Mapping P2X and P2Y receptor proteins in striatum and substantia nigra: An immunohistological study

Abstract

Our work aimed to provide a topographical analysis of all known ionotropic P2X(1-7) and metabotropic P2Y(1,2,4,6,11-14) receptors that are present in vivo at the protein level in the basal ganglia nuclei and particularly in rat brain slices from striatum and substantia nigra. By immunohistochemistry-confocal and Western blotting techniques, we show that, with the exception of P2Y(11,13) receptors, all other subtypes are specifically expressed in these areas in different amounts, with ratings of low (P2X(5,6) and P2Y(1,6,14) in striatum), medium (P2X(3) in striatum and substantia nigra, P2X(6,7) and P2Y(1) in substantia nigra) and high. Moreover, we describe that P2 receptors are localized on neurons (colocalizing with neurofilament light, medium and heavy chains) with features that are either dopaminergic (colocalizing with tyrosine hydroxylase) or GABAergic (colocalizing with parvalbumin and calbindin), and they are also present on astrocytes (P2Y(2,4), colocalizing with glial fibrillary acidic protein). In addition, we aimed to investigate the expression of P2 receptors after dopamine denervation, obtained by using unilateral injection of 6-hydroxydopamine as an animal model of Parkinson's disease. This generates a rearrangement of P2 proteins: most P2X and P2Y receptors are decreased on GABAergic and dopaminergic neurons, in the lesioned striatum and substantia nigra, respectively, as a consequence of dopaminergic denervation and/or neuronal degeneration. Conversely, P2X(1,3,4,6) on GABAergic neurons and P2Y(4) on astrocytes augment their expression exclusively in the lesioned substantia nigra reticulata, probably as a compensatory reaction to dopamine shortage. These results disclose the presence of P2 receptors in the normal and lesioned nigro-striatal circuit, and suggest their potential participation in the mechanisms of Parkinson's disease.

Fig. 1

P2X and P2Y receptor proteins in rat striatum. Transverse sections through the striatum of adult rats were processed for double immunofluorescence studies. Rabbit polyclonal antisera against P2X_1,2,4 and P2Y₄ receptors (red Cy3 immunofluorescence) were used in combination with antibodies against neuronal or glial markers (green Cy2 immunofluorescence). Panel A P2X₁: confocal images illustrate clear colocalization of P2X₁ receptor with neurofilament-light protein (NF-L). The merged field of inset a shows absence of colocalization between the neuronal GABAergic marker calbindin (green) (a calcium-binding protein expressed mainly in medium spiny neurons of the striatum) and P2X₁ receptor (red). The merged field of inset b shows the merged field of P2X₁ (red) and MBP (green) overlapping immunoreactivities at higher magnification. Panel B P2X₂: double immunofluorescence demonstrates that P2X₂ receptor immunoreactivity (red) colocalizes with calbindin protein (green). The insets e and f show colocalization with the neuronal GABAergic marker parvalbumin (green) (a calcium-binding protein that is expressed in interneurons of the striatum). Panel C P2X₄: red immunofluorescence for P2X₄ protein merges with the green signals of the three types of neurofilament proteins: NF-L (inset g, merged field), NF160 (inset i, merged field) and SMI 32 and, moreover, with parvalbumin (inset h, merged field) and calbindin (inset i, merged field). Panel D P2Y₄: red P2Y₄ immunoreactivity is present on calbindin-positive neurons (green), on parvalbumin-positive neurons (green) (insets n–q), and on GFAP-positive astrocytes (inset o, merged field). Western blot analysis also confirms the presence of receptor proteins P2X_1,2,4 (insets c in panel A, d in panel B, m in panel C, respectively) and P2Y₄ (inset r in panel D) in striatum. Specificity of the P2 receptor signals was assessed by incubations of the primary antisera with the corresponding neutralizing immunogenic peptides (μg protein ratio 1:1 between peptide and antiserum). Scale bars are 10 μm in A; 100 μm in inset a; 2 μm in inset b; 50 μm in B and in insets e and f; 20 μm in C; 10 μm in insets h, i, l; 5 μm in inset g; 50 μm in D; 5 μm in inset n; and 20 μm in inset o. Similar results were obtained in at least four independent experiments

Fig. 2

P2X and P2Y receptor proteins in rat substantia nigra. Double immunofluorescence visualized by confocal analysis was performed in transverse sections through the substantia nigra of adult rats. Strong signals for ionotropic P2X_2,5 and metabotropic P2Y_6,14 (red Cy3 immunofluorescence) are present on dopaminergic neurons (TH-positive, green Cy2 immunofluorescence) of substantia nigra pars compacta (SNC), whereas in substantia nigra pars reticolata (SNR) P2X/Y immunoreactivity is limited to sparse neuronal bodies. Western blot analysis confirms the presence in substantia nigra of receptor proteins P2X_2,5 (insets a in panel A, and b in panel B, respectively) and P2Y_6,14 (insets c in panel C, and d in panel D, respectively). Specificity of the P2 receptor signals was assessed by incubation of the primary antisera with the corresponding neutralizing immunogenic peptides (μg protein ratio 1:1 between peptide and antiserum).Scale bars in all panels are 50 μm. Similar results were obtained in at least four independent experiments

Fig. 3

6-Hydroxydopamine modulates the expression of selected P2 receptor proteins in striatum and substantia nigra. Staining of rat substantia nigra after 6-hydroxydopamine treatment. Panel A Conventional microscopy images of Nissl staining shows several dopaminergic neurons (arrows) in the contralateral control hemisphere, which are lost (asterisks) in the ipsilateral lesioned hemisphere. Specific ipsilateral dopaminergic lesion of substantia nigra pars compacta (SNC) was also visualized by confocal TH-immunostaining (green) (insets a, b). Panel B Confocal merged yellow images show upregulation of P2X₁ receptor protein (red) in parvalbumin-positive GABAergic neurons (green) in the lesioned side of substantia nigra pars reticolata (SNR) of 6-hydroxydopamine-treated rats. Panel C Confocal merged yellow images show a drastic increase in GFAP-positive astrocytes (green) in the lesioned side of 6-hydroxydopamine-treated rats and, correspondingly, an augment of P2Y₄ signal (red) (inset c). Scale bars are 100 μm in A, B and in insets a, b; 20 μm in C; and 10 μm in inset c. Similar results were obtained in at least three independent experiments