P2 receptor-types involved in astrogliosis in vivo

Abstract

1. In the nucleus accumbens (NAc) of rats, the involvement of P2X and P2Y receptors in the generation of astrogliosis in vivo, was investigated by local application of their respective ligands. The agonists used had selectivities for P2X1,3 (alpha,beta-methylene adenosine 5'-triphosphate; alpha,beta-meATP), P2Y1,12 (adenosine 5'-O-(2-thiodiphosphate; ADP-beta-S) and P2Y2,4,6 receptors (uridine 5'-O-(3-thiotriphosphate; UTP-gamma-S). Pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (PPADS) was used as a non-selective antagonist. The astroglial reaction was studied by means of immunocytochemical double-labelling with antibodies to glial fibrillary acidic protein (GFAP) and 5-bromo-2'-deoxyuridine (BrdU). 2. The agonist-induced changes in comparison to the artificial cerebrospinal fluid (aCSF)-treated control side reveal a strong mitogenic potency of ADP-beta-S and alpha,beta-meATP, whereas UTP-gamma-S was ineffective. The P2 receptor antagonist PPADS decreased the injury-induced proliferation when given alone and in addition inhibited all agonist effects. 3. The observed morphogenic changes included hypertrophy of astrocytes, elongation of astrocytic processes and up-regulation of GFAP. A significant increase of both GFAP-immunoreactivity (IR) and GFA-protein content (by using Western blotting) was found after microinfusion of alpha,beta-meATP or ADP-beta-S. In contrast, UTP-gamma-S failed to increase the GFAP-IR. The morphogenic effects were also inhibited by pre-treatment with PPADS. 4. A double immunofluorescence approach with confocal laser scanning microscopy showed the localisation of P2X3 and P2Y1 receptors on the GFAP-labelled astrocytes. 5. In conclusion, the data suggest that P2Y (P2Y1 or P2Y12) receptor subtypes are involved in the generation of astrogliosis in the NAc of rats, with a possible minor contribution of P2X receptor subtypes.

Figure 1

Horizontal section of the rat brain including the nucleus accumbens (NAc). The schematic localization of the needle tracts and the areas in which the cells were counted (1: core 1; 2: core 2; 3: ventral shell; 4: piriform cortex; according to Franke et al., 1999a) are shown.

Figure 2

Glial fibrillary acidic protein (GFAP)-/bromodeoxyuridine (BrdU)-double stained cells in the NAc of the rat (GFAP: brown, cytoplasm and processes; BrdU: dark-blue to violet, nuclei). (A,B) Effects of α,β-meATP on astrocytes in the NAc of rats after a postinjection time of 4 days. Asterisks mark examples of intensive elongation of astrocytic processes. (C,D) Single- (arrowhead) and double-stained cells (arrow) 4 days after ADP-β-S-application. (C) Artificial cerebrospinal fluid (aCSF)-treated control side. (D) ADP-β-S-treated side (scale bar: 20 μm).

Figure 3

Effects of ADP-β-S, PPADS plus ADP-β-S and PPADS alone (A), α,β-meATP, PPADS plus α,β-meATP and PPADS alone (B), and UTP-γ-S, PPADS plus UTP-γ-S and PPADS alone (C) on the number of GFAP-positive cells and the number of GFAP-/BrdU-double stained cells in the NAc of rats after a postinjection time of 4 days. Rats received at first an ipsilateral injection of aCSF, followed by agonist (0.1 nmol), or PPADS alone (0.03 nmol) or at first PPADS (0.03 nmol), followed by a mixture of PPADS (0.03 nmol) and agonist (0.1 nmol). The contralateral NAc received two injections of aCSF as a control. The values are expressed as a percentage of controls and represent the mean±s.e.m. of six animals per group (^*P<0.05, versus aCSF group; +P<0.05, agonist versus PPADS/agonist group; #P<0.05, PPADS versus PPADS/agonist group).

Figure 4

Comparison of the sum of GFAP-/BrdU-double stained cells in various areas of the NAc 4 days after microinjection of P2 receptor agonists. Similar results with 2-MeSATP are included from a previous publication (Franke et al., 1999a). Values are expressed as a percentage of controls and represent the mean±s.e.m. of six animals per group (^*P<0.05, versus aCSF group). The differences in the effects of ADP-β-S, 2-MeSATP and α,β-meATP are statistically significant (P<0.05).

Figure 5

GFA-protein was detected by immunoblotting followed by measurement of chemiluminescence on nitrocellulose filter. The protein content was determined after ADP-β-S microinfusion in comparison with the aCSF-treated control side following a postinjection time of 4 days. (A) Quantification of the ADP-β-S-induced GFA-protein content in comparison with that induced by ADP-β-S plus PPADS or PPADS alone. The values are expressed as a percentage of controls and represent the mean±s.e.m. of three animals per group (^*P<0.05, versus aCSF-treated side). (B) Immunoblot analysis of preparations of the aCSF- (lanes 1 – 3) and ADP-β-S-treated sides (lanes 4 – 6) using GFAP antibodies. Each lane contains 0.25 μg protein.

Figure 6

Confocal images of double immunofluorescence for GFAP (A,D,G,J; Cy2-green fluorescence) and P2 receptor-subtypes (B,E,H,K; Cy3-red immunofluorescence) to characterize the receptor-localization on astrocytes in the NAc of rats. ACSF (A – C, G – I), α,β-meATP (D – F) or ADP-β-S (J – L) were microinfused 4 days before preparation. Double labelling for GFAP and the P2X₃ receptor subtype is shown in C,F and for the P2Y₁ receptor-subtype in I,L (scale bar: 10 μm (A – L). The expression of P2X₃ receptors in GFAP-immunoreactive astrocytes after aCSF microinfusion has been demonstrated earlier (Franke et al., 2001).