Dynamic changes in striatal dopamine D2 and D3 receptor protein and mRNA in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) denervation in baboons

Abstract

Loss of nigrostriatal neurons leads to striatal dopamine deficiency and subsequent development of parkinsonism. The effects of this denervation on D2-like receptors in striatum remain unclear. Most studies have demonstrated increases in striatal dopamine D2-like receptors in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated denervation, but others have found either decreases or no change in binding. To clarify the response to denervation, we have investigated the time-dependent changes in dopamine D2, D3, and D4 receptor protein and mRNA levels in unilaterally MPTP-lesioned baboons. MPTP (0.4 mg/kg) was infused into one internal carotid artery, producing a contralateral hemi-parkinsonian syndrome. After MPTP treatment, the animals were maintained for 17-480 d and then euthanized. MPTP decreased ipsilateral dopamine content by >90%, which did not change with time. Ipsilateral D2-like receptor binding in caudate and putamen initially decreased then increased two- to sevenfold over the first 100 d and returned to near baseline levels by 480 d. Relative levels of D2 mRNA were essentially unchanged over this period. D4 mRNA was not detected. In contrast, D3 mRNA increased sixfold by 2 weeks and then decreased. At the peak period of increase in binding sites, all D2-like receptors were in a micromolar affinity agonist-binding state, implying an increase in uncoupled D2 but not D3 receptor protein. Taken together, these data suggest that MPTP-induced changes in D2-like dopamine receptors are complex and include translational or post-translational mechanisms.

Fig. 1.

Time course of changes in striatal D₂-like receptor binding and dopamine content after MPTP lesioning. Animals were killed at the indicated times after unilateral MPTP administration, and samples were prepared for receptor number and dopamine content measurements as described in Materials and Methods. Values shown for receptor number are the mean ± SEM of two to four independent saturation binding experiments repeated in triplicate except for the putamen day 45 study in which tissue was only available for a single determination in triplicate. Samples from the injected (filled circles) and noninjected (open circles) regions were assayed simultaneously for each animal. The day 0 values represent the mean ± SE of five untreated control baboons. The ratio of dopamine content (filled squares) represents the ratio of tissue values from the injected side divided by the noninjected side. Actual dopamine contents are described in the text.

Fig. 2.

Detection of dopamine D₂ and D₃ but not D₄ mRNA in baboon putamen. Total cellular RNA was purified, and 1 μg aliquots were reverse-transcribed with oligonucleotides complementary to the individual RNAs. The resulting cDNAs were used for amplification with the secondary primers indicated in Materials and Methods. All RNA samples were standardized by using complementary oligonucleotide primers to the 18S fragment of rRNA. For comparison, plasmids containing the rat dopamine D₂, D₃, or D₄ cDNA sequences were amplified under identical conditions. Oligonucleotides were end-labeled for quantification. All products were separated on 12% polyacrylamide gels, and the gels were dried and radioactivity was quantified as described in Materials and Methods. For each receptor subtype, the results of amplification of baboon putamen (baboon) and rat cDNA containing plasmid (+) are shown as well as the negative control of no added DNA (−). The expected size products for D₂ and D₃ are found, whereas no product is found for D₄.

Fig. 3.

Demonstration that dopamine D₄oligonucleotides can detect baboon dopamine D₄ DNA sequences. The D₄-specific oligonucleotides used in Figure2 were paired with two other D₄-specific oligonucleotides that did not cross an exon boundary (see Materials and Methods for sequences). Ten nanogram aliquots of baboon or human genomic DNA, or plasmid containing the rat D₄ cDNA (+), were amplified as described in Materials and Methods using end-labeled oligonucleotides. A no-DNA-added control (−) was also included. When paired with a complementary oligonucleotide not crossing an exon boundary, both o-513 and o-515 detected the same size products in baboon and human genomic DNA as well as rat cDNA for the D₄ receptor.

Fig. 4.

Changes in dopamine D₂ and D₃ receptor mRNA levels after MPTP treatment. Levels of mRNA expression for D₂ and D₃ receptors are shown as a function of days after MPTP treatment for ipsilateral putamen compared to control tissues. Total cellular RNA was purified and specific mRNAs amplified using RT-PCR as described in Materials and Methods. All samples were standardized by comparison of the level of 18S ribosomal RNA. All values shown are mean ± SE of three to six independent determinations. For comparison with the time course of mRNA expression, changes in putamen D₂-like receptor binding are also shown. These are the same data as in Figure 1 for the injected side of the putamen. Dashed lines in all three panels indicate control animal levels (mean of 5 control animals). Significant pairwise comparisons (to controls) are indicated byasterisks.

Fig. 5.

Demonstration of an increase in low-affinity dopamine D₂ receptor number in MPTP-treated putamen. Putamen membranes from control [(−)MPTP] and 101 d after MPTP [(+)MPTP]-treated animals were assayed for the ability of 7-OH-DPAT to compete with 1 nm[³H]spiperone binding as described in Materials and Methods. Results of a representative experiment are shown for quinpirole competition in the presence (open circles) and absence (filled circles) of 10 μm GTP. In control membranes, quinpirole competition in the absence of Gpp(NH)p is most compatible with the presence of approximately equal concentrations of high (83 nm) and low (3.2 μm) binding sites. The resolved components are displayed as solid lines in the figure. In the presence of Gpp(NH)p, the competition was best fit by a single class of binding sites (K_i = 4.2 μm;dashed line). For MPTP-treated putamen membranes, quinpirole competition was best described by a single binding site of low affinity in the presence or absence of Gpp(NH)p (filled circle, K_i = 1.5 μm; open circle,K_i = 0.6 μm). See Materials and Methods for details of data analysis.