Temporal changes of CB1 cannabinoid receptor in the basal ganglia as a possible structure-specific plasticity process in 6-OHDA lesioned rats

Abstract

The endocannabinoid system has been implicated in several neurobiological processes, including neurodegeneration, neuroprotection and neuronal plasticity. The CB1 cannabinoid receptors are abundantly expressed in the basal ganglia, the circuitry that is mostly affected in Parkinson's Disease (PD). Some studies show variation of CB1 expression in basal ganglia in different animal models of PD, however the results are quite controversial, due to the differences in the procedures employed to induce the parkinsonism and the periods analyzed after the lesion. The present study evaluated the CB1 expression in four basal ganglia structures, namely striatum, external globus pallidus (EGP), internal globus pallidus (IGP) and substantia nigra pars reticulata (SNpr) of rats 1, 5, 10, 20, and 60 days after unilateral intrastriatal 6-hydroxydopamine injections, that causes retrograde dopaminergic degeneration. We also investigated tyrosine hydroxylase (TH), parvalbumin, calbindin and glutamic acid decarboxylase (GAD) expression to verify the status of dopaminergic and GABAergic systems. We observed a structure-specific modulation of CB1 expression at different periods after lesions. In general, there were no changes in the striatum, decreased CB1 in IGP and SNpr and increased CB1 in EGP, but this increase was not sustained over time. No changes in GAD and parvalbumin expression were observed in basal ganglia, whereas TH levels were decreased and the calbindin increased in striatum in short periods after lesion. We believe that the structure-specific variation of CB1 in basal ganglia in the 6-hydroxydopamine PD model could be related to a compensatory process involving the GABAergic transmission, which is impaired due to the lack of dopamine. Our data, therefore, suggest that the changes of CB1 and calbindin expression may represent a plasticity process in this PD model.

Figure 1. Temporal changes of TH levels in the striatum of PD-induced rats.

TH levels in striatum of rats submitted to unilateral intrastriatal injections of 6-OHDA after 1, 5, 10, 20, and 60 days post-lesion (DPL). (A) Digital images of coronal sections of the dorsolateral striatum shows a decrease in the expression of TH on the 6-OHDA side, with a peak in 10 DPL (B) Digital images of coronal sections of the dorsolateral striatum at 60 DPL; the arrow indicates the site of infusion. Note the emergence of TH+ cells in the experimental side. (C) Semi-quantitative analysis of TH staining represented as percentage. Control side (dashed line). Statistical analysis by ANOVA (one way) with Tukey post-test. * p <0.05, ** p <0.01 and *** p <0.001.

Figure 2. Temporal changes of TH expression in the SNPc of PD-induced rats.

TH expression in SNpc of rats submitted to unilateral intrastriatal injections of 6-OHDA after 1, 5, 10, 20 and 60 days post-lesion (DPL). (A) Digital images of coronal sections of the SNpc show a progressive decreased TH expression in 6-OHDA side. (B) Semi-quantitative analysis of TH staining represented as percentage. Control group (dashed line). Statistical analysis by ANOVA (one way) with Tukey post-test. * p <0.05, ** p <0.01 and *** p <0.001.

Figure 3. Temporal changes of CB1 expression in the EGP of PD-induced rats.

CB1 expression in external globus pallidus (EGP) of rats submitted to unilateral intrastriatal injections of 6-OHDA after 1, 5, 10, 20, and 60 days post-lesion (DPL). Digital images of coronal sections of the EGP show an increase of CB1 expression in the experimental side at earlier time points.

Figure 4. Temporal changes of CB1 expression in the IGP of PD-induced rats.

CB1 expression in internal globus pallidus (IGP) of rats submitted to unilateral intrastriatal injections of 6-OHDA after 1, 5, 10, 20 and 60 days post-lesion (DPL). Digital images of coronal sections of the IGP show an increase in the CB1 expression in the experimental side at 1 DPL. From 5 to 60 DPL, there was a gradual decrease in the CB1 expression.

Figure 5. Temporal changes of CB1 expression in the SNpr of PD-induced rats.

CB1 expression in the substantia nigra pars reticulata (SNpr) of rats submitted to unilateral intrastriatal injections of 6-OHDA after 1, 5, 10, 20 and 60 days post-lesion (DPL). Digital images of coronal sections of the SNpr show a decrease in the CB1 expression in the experimental side from 5 DPL to 60 DPL.

Figure 6. Semi-quantitative analysis of CB1 staining in basal ganglia of PD-induced rats.

The quantification is represented by percentage in (A) striatum, (B) external globus pallidus (EGP), (C) internal globus pallidus (IGP) and (D) substantia nigra pars reticulata (SNpr) of rats submitted to unilateral intrastriatal injections of 6-OHDA after 1, 5, 10, 20, and 60 days post-lesion (DPL). Comparison between the percentage of experimental sides calculated from the control side to each group (dashed line). Statistical analysis by ANOVA (one way) with Tukey post-test. *p <0.05, **p<0.01 and *** p <0.001.

Figure 7. Scheme of CB1 expression in basal ganglia of PD-induced rats.

Schematic representation of the CB1 receptors expression in the striatum, external globus pallidus (EGP), internal globus pallidus (IGP) and substantia nigra pars reticulata (SNpr). This scheme represents control side (A) and intrastriatal 6-OHDA injected side (B). Figure in A depicts the dopaminergic projection (full gray arrows) of the substantia nigra pars compact (SNpc) to the striatum, and GABAergic projections (full black arrows) of the striatum to EGP, IGP and SNpr, where CB1 receptors are found (gray squares). Figure in B represents the decreased dopaminergic (dotted gray arrows) and GABAergic (dotted black arrows) projections, the increased GABAergic projections (thicker full black arrows), increased CB1expression (bigger gray square), and the decreased CB1 expression (smaller gray square) after 6-OHDA injection. Our hypothesis is that the decrease of CB1 expression in the direct pathway (SNpr and IGP) could increase the GABAergic transmission, and the increase of CB1 expression in indirect pathway (EGP) could decrease the GABAergic transmission, which could together contribute to a restoration of normal thalamic activation. Both events may be a plastic response of the cannabinoid system due to the lack of dopamine.