Brain Iron Deficiency Changes the Stoichiometry of Adenosine Receptor Subtypes in Cortico-Striatal Terminals: Implications for Restless Legs Syndrome

Abstract

Brain iron deficiency (BID) constitutes a primary pathophysiological mechanism in restless legs syndrome (RLS). BID in rodents has been widely used as an animal model of RLS, since it recapitulates key neurochemical changes reported in RLS patients and shows an RLS-like behavioral phenotype. Previous studies with the BID-rodent model of RLS demonstrated increased sensitivity of cortical pyramidal cells to release glutamate from their striatal nerve terminals driving striatal circuits, a correlative finding of the cortical motor hyperexcitability of RLS patients. It was also found that BID in rodents leads to changes in the adenosinergic system, a downregulation of the inhibitory adenosine A₁ receptors (A₁Rs) and upregulation of the excitatory adenosine A_2A receptors (A_2ARs). It was then hypothesized, but not proven, that the BID-induced increased sensitivity of cortico-striatal glutamatergic terminals could be induced by a change in A₁R/A_2AR stoichiometry in favor of A_2ARs. Here, we used a newly developed FACS-based synaptometric analysis to compare the relative abundance on A₁Rs and A_2ARs in cortico-striatal and thalamo-striatal glutamatergic terminals (labeled with vesicular glutamate transporters VGLUT1 and VGLUT2, respectively) of control and BID rats. It could be demonstrated that BID (determined by measuring transferrin receptor density in the brain) is associated with a selective decrease in the A₁R/A_2AR ratio in VGLUT1 positive-striatal terminals.

Keywords: adenosine A1 receptor; adenosine A2A receptor; brain iron deficiency; cortico-striatal terminals; restless legs syndrome; striatum; thalamo-striatal terminals.

Figure 1

Increased density of transferrin receptor (TrfR) in the cerebral cortex of rats with BID as compared with controls (CTRL). (A) Representative blot with 10 µg proteins obtained from total cortical homogenates of three pairs of CTRL and rats with BID. (B) TrfR density values were compared to their respective β-actin density values after reprobing the stripped membranes and the average of TrfR/β-actin ratios from CTRL rats were taken as 100%. Statistical comparisons between rats with BID and controls were made with two-tailed unpaired Student’s t-test (* = p < 0.05).

Figure 2

Localization of A₁R and A_2AR in striatal terminals (A) Representative flow synaptometry dual parameter dot-plot of striatal synaptosomes for size (forward scatter; proportional to the particle size) and for complexity/granularity (side scatter). The logarithmic scales of the x and the y axes represent signal intensity in arbitrary units. Red dots represent FITC-labeled synaptosomes, while blue dots are size calibration beads. (B) Representative fluorescence histogram documenting the selectivity of anti-synaptophysin labeling. Specific signal (M2 region) for single-labeled synaptosomes was calculated by subtracting the percentage of labeling by the secondary antibodies alone (histogram filled with green representing synaptosomes incubated only with FITC-conjugated anti-rabbit antibody) from the percentage of labeling by the antibody of interest (histogram in red color). M1 region represents the unlabeled synaptosomes. Note that similar controls were also carried out for the other primary antibodies. (C) Striatal presynaptic frequency (as % of synaptophysin positive terminals, SYN⁺) of vesicular glutamate transporters 1 and 2 (VGLUT1/2). (D₁) Percentage of A₁R⁺ cortico-striatal terminals, (D₂), percentage of A_2AR⁺ cortico-striatal terminals, and (D₃) ratio between the frequency of inhibitory A₁R excitatory and the A_2AR. The inter-animal variability of A₁R and A_2AR labelling masks the difference between control and BID in panels D₁ and D₂, while in panel D₃ there is an intra-animal normalization of A_2AR labelling to A₁R labelling, better illustrating the effect of BID. For panels D₁–D₃, all raw data with the corresponding statistical analyses can be accessed in Supplementary Table S1. Bars represent mean + S.E.M. of n = 8–11 animals. Statistical comparisons were made with a two-tailed unpaired Student’s t-test (n.s. = not significant; * = p < 0.05).

Figure 3

Localization of A₁Rs and A_2ARs in cortico-striatal terminals. (A₁–A₃) Representative dot-plots showing the colocalization of VGLUT1, A₁Rs and A_2ARs in the upper right quadrants in striatal synaptosomes obtained from a control rat (dark blue) and a BID animal (cyan). Logarithmic scales of the x and the y axes represent the intensity of fluorescence in arbitrary units. Percentage of cortico-striatal terminals (VGLUT1 positive) terminals that are positive for (B₁) A₁R⁺, (B₂) A_2AR⁺ and (B₃) ratio between the inhibitory A₁R and the excitatory A_2AR. Bars represent mean + S.E.M. of n = 8–11 animals. Statistical comparisons between rats with BID and controls were made with a two-tailed unpaired Student’s t test (n.s. = not significant; * = p < 0.05).

Figure 4

Localization of A₁Rs and A_2ARs in thalamo-striatal terminals. Percentage of thalamo-striatal terminals (VGLUT2 positive) terminals that are positive for (A₁) A₁R⁺, (A₂) A_2AR⁺ and (A₃) ratio between the inhibitory A₁R and the excitatory A_2AR. Bars represent mean + S.E.M. of n = 8–11 animals. Statistical comparisons between rats with BID and controls were made with a two-tailed unpaired Student’s t-test (n.s. = not significant).