Gene expression deficits in a subclass of GABA neurons in the prefrontal cortex of subjects with schizophrenia

Abstract

Markers of inhibitory neurotransmission are altered in the prefrontal cortex (PFC) of subjects with schizophrenia, and several lines of evidence suggest that these alterations may be most prominent in the subset of GABA-containing neurons that express the calcium-binding protein, parvalbumin (PV). To test this hypothesis, we evaluated the expression of mRNAs for PV, another calcium-binding protein, calretinin (CR), and glutamic acid decarboxylase (GAD67) in postmortem brain specimens from 15 pairs of subjects with schizophrenia and matched control subjects using single- and dual-label in situ hybridization. Signal intensity for PV mRNA expression in PFC area 9 was significantly decreased in the subjects with schizophrenia, predominantly in layers III and IV. Analysis at the cellular level revealed that this decrease was attributable principally to a reduction in PV mRNA expression per neuron rather than by a decreased density of PV mRNA-positive neurons. In contrast, the same measures of CR mRNA expression were not altered in schizophrenia. These findings were confirmed by findings from cDNA microarray studies using different probes. Across the subjects with schizophrenia, the decrease in neuronal PV mRNA expression was highly associated (r = 0.84) with the decrease in the density of neurons containing detectable levels of GAD67 mRNA. Furthermore, simultaneous detection of PV and GAD67 mRNAs revealed that in subjects with schizophrenia only 55% of PV mRNA-positive neurons had detectable levels of GAD67 mRNA. Given the critical role that PV-containing GABA neurons appear to play in regulating the cognitive functions mediated by the PFC, the selective alterations in gene expression in these neurons may contribute to the cognitive deficits characteristic of schizophrenia.

Figures

Figure 1.

Schematic representation of the sampling strategy for grain analysis of mRNA expression. A, Camera lucida drawing of the dorsal PFC, with gray shading indicating the boundaries of a typical contour used for sampling. A sampling grid was randomly super imposed on this contour to designate sampling frames (small filled squares). Orientation is indicated in the bottom left: L, lateral; D, dorsal; M, medial; V, ventral. B, C, Representative bright-field and dark-field images, respectively, of a sampling site for PV mRNA grain analysis. In the bright-field image (B), Nissl-stained neuronal nuclei were identified and included for study according to unbiased inclusion and exclusion rules (broken and solids lines indicate inclusion and exclusion boundaries, respectively). Circles of 22 μm diameter were centered over every neuronal nucleus, and the number of grains within the circle was counted in dark-field image (C) of the same sampling frame. Scale bars, 50 μm.

Figure 2.

Representative photomicrograph of dual-label in situ hybridization verifying the simultaneous detection of GAD67 mRNA by DIG- and 35S-labeled riboprobes in the same neurons. The DIG- and 35S-labeled probes recognize different regions of the mRNA molecule (see Materials and Methods), and their specific hybridization was visualized as color reaction product and silver grain accumulation, respectively. Note the coexistence of both types of signals in all labeled profiles. Scale bar, 50 μm.

Figure 3.

Distribution of silver grain clusters representing PV and CR mRNA-positive neurons. Three serial sections of PFC area 9 of a control subject (604) were stained for Nissl substance (A) or hybridized with antisense riboprobes for PV (B, D) or CR (C, E) mRNAs and then stained for Nissl substance. Note that the density of PV mRNA-positive neurons appears greatest in layers III and IV (B), whereas the density of CR mRNA-positive neurons is higher in layers II and superficial III (C). Representative high-magnification photomicrographs illustrate the expression of PV (D) and CR (E) mRNAs in deep layers III and layer II, respectively. For both probes, silver grains accumulated around neuronal nuclei. The size of grain clusters for PV mRNA appears larger than those for CR mRNA, reflecting the difference in somal sizes of PV- and CR-containing neurons (Gabbott and Bacon, 1996). Roman numerals indicate cortical layers, and WM indicates white matter. Scale bar: (in C), A–C, 300 μm; (in E), D, E, 50 μm.

Figure 4.

Representative film autoradiograms showing signals for PV and CR mRNAs in PFC area 9 of subject pairs 3 (A–D) and 5 (E–H). The densities of hybridization signals are presented in pseudocolor manner according to the scale in bottom right. In both pairs, the PV mRNA signals appear to be weaker in subjects with schizophrenia (B, F) than in matched controls (A, E), whereas in the same region of the adjacent sections, CR mRNA signals do not appear to differ between subjects with schizophrenia (D, H) and matched controls (C, G). Solid and broken white lines indicate the pial surface and the border between white matter and gray matter, respectively. Scale bars: (apply to both sections from a given subject), 1 mm.

Figure 5.

Pair-wise comparisons of film optical density (A, B), grain density per positive neuron (C, D), and positive neuron density (E, F) for parvalbumin (A, C, E) and calretinin (B, D, F) mRNA expression. Mean levels of expression for each subject group are indicated by the horizontal bars.

Figure 6.

Mean ± SD differences from control subjects in film optical density (A) and grain density per positive neuron (B) for parvalbumin mRNA across subject pairs grouped by sex (male, n = 12; female, n = 3), substance abuse history (yes, n = 8; no, n = 7), and the diagnosis of schizoaffective disorder (yes, n = 5; no, n = 10).

Figure 7.

Comparison by cortical layer of mean ± SD film optical density for PV (A) and CR (B) mRNA expression between the subject groups. Asterisks indicate statistical significance (*p = 0.012; **p = 0.005).

Figure 8.

Representative photomicrographs of dual-label in situ hybridization showing simultaneous detection of PV and GAD67 mRNAs in PFC area 9 of subject pair 14. Silver grain clusters represent PV mRNA, which was detected by a 35S-labeled probe, whereas GAD67 mRNA was visualized as color reaction product by a digoxigenin-labeled probe. We detected single-labeled GAD67 mRNA-positive profiles (solid arrowheads), single-labeled PV mRNA-positive silver grain clusters (open arrowheads), and double-labeled profiles (double arrowheads). In the control subject, GAD67 mRNA signals were detected in all PV mRNA-positive grain clusters (A), whereas in the schizophrenia subject, GAD67 mRNA signals were undetectable in some of the PV mRNA-positive grain clusters (B). Scale bar: (in A), A, B, 50 μm.

Figure 9.

Representative autoradiograms showing signals for PV mRNA in the PFC of control (A) and haloperidol-treated (B) monkeys. The densities of hybridization signals are presented in pseudocolor manner according to the scale in bottom right. Note that the signal distribution and intensity appear to be unchanged in the PFC of the haloperidol-treated monkey. Solid and broken white lines indicate the pial surface and the border between gray and white matter, respectively. PS, Principal sulcus. Pairs of large and small white arrowheads indicate the quantified regions in areas 9 and 46, respectively. Scale bars, 1 mm.