Pharmacological analysis demonstrates dramatic alteration of D1 dopamine receptor neuronal distribution in the rat analog of L-DOPA-induced dyskinesia

Abstract

We have associated behavioral, pharmacological, and quantitative immunohistochemical study in a rat analog of l-DOPA-induced dyskinesia to understand whether alterations in dopamine receptor fate in striatal neurons may be involved in mechanisms leading to movement abnormalities. Detailed analysis at the ultrastructural level demonstrates specific alterations of dopamine D(1) receptor (D(1)R) subcellular localization in striatal medium spiny neurons in l-DOPA-treated 6-hydroxydopamine-lesioned rats with abnormal involuntary movements (AIMs). This includes exaggerated D(1)R expression at the plasma membrane. However, D(1)R retains ability of internalization, as a challenge with the potent D(1)R agonist SKF-82958 induces a strong decrease of labeling at membrane in animals with AIMs. Since a functional cross talk between D(1)R and D(3)R has been suggested, we hypothesized that their coactivation by dopamine derived from l-DOPA might anchor D(1)R at the membrane. Accordingly, cotreatment with l-DOPA and the D(3)R antagonist ST 198 restores normal level of membrane-bound D(1)R. Together, these results demonstrate that AIMs are related to abnormal D(1)R localization at the membrane and intraneuronal trafficking dysregulation, and suggest that strategies aiming at disrupting the D(1)R-D(3)R cross talk might reduce l-DOPA-induced dyskinesia by reducing D(1)R availability at the membrane.

Figure 1.

Detection of D₁R immunoreactivity at light and electron microscopic levels in striatal medium spiny neurons of 6-OHDA unilaterally lesioned rats. All animals bear a 6-OHDA lesion. A–E, Light microscopy, Avidin–biotin DAB Nickel technique (scale bar, 10 μm). A, Control (benserazide only); B, l-DOPA treated without AIMs; C, l-DOPA-treated with AIMs; D, SKF-82958 treated; E, combination of l-DOPA and ST 198. D₁R immunoreactivity is mostly located at plasma membrane of cell bodies and in the neuropil in all situations, except after SKF-82958 where immunoreactivity has prominent cytoplasmic localization. F–I, Electron microscopy, Immunogold technique (scale bar, 0.5 μm). F, Control (benserazide only); G, l-DOPA treated without AIMs; H, l-DOPA treated with AIMs; I, combination of l-DOPA and SKF-82958. D₁R immunoreactivity shows prominent localization at plasma membrane in all situations (F–H, arrows) but after combination of l-DOPA and SKF-82958 (I) where D₁R immunoreactivity is prominently localized in cytoplasmic organelles (er, endoplasmic reticulum; Go, Golgi apparatus). Insets in I show labeling at the periphery of vesicles (*) and multivesicular bodies (**).

Figure 2.

Effect of l-DOPA during D₁R subcellular distribution in striatal neurons. Ratio L/NL (mean ± SEM) represents the number of immunoparticles in lesioned side (L) versus nonlesioned (NL) side at the plasma membrane [Kruskal–Wallis (KW) = 7.423; p < 0.05], in the cytoplasm [KW = 4.269; not significant (ns; A)], and in organelles (B) (er, endoplasmic reticulum; KW = 0.199, ns; Golgi, KW = 0.105, ns; vesicles, KW = 0.675, ns). * indicates a significant difference between related groups; p < 0.05; Mann–Whitney post hoc test.

Figure 3.

Relationship between D₁R subcellular distribution and AIMs: effect of D₁R agonist. Data (mean ± SEM) were collected on the 6-OHDA-lesioned side. A, Experimental details of the six groups showing the pharmacological treatments and their behavioral consequences. Number of D₁R immunoparticles (B) per neuronal cell body (F_(1,23) = 11.6, p < 0.01), per 100 μm of plasma membrane (F_(1,23) = 5.5, p < 0.05) (C), and per 100 μm² of cytoplasm (F_(1,23) = 34.4, p < 0.001) (D). The subcellular distribution in cytoplasmic organelles is further broken down into (Di) endoplasmic reticulum (F_(1,22) = 39.4, p < 0.001), (Dii) Golgi apparatus (F_(1,23) = 8.3, p < 0.01), (Diii) vesicles (F_(1,23) = 8.4, p < 0.01), and (Div) multivesicular bodies (F_(1,23) = 4.9, p < 0.05). *indicates a significant difference between related groups; p < 0.05, Mann–Whitney post hoc test.

Figure 4.

Effect of D₃R antagonist ST-198 during D₁R subcellular distribution in striatal neurons. Ratio L/NL (mean ± SEM) represents the number of immunoparticles in lesioned side (L) versus nonlesioned (NL) side at the plasma membrane [Kruskal–Wallis (KW) = 7.423, p < 0.05], in the cytoplasm [KW = 3.5, not significant (ns)] (A), and in organelles (B) (er, endoplasmic reticulum; KW = 0.167, ns; Golgi, KW = 0.437, ns; vesicles, KW = 0.716, ns). *indicates a significant decrease compared with l-DOPA-treated dyskinetic rats; p < 0.05, Mann–Whitney post hoc test.