doi:10.1016/j.brainres.2006.10.049 
Copyright © 2006 Elsevier B.V. All rights reserved.
Research Report
Immunohistochemical localization of dopamine receptor subtypes (D1R–D5R) in Alzheimer's disease brain
Ujendra Kumara, 
, 
and Shutish C. Patelb
aFaculty of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, 2146 East Mall, University of British Columbia, Vancouver, BC, Canada V6T 1Z3
bVA Connecticut Healthcare System and New England Biomedical Research Center, Newington, CT 06111, USA
Accepted 25 October 2006.
Available online 19 December 2006.
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Abstract
Among the neurotransmitter abnormalities that have been investigated in Alzheimer's disease (AD), deficits in the cholinergic system have been the most intensively studied. Another key neurotransmitter system involved with emotion and cognition is the dopaminergic system. Here we have investigated alterations in all five dopamine receptor subtypes in AD brain. Using antipeptide rabbit antibodies for each of the five dopamine receptors (D1–D5) we mapped the distribution of these receptors in postmortem AD and age-matched control brains in the frontal cortex, utilizing biotin–avidin immunocytochemistry. All five DR subtypes were expressed as cell surface and cytoplasmic proteins. Receptor-specific changes in control and AD brain were identified as follows: D4R and D3R were the predominant receptor subtypes in age-matched controls followed by D2R and D1R; D5R is the least expressed receptor subtype. In AD brain, D2R and D5R are well expressed in comparison to D1R, D3R and D4R. Expression of D1R, D3R and D4R was severely reduced in AD cortex. D2R expression is moderately reduced in the frontal cortex of AD brain. D5R is the only receptor subtype whose expression is increased in AD frontal cortex. Furthermore, in AD, we found comparable expression of D3R in astrocytes, whereas D5R-like immunoreactivity is significantly increased in astrocytes, in comparison to normal frontal cortex, where it was predominantly neuronal. These results demonstrate subtype-specific changes in dopamine receptors in AD that may be important in disease pathophysiology and that may also serve as potential targets for therapeutic intervention in AD.
Keywords: Alzheimer's disease; β-amyloid; Cortex; Dopamine; Dopamine receptor; Neurofilament tangle; Senile plaques
Abbreviations: AD, Alzheimer's disease; APP, amyloid precursor protein; DA, dopamine; DRs, dopamine receptors; CNS, central nervous system; NFT, neurofilament tangles; PD, Parkinson's disease; PET, positron emission tomography; SP, senile plaques
Fig. 1. Photomicrographs illustrating the specificity of dopamine receptor subtype antibodies using western blot analysis and immunocytochemistry. (A) Membrane protein (50 μg) prepared from rat brain was fractionated by SDS-PAGE and probed with antigen preabsorbed antibodies (lane i) or affinity-purified DR antibodies (lane ii of respective antibody) as described in Experimental procedures. DR1–5 were well expressed at the expected size of 65/49 kDa, 63 kDa, 65/51 kDa, 49 kDa and 51 kDa respectively. Note the complete blockade of receptor-specific immunosignal in the presence preimmune serum. (B) Cortical sections from control brain were incubated in the presence of corresponding preimmune serum of each DR subtypes and processed for peroxidase immunocytochemistry. Except for background staining, no specific signals were seen for any of the DR subtypes (a–e) respectively. Note the similar results in the absence of first antibody (f). Scale bar, 25 μM.
Fig. 2. Photomicrographs illustrating the distribution of dopamine receptor subtype 1 (D1R) in age-matched control and AD brain frontal cortex. In the frontal cortex of control brain, D1R-like immunoreactivity predominantly expressed in cytoplasm as well as at the cell surface (A and B). D1R-like immunoreactivity is lost in most of the neurons in AD brain (C and D; arrows) while well expressed in age-matched control brain (B; arrows). In AD brain sections, virtually no immunoreactivity was detected (D). Note the morphological distinction of neurons expressing D1R-like immunoreactivity and the presence of neurons but lacking receptor-like immunoreactivity (B; arrowhead). Scale bar = 25 μM (A and C), 5 μM (B and D).
Fig. 3. Representative photomicrographs demonstrating the distribution of dopamine receptor subtype D2R in control and AD brain tissue sections. D2R-like immunoreactivity is expressed in a comparable manner in control (A and B) and AD brain (C and D). Note the significant accumulation of receptor-like immunoreactivity in perinuclear zone of control brain and the loss of receptor-like immunoreactivity in perinuclear area in neurons in AD cases (C and D). In both cases, comparable receptor-like immunoreactivity was seen in neuronal dendrites (C and D; arrowheads). Scale bar = 25 μM (A and C), 5 μM (B and D).
Fig. 4. Photomicrographs demonstrating the distribution of dopamine receptor subtype D3R in control and AD brain tissue sections. In control, frontal cortical brain region receptor-like immunoreactivity is well expressed as membrane and cytoplasmic protein (B). Within the same field, neurons negative to receptor are also present (B; arrowhead). D3R-like immunoreactivity is lost in a majority of neurons in AD brain (C and D). Scale bar = 25 μM (A and C), 5 μM (B and D).
Fig. 5. Photomicrographs demonstrating the distribution of dopamine receptor subtypes D4R in control and AD brain tissue sections. In control brain, D4R-like immunoreactivity is predominantly expressed in neuronal cell body (B; arrows) and nerve fibers (B; arrowheads) throughout the cortical regions. Unlike control brain tissue, receptor-like immunoreactivity was virtually lost in all the neurons. No receptor-like immunoreactivity was seen in nerve fibers. Rarely weakly receptor-positive neurons were observed (D; arrow). Scale bar = 25 μM (A and C), 5 μM (B and D).
Fig. 6. Photomicrographs demonstrating the distribution of dopamine receptor subtypes D5R in control and AD brain tissue sections. D5R1-like immunoreactivity is weakly expressed in control brain (A and B). In comparison to control brain, the numbers of neurons exhibiting D5R-like immunoreactivity significantly increase in AD brain (C and D). Arrowheads, in panel B, indicate neurons lacking receptor-like immunoreactivity. Scale bar = 25 μM (A and C), 5 μM (B and D).
Fig. 7. Photomicrographs illustrating the expression of dopamine receptors in astrocytes in control and AD brain tissue sections. In control, cortical brain regions D3R and D5R poorly expressed (A and C). In comparison to control, there is a slight increase in D3R-like immunoreactivity in astrocytes in AD brain (B; arrowheads). Note a strong D5R-like immunoreactivity in astrocytes in AD cases in comparison to control brain (D; arrowheads). Scale bar = 25 μM (A and C), 5 μM (B and D).
Fig. 8. Quantification of DR-immunoreactive neurons in frontal cortex. Receptor-positive neurons were counted from 8 to 10 randomly selected areas of cortex from age-matched control and AD brain tissues in three cases. Numbers of immunoreactive neurons are presented per mm2 for each receptor subtype. Histograms represent the neurons positive to a given DR subtype. Mean ± SE (n = 3). P < 0.05 vs. control brain.
Table 1.
Semiquantitative analysis of dopamine receptor subtypes (D1R–D5R) immunoreactivity in control and AD frontal cortex
Values are ranked ++++ (strongly immunopositive) to − (negative). Immunoreactivity was ranged on the basis of intensity of receptor-like immunoreactivity.
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