NMDA receptor subunit expression in GABAergic interneurons in the prefrontal cortex: application of laser microdissection technique

Abstract

The selective involvement of a subset of neurons in many psychiatric disorders, such as gamma-aminobutyric acid (GABA)-ergic interneurons in schizophrenia, creates a significant need for in-depth analysis of these cells. Here we introduce a combination of techniques to examine the relative gene expression of N-methyl-d-aspartic acid (NMDA) receptor subtypes in GABAergic interneurons from the rat prefrontal cortex. Neurons were identified by immunostaining, isolated by laser microdissection and RNA was prepared for reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. These experimental procedures have been described individually; however, we found that this combination of techniques is powerful for the analysis of gene expression in individual identified neurons. This approach provides the means to analyze relevant molecular mechanisms that are involved in the neuropathological process of a devastating brain disorder.

Figure 1

NovaRed stained PV-ir interneurons in mPFC before and after LMD. A, photograph of Cresyl violet-stained frontal cortex with dorsal to the right and ventral to the left of the image. B, higher magnification (squared area in A) of Nissl staining showing the laminar architecture of mPFC. Dashed lines delineate cortical layers. C and D, photographs of NovaRed-staining at low magnifications showing the area of interest in the mPFC. E and F, photographs from D (squared area) showing the PV-ir interneurons (arrows point to the cells of interest) before (E) and after (F) laser cut. The PV-ir interneurons were stained in brown/red color. Scale bar in A = 1600 μm for A, 400 μm for B and C, 200 μm for D, and 50 μm for E and F.

Figure 2

Measurements RNA integrity number in isolated RNA from a LMD PV-ir sample with Agilent BioAnalyzer. A, gel electrophoresis image for sample exhibited in B. B, electropherogram of RNA isolated from LMD picked PV-ir interneurons. 5S covers the small rRNA fragments (5S and 5.8S rRNA and tRNA), while 18 S and 28 S cover the 18S peak and 28S peak. C, summary graph showing the RIN (8.63 ± 0.16, n = 9) as well as 28/18S ratio (1.90 ± 0.15) and 18S/baseline value (7.23 ± 1.17).

Figure 3

RT-PCR amplification of PV (150 bp) from different samples. Lanes 1 to 6 indicate the cells expressing PV captured from six different animals (A–F). The right lane was the molecular weight marker with 100 bp intervals.

Figure 4

Gene expressions in PV-ir interneurons versus PV-negative tissues. A and B, photographs showing the PV-ir interneurons (arrows) before (A) and after (B) laser cut. C and D, section of NovaRed staining showing the PV-negative areas (red cycles in C) and the corresponding holes (D) cut as PV-negative tissue sample. Arrows point to the PV-ir interneurons. E, electrophoresis of the three different genes in the PV-ir interneurons