and methods. PC12 cell line was used as a positive control for DRD3 expression (bottom panels). Cells were immunostained with anti-DRD3 antibody (left panels) or an isotype-matched control (right panels) primary antibody followed by an Alexa488-coupled secondary antibody and the immunofluorescence (green) associated was analysed by fluorescence microscopy. Nuclei were stained with DAPI (blue). Representative photomicrographs are shown. A higher magnification section is inserted in the bottom left corner of each image. Bar, 10 μm Full size image

To evaluate whether DRD3 immunoreactivity is also associated to astrocytes in vivo upon inflammatory conditions, WT mice were treated with systemic LPS (5 mg/kg) or vehicle (PBS) and 3 h later sacrificed and the immunoreactivity for DRD3 and GFAP were analysed in brain slices, since previous results have shown that neuroinflammation is already evident in these conditions [36]. For this purpose, we first analysed GFAP expression in different areas of the brain in mice treated with LPS (Additional file 1: Figure S1). Despite high GFAP expression was observed in different areas of the brain (Additional file 1: Figure S1), subventricular zone was chosen to further analysis of the co-expression of GFAP and DRD3, as individual GFAP+ cells were clearly identified in this region. To confirm the specificity of the immunoreactivity associated to the anti-DRD3 antibody used in this analysis, this antibody was pre-incubated with the immunogen used to develop the antibody, the peptide DRD3 15-29 . Of note, we have recently demonstrated how the immunoreactivity associated to this anti-DRD3 antibody is lost in DRD3-deficient cells [25]. The results show that DRD3-immunoreactivity is in part associated to GFAP-immunoreactivity (Fig. 2a, top panels). Furthermore, when the specific immunoreactivity was abrogated by pre-incubating the anti-DRD3 antibody with the DRD3 15-29 peptide, most DRD3-immunoreactivity disappeared (Fig. 2a, top panels). In addition, to quantify the degree of colocalization, we used the intensity correlation analysis method [37]. As observed in pseudocolored images (Fig. 2a, bottom panels), DRD3 intensity variations synchronize better with stronger GFAP intensities (GFAP+ cells) than with background random intensities in surrounding structures. Interestingly, the quantification of intensity correlation quotients (ICQ) shows a significant increase in the colocalization of DRD3 and GFAP when mice were treated with systemic LPS in comparison with those mice treated with PBS (Fig. 2b). Moreover, the degree of colocalization of GFAP and DRD3 immunostaining in brain samples obtained from LPS-treated mice was significantly lower when the anti-DRD3 antibody was pre-incubated with the peptide DRD3 15-29 (Fig. 2b), indicating that GFAP+ cells colocalize with the specific DRD3 immunoreactivity in LPS-treated mice. Of note, the degree of colocalization of DRD3 and GFAP immunoreactivity was not significantly different in the presence or in the absence of the peptide DRD3 15-29 in samples obtained from PBS-treated mice (Fig. 2b), suggesting that there is no significant DRD3 colocalizing with GFAP+ cells in steady-state. Together, these results suggest that, at least in part, specific DRD3 immunoreactivity is associated with astrocytes in the mouse brain upon systemic inflammation.

Fig. 2 DRD3 specific immunoreactivity is associated to astrocytes in the mouse brain upon systemic inflammation. Wild-type mice received an i.p. injection of PBS or LPS (5 mg/kg) and 3 h later were sacrificed and immunofluorescence analysis was performed in brain slices. To determine the specific DRD3 immunostaining, a polyclonal anti-DRD3 antibody was pre-incubated or not with the peptide DRD3 15-29 (CGAENSTGVNRARPH) used as immunogen to develop the antibody. Afterward, brain sections were incubated with untreated anti-DRD3 or with anti-DRD3 pre-incubated with peptide DRD3 15-29 , followed by incubation with the AlexaFluor488-coupled secondary antibody (green). Samples were subsequently immunostained with anti-GFAP-PE (red) antibody and nuclei were stained with DAPI (blue). a Representative images of the subventricular zone are shown (top panels; bar, 5 μm). Pseudocolored images showing areas in which DRD3-associated intensity correlates positively with GFAP-associated intensity are shown in bottom panels (bar, 5 μm). b Intensity correlation analysis of confocal microscopy images obtained from DRD3 and GFAP immunostaining. Colocalization intensity was determined as the intensity correlation quotients (ICQ). Data from four mice per group is shown. Values are the mean ± SEM. *p < 0.05; **p < 0.01; by two-way ANOVA followed by Sidak’s post-hoc test Full size image

