Decreased parvalbumin mRNA expression in dorsolateral prefrontal cortex in Parkinson's disease

Abstract

It has recently been shown that expression of the rate-limiting GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) is decreased in Brodmann area 9 (BA9) of the dorsolateral prefrontal cortex (DLPFC) in Parkinson's disease (PD) compared to control brains (Lanoue, A.C., Dumitriu, A., Myers, R.H., Soghomonian, JJ., 2010. Exp. Neurol. 206 (1), 207-217). A subpopulation of cortical GABAergic interneurons expresses the calcium-binding protein parvalbumin and plays a critical role in the control of pyramidal neuron excitability and the generation of cortical gamma frequency oscillations. In view of its key role in the physiology of the cerebral cortex, we sought to determine whether the expression of parvalbumin and the number of parvalbumin-expressing neurons are altered in BA9 of PD brains. First, isotopic in situ hybridization histochemistry was used to examine mRNA expression of parvalbumin on post-mortem brain sections. Second, immunohistochemistry and design-based stereology were used to determine the density of parvalbumin-positive interneurons in BA9. Quantification of mRNA labeling at the single cell level showed a significant decrease in parvalbumin expression in PD cases. In contrast, neuronal density of parvalbumin-positive neurons was not significantly different between PD and controls. Results confirm that the GABAergic system is altered in the DLPFC in PD and identify the contribution of parvalbumin-expressing neurons in these alterations. We speculate that these effects could contribute to altered cortical excitability and oscillatory activity previously documented in PD.

Keywords: GABA; Gene expression; Parkinson′s disease; Parvalbumin; Prefrontal cortex; mRNA.

Figure 1

Bright-field photomicrographs illustrating immunoreactivity for parvalbumin in a normal control (A) and a PD case (B). Arrows indicate parvalbumin-positive neurons. Stars indicate unlabeled soma surrounded by immunolabeled puncta. Scale bar: 20 μm.

Figure 2

Bright-field photomicrographs of adjacent sections of BA9 from a control case. Sections were labeled by immunohistochemistry with an antibody against parvalbumin (A, B) or Nissl-stained (C). In order to provide better visualization of the distribution of parvalbumin-labeled neurons in panel A, black dots were drawn over labeled neurons on the same photomicrograph in panel B. Cortical layers are demarcated in an adjacent Nissl-stained section (C) with black dotted lines. Note that parvalbumin-positive neurons appear in layers II-VI and their density is greatest in layer IV. Roman numerals indicate cortical layers and WM indicates white matter. Scale bar: 200 μm.

Figure 3

(A) Photograph from an X-ray film illustrating the distribution of parvalbumin mRNA positive neurons. The dotted lines delineate the pial and white matter boundaries and silver grain clusters representing parvalbumin mRNA positive neurons are represented by black dots between these dotted lines. Note that parvalbumin mRNA positive neurons are concentrated in the middle layers. (B, C) Bright-field photomicrographs illustrating parvalbumin mRNA labeling on emulsion radioautographs in BA9. Arrows indicate parvalbumin mRNA-labeled neurons. Arrowheads illustrate examples of unlabeled neuronal profiles. B: Control case. C: PD case. Scale bar: A=1 mm; B, C=20 μm. WM=white matter.

Figure 4

Relative levels of parvalbumin mRNA labeling per single neuron in BA9. Labeling was measured by computerized image analysis of emulsion radioautographs. Values are the mean of the mean number of pixels per profile and are expressed as percent of controls. Data are from 11 control and 15 PD cases (*p<0.05 vs. controls; two-tailed unpaired t-test).

Figure 5

Histograms of relative frequency distribution of parvalbumin mRNA labeling in BA9 of all control (A) and PD cases (B). Parvalbumin mRNA labeling was expressed as a number of pixels. In control cases (A), the median number of pixels per neuron is 40.5 and the mean is 59.7 pixels, while in PD cases (B), the median number of pixels per neuron is 32.0 and the mean is 45.5 pixels per neuron. This illustrates that the level of labeling per neuron is shifted to the left in the PD cases. The level of labeling was significantly decreased in the PD cases (data not shown; p<0.0001; two-tailed unpaired t-test). Med.: median.

Figure 6

Linear regression analyses between (A) age at death, (B) pH, (C) PMI or (D) disease duration and mean parvalbumin mRNA labeling per neuron measured on emulsion radioautographs. Analyses include all 11 control and 15 PD samples from the in situ hybridization study for parvalbumin. There was no significant correlation between parvalbumin mRNA labeling and (A) age at death (Pearson correlation coefficient r=−0.1576, p=0.4420), (B) pH (Pearson correlation coefficient r=0.2940, p=0.1449), (C) PMI (Pearson correlation coefficient r=−0.1746, p=0.3937) or disease duration (Pearson correlation coefficient r=−0.1325, p=0.6516).