The inhibition of DRD3 signalling attenuates microglial activation in the brain of mice undergoing systemic inflammation induced by LPS

To determine the in vivo relevance of DRD3 in glial activation, we next performed a set of experiments in which neuroinflammation was induced by the systemic administration of LPS [36] in mice harbouring the genetic deficiency of DRD3 or undergoing pharmacological inhibition of DRD3 signalling. For this purpose, a DRD3 selective antagonist PG01037 was i.p. administered at 30 mg/kg, a drug that has been previously proven to cross the blood-brain barrier and a dose that exerts a therapeutic effect attenuating neurodegeneration in two different mouse models of Parkinson’s disease [31]. Accordingly, WT or DRD3KO mice were pre-treated or not with PG01037 and then systemic inflammation was induced by a single injection of LPS (5 mg/kg). Since previous studies have shown that systemic LPS administration triggers the loss of dopaminergic neurons of the nigrostriatal pathway [36, 39], we focused the analysis of neuroinflammation in the midbrain of mice treated with LPS. To validate this animal model of neuroinflammation in our hands, we first determined whether inflammatory cytokines were elevated in the brain of LPS-treated animals. Accordingly, we found that transcripts for TNF-α and IL-1β were strongly increased in the midbrain and striatum of mice 24 h after systemic LPS administration (Additional file 1: Figure S2A and B). To determine the activation of microglia, we first performed an immunohistochemical analysis of Iba1 3 h after LPS-treatment as described before [36]. The results show that after LPS treatment, WT mice present a significant increase in the percentage of microglial cells with typical activated phenotype, including amoeboid shape and high density of Iba1 expression (Fig. 3a, b). Conversely, DRD3-deficient mice presented a significant reduction in the percentage of activated microglia in steady-state conditions, and the degree of microglial activation was not changed after LPS treatment (Fig. 3a, b). Thus, these results suggest that DRD3 deficiency results in attenuated microglial activation upon systemic LPS treatment. As a complementary approach to evaluate the role of DRD3 signalling in the acquisition of inflammatory phenotype by microglial cells, we determined the M1 and M2 phenotypes acquired by microglia 24 h after LPS treatment in WT and DRD3KO mice. To this end, we evaluated surface markers defining the M1 (CD16/32+ CD206−) and M2 (CD16/32+ CD206+) phenotypes in the gate of microglial cells (CD11b+ CD45+) by flow cytometry (Fig. 4a, b) as described before [6]. The results show that the percentage of M1 microglia was not affected by genetic deficiency or pharmacologic antagonism of DRD3-signalling (Fig. 4c and Additional file 1: Figure S3). Nevertheless, the percentage of M2 phenotype in microglial cells was significantly reduced upon DRD3 antagonism in LPS-treated WT mice (Fig. 4c). Accordingly, the percentage of M2 microglia was lower in LPS-treated DRD3KO mice in comparison to LPS-treated WT mice (Fig. 4c). As expected, DRD3-antagonism had no effect in the extent of M1 or M2 percentages in microglia of DRD3-deficient mice (Fig. 4c). According to the changes observed in M2 microglia, genetic deficiency or pharmacologic antagonism of DRD3 signalling resulted in increased M1-to-M2 ratio in microglial cells upon LPS-induced neuroinflammation (Fig. 4c). To further characterize the effect of DRD3 signalling in microglial activation upon LPS-induced neuroinflammation, we also evaluated the density of surface expression of key molecular markers associated to microglial activation, including CD11b (also known as Mac-1), CD45, CD16/32 and CD206. As expected, surface density of CD11b and CD45 was increased upon LPS treatment; however, neither DRD3 antagonism in WT mice nor DRD3 deficiency affected these parameters (Additional file 1: Figure S4). Similarly, no relevant changes in surface density of CD16/32 and CD206 were observed in microglia upon genetic-deficiency or pharmacologic antagonism of DRD3 signalling (Additional file 1: Figure S4).

Fig. 3 DRD3 deficiency results in attenuated microglial activation upon systemic LPS treatment. Wild-type (WT) or DRD3 knockout (DRD3KO) mice received an i.p. injection of LPS (5 mg/kg) or PBS. After 3 h, mice were sacrificed, and microglial activation was analysed by immunohistochemical analysis of Iba1 in the striatum. a Representative overview images at low magnification (× 20) are shown. High magnification (× 100) images are inserted in the bottom-right corner of each overview image. b Quantification of the density of Iba1high cells with ameboid shape. Data from five to six mice per group is shown. Values are the mean ± SEM. *p < 0.05; ***p < 0.001; ****p < 0.0001 by two-way ANOVA followed by Sidak’s post-hoc test Full size image

Fig. 4 Genetic deficiency or pharmacologic antagonism of DRD3-signalling attenuates the acquisition of M2-phenotype by microglial cells in the midbrain of mice undergoing systemic inflammation induced by LPS. Wild-type (WT) or DRD3 knockout (DRD3KO) mice were pre-treated or not with an i.p. injection of a DRD3-selective antagonist (PG01037; 30 mg/kg) and 1 h later received an i.p. injection of LPS (5 mg/kg) or PBS. Twenty-four hours after LPS administration, the midbrain/striatum structures were isolated, disaggregated, and M1 and M2 phenotypes were analysed in microglial cells by flow cytometry. a Schematic illustration of the experimental design. b Gating strategy used to analyse the M1 (CD16/32+CD206- cells) and M2 (CD16/32+CD206+ cells) phenotypes in living (ZAq−) microglial cells (CD11b+ CD45+). c Quantification of the frequencies of M1 (left panel) and M2 (middle panel) phenotypes and the M1-to-M2 ratio (right panel). Data from five mice per group is shown. Each symbol represents a WT (white) or a DRD3KO (black) animal. In each experimental group, the line and error bars represent the mean ± SEM, respectively. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc test. Black asterisks represent significant differences between treatments, whilst grey asterisks represent significant differences between genotypes Full size image

To further analyse the effect of DRD3 signalling in the dynamic of microglial activation, the acquisition of M1 and M2 phenotypes by microglial cells was evaluated at earlier time-points after LPS-induced neuroinflammation. For this purpose, we determined the percentage of M1 and M2 microglia 4 h after systemic LPS administration, as it has been shown that a number of pro- and anti-inflammatory cytokines produced by microglial cells peaked at this time-point after LPS-induced neuroinflammation [40]. Moreover, consistently 4 h after systemic LPS treatment constitutes a time-point in which neuroinflammation is already appreciated in our hands (Additional file 1: Figure S2B). The results show a similar extent of increased percentage of M1 phenotype and decreased percentage of M2 microglia in WT and DRD3KO mice upon LPS treatment (Additional file 1: Figure S5A). Nevertheless, according to the effect of DRD3 signalling observed at 24 h after LPS treatment (Fig. 4c), the M1-to-M2 ratio was significantly higher in DRD3-deficient mice even after 4 h of LPS-induced neuroinflammation (Additional file 1: Figure S5A). Thus, these results together suggest that DRD3 signalling would be affecting the inflammatory behaviour of microglial cells upon LPS-induced inflammation.

DRD3 deficiency results in unresponsiveness of astrocytes upon LPS-induced neuroinflammation

Since astrocytes might strongly regulate microglial behaviour [41] and astrocytes but not microglia express DRD3 (Fig. 1), we also aimed to evaluate whether DRD3 signalling affects the phenotype of astrocytes upon LPS-induced neuroinflammation. To determine the acquisition of activated phenotype by astrocytes, we performed immunohistochemical analyses of GFAP+ cells of WT and DRD3-deficient mice 3 h after LPS-treatment. The results show similar extent of astrogliosis of WT and DRD3KO mice both in control conditions or after LPS treatment (Fig. 5a, b). Despite we observed just a slight increase of GFAP immunoreactivity after LPS-treatment, only WT, but not DRD3KO mice, presented a significant increase of astrogliosis after LPS treatment (Fig. 5a, b). To gain deeper insight in the involvement of DRD3 signalling in astrocyte activation, 24 h after LPS treatment, we evaluated molecular markers defining the inflammatory (inducible nitric oxide synthase; iNOS) and anti-inflammatory (arginase 1; Arg-1) phenotypes in the gate of astrocytes (GFAP+) by flow cytometry (Fig. 6a) as describe before [42]. Interestingly, whereas the percentage of inflammatory phenotype of astrocytes (iNOS+ GFAP+ cells) was increased in WT mice, it was unaltered in DRD3-deficient mice after systemic LPS treatment (Fig. 6b). However, the frequency of anti-inflammatory phenotype of astrocytes was not affected in WT or DRD3KO mice at this time-point (Fig. 6b). Accordingly, the inflammatory-to-anti-inflammatory ratio of astrocytes was exclusively increased in WT mice but not in DRD3-deficient mice 24 h after LPS-induced neuroinflammation (Fig. 6b). Of note, neither inflammatory nor anti-inflammatory phenotypes were affected by DRD3 signalling at an earlier time-point (4 h) after LPS-induced neuroinflammation (Additional file 1: Figure S5B). To further characterize the effect of DRD3 signalling in astrocyte activation upon LPS-induced neuroinflammation, we also evaluated the density of expression of astrocytic molecular markers, including GFAP, iNOS and Arg-1; however, we found no differences between WT and DRD3KO mice (Additional file 1: Figure S6). Thereby, taken together, these results (Fig. 6 and Additional file 1: Figure S5B and S6) indicate that the genetic deficiency of DRD3 signalling results in an unresponsive phenotype of astrocytes upon LPS-induced neuroinflammation.

Fig. 5 DRD3 deficiency results in unresponsiveness of astrocytes upon systemic LPS treatment. Wild-type (WT) or DRD3 knockout (DRD3KO) mice received an i.p. injection of LPS (5 mg/kg) or PBS. After 3 h, mice were sacrificed, and astrogliosis was analysed by immunohistochemical analysis of GFAP in the striatum. a Representative overview images at low magnification are shown (bar, 100 μm). b Quantification of GFAP immunoreactivity density. Data from five to six mice per group is shown. Values are the mean ± SEM. *p < 0.05 by two-way ANOVA followed by Sidak’s post-hoc test Full size image

Fig. 6 DRD3 deficiency results in an unresponsive phenotype of astrocytes in the midbrain of mice undergoing systemic inflammation induced by LPS. WT or DRD3KO mice were treated with an i.p. injection of LPS (5 mg/kg) or PBS (control). Twenty-four hours later, the midbrain/striatum structures were isolated, disaggregated and the inflammatory and anti-inflammatory phenotypes of astrocytes were analysed by flow cytometry. a Gating strategy used to analyse the inflammatory (iNOS+ cells) and anti-inflammatory (Arg1+ cells) phenotypes in living (ZAq−) astrocytes (GFAP+ cells). b Representative contour-plots indicating the percentage of pro-inflammatory glia (red numbers) and anti-inflammatory glia (blue numbers). c Quantification of the frequencies of inflammatory (left panel) and anti-inflammatory (middle panel) phenotypes and the inflammatory-to-anti-inflammatory ratio (right panel). Data from eight mice per group is shown. Each symbol represents a WT (white) or a DRD3KO (black) animal. In each experimental group, the line and error bars represent the mean ± SEM, respectively. *p < 0.05; **p < 0.01 by one-way ANOVA followed by Tukey’s post-hoc test Full size image

Deficiency of DRD3 signalling results in exacerbated production of the anti-inflammatory mediator Fizz1 and decreased expression of the pro-inflammatory enzyme iNOS

To gain a deeper insight of the role of DRD3-signalling in neuroinflammation, we next aimed to evaluate a set of inflammatory and anti-inflammatory molecules in the brain of WT and DRD3-deficient mice upon systemic LPS treatment. For this purpose, we induced neuroinflammation by the i.p. administration of LPS in WT and DRD3KO mice and after 24 h the total RNA was isolated and quantitative RT-PCR analysis was performed to determine the extent of transcription of the pro-inflammatory mediators iNOS, IL-1β and TNF-α and of the anti-inflammatory molecules Arg-1 and Fizz1. The results show that DRD3 deficiency resulted in a selective and strong decrease of iNOS transcription upon LPS treatment (Fig. 7a). Furthermore, we noticed an increased transcription of Fizz1 in DRD3KO mice in basal conditions (Fig. 7b). Thus, these results indicate that despite DRD3 deficiency results in an exacerbated M1-to-M2 ratio in microglia (Figs. 4 and Additional file 1: S5A) and unresponsive phenotype in astrocytes (Figs. 5 and 6), the overall effect of the lack of DRD3 in LPS-induced neuroinflammation is an increased production of an anti-inflammatory mediator and the reduction in the extent of expression of a pro-inflammatory enzyme.

Fig. 7 DRD3 deficiency results in altered expression of inflammatory and anti-inflammatory mediators in the midbrain of mice basally and undergoing systemic inflammation induced by LPS. WT (white) or DRD3KO (black) mice were treated with an i.p. injection of LPS (5 mg/kg) or PBS (control). Twenty-four hours later, the midbrain/striatum structures were isolated, disaggregated and the RNA was extracted and analysed by quantitative RT-PCR. Inflammatory (a) and anti-inflammatory mediators were evaluated (b). Gapdh transcript was used as a house keeping for normalization. Data from three to eight mice per group is shown. Values are the mean ± SEM. *p < 0.05 by two-way ANOVA followed by Sidak’s post-hoc test Full size image

Deficiency of DRD3 signalling in glial cells leads to enhanced expression of pro-inflammatory cytokines and exacerbated production of the anti-inflammatory mediator Fizz1 in response to inflammatory and anti-inflammatory stimuli respectively

Since experiments commented above were carried out in WT or global DRD3KO mice, the effects observed could be due to the deficiency of DRD3 signalling in astrocytes or other kind of cells that normally express DRD3. To analyse the precise role of DRD3 signalling confined to glial cells in the production of mediators in response to inflammatory or anti-inflammatory environments, we next performed experiments in primary cultures of mixed glial cells containing both, astrocytes and microglia. For this purpose, primary cultures of mixed glial cells were generated from the midbrain/striatum of WT or DRD3-deficient newborn mice and challenged with pro-inflammatory or anti-inflammatory environments given by LPS or IL-4 respectively, and the extent of transcripts codifying for a panel of cytokines, enzymes and transcription factors related with the regulation of neuroinflammation was determined by quantitative RT-PCR (qRT-PCR). The results show that DRD3 deficiency in glial cells resulted in a selective increase in the transcription of the gene codifying for IL-1β and without effect in other mediators, enzymes or transcription factors evaluated in response to LPS (Fig. 8a–c). Conversely, the lack of DRD3 signalling in glial cells resulted in exacerbated transcription of the anti-inflammatory mediator Fizz1, without detectable effects in other mediators evaluated in response to an anti-inflammatory environment given by IL-4 (Fig. 8a–c).

Fig. 8 DRD3 deficiency results in a selective and strong exacerbation in the production of fizz1 in mixed glial culture in response to IL-4. Mixed glial cultures were generated from the midbrain/striatum structures obtained from WT (white) or DRD3KO (black) mice, which were left untreated (control) or treated with LPS (1 μg/ml) or IL-4 (25 ng/ml). Twenty-four hours later, the RNA was extracted and analysed by quantitative RT-PCR. Inflammatory (a), anti-inflammatory mediators (b) and neurotrophic factors (c) were evaluated. Gapdh transcript was used as a house keeping for normalization. Data from three (nrf2, bdnf, igf1, nt3), five (ym1), six (il1b, fizz1, gdnf) and seven (inos, arg1) independent experiments is shown. Values are the mean ± SEM. *p < 0.05 by two-way ANOVA followed by Sidak’s post-hoc test Full size image

To confirm the most relevant differences obtained in mixed glial cultures at the level of protein, we next evaluated the production of IL-1β and Fizz1 in the supernatant of the cultures after 24 h of LPS or IL-4 treatment. The results show that DRD3 deficiency in glial cells leads to approximately three-fold higher production of Fizz1 in the supernatant in response to an anti-inflammatory environment given by IL-4 (Fig. 9a, left panel), thus confirming the results obtained at the levels of fizz1 transcripts (Fig. 8b). Conversely, the concentration of IL-1β was undetectable under all conditions tested here (data not shown). To evaluate another relevant pro-inflammatory cytokine associated to neuroinflammation, we evaluated the production of TNFα in the supernatant of mixed glial cultures. Interestingly, the results show that DRD3 deficiency in glial cells leads to increased secretion of TNFα within the supernatant of the culture in response to a pro-inflammatory environment given by LPS treatment (Fig. 9a, middle panel). In addition, we determined the expression of the classic astrocytic pro-inflammatory marker iNOS in the GFAP+ population. In agreement with results obtained at the level of inos transcription (Fig. 8a), we did not observe differences in the extent of iNOS expression at the level of protein between astrocytes from both genotypes at any of the conditions tested (Fig. 9a, right panel). Thus, together, these results suggest that DRD3 signalling confined to astrocytes plays a dual role limiting the production of inflammatory cytokines in response to a pro-inflammatory environment, but also attenuating Fizz1 secretion by glial cells in response to anti-inflammatory cues.

Fig. 9 DRD3 signalling induces a pro-inflammatory profile in astrocytes. a Mixed glial cultures were generated from the midbrain/striatum structures obtained from WT (white) or DRD3KO (black) mice, which were left untreated (control) or treated with LPS (1 μg/ml) or IL-4 (25 ng/ml). Twenty-four hours later, the secretion of Fizz1 and TNF-α was determined in the supernatant of cultures by ELISA (left and middle panels) and the extent of iNOS expression (right panel) was determined by flow cytometry. Values are the mean ± SEM from triplicates. *p < 0.05; ****p < 0.00001 by two-way ANOVA followed by Sidak’s post-hoc test. a Astrocytes were generated from the midbrain/striatum structures obtained from WT mice. Cells were left untreated (control) or treated with dopamine (100 nM), a DRD3-selective agonist (PD128907, 20 nM) or LPS (1 μg/ml) for 24 h and the expression of iNOS was quantified in the GFAP+ population by flow cytometry. Numbers represent the frequency of cells iNOS+. Values are the mean ± SEM from triplicates. *p < 0.05; **p < 0.01 by one-way ANOVA followed by Tukey’s post-hoc test. c Astrocytes were generated from the midbrain/striatum structures obtained from WT mice. The cells were left untreated (control) or treated with LPS (1 μg/ml) or IL-4 (25 ng/ml) in the absence or presence of dopamine (100 nM) or PD128907 (20 nM) for 24 h. Afterwards, the RNA was extracted and the levels of drd3 transcripts were evaluated by quantitative RT-PCR. Gapdh transcript was used as a house keeping for normalization. Values are the mean ± SEM from triplicates. **p < 0.01 by two-way ANOVA followed by Sidak’s post-hoc test. Representative data from one out of two independent experiments is shown Full size image

To gain a further mechanistic insight of the role of DRD3 signalling in the pro-inflammatory response of astrocytes, we addressed the question of whether the direct DRD3 stimulation on astrocytes affects the pro-inflammatory profile of these cells and whether this signalling exerts regulation in DRD3 expression. For this purpose, we generated cultures of astrocytes isolated from the midbrain/striatum of WT mice and were treated with dopamine 100 nM (a concentration that selectively stimulates DRD3) or a DRD3-selective agonist (PD128097) for 24 h and the expression of the pro-inflammatory marker iNOS was evaluated by flow cytometry. As a positive control of inflammatory signal, astrocytes were treated with LPS. The results show that both dopamine and PD128907 induced a significant increase of iNOS expression, in a similar extent to that induced by LPS (Fig. 9b). Thus, these results indicate that DRD3 stimulation in astrocytes induces a pro-inflammatory profile. To determine whether DRD3 stimulation in astrocytes exerts regulation on DRD3 expression, next we treated astrocytes with dopamine or PD128907 in the absence of an additional challenge or in response to a pro-inflammatory environment given by LPS treatment or in response to an anti-inflammatory environment given by IL-4 treatment, and the levels of drd3 transcription were evaluated by qRT-PCR. The results show that DRD3 stimulation by dopamine or PD128907 did not exert a significant regulation on drd3 transcription under any of the conditions tested (Fig. 9c). However, the drd3 transcription was significantly reduced in response to a pro-inflammatory environment given by LPS treatment (Fig. 9c). Drd3 transcription was not affected in response to an anti-inflammatory environment given by IL-4 treatment (Fig. 9c). Taken together, these results indicate that DRD3 stimulation induces pro-inflammatory signals in astrocytes similar to those exerted by LPS; however, only a classic pro-inflammatory challenge such as that induced by LPS, but not that induced by DRD3 signalling, exerts downregulation of drd3 transcription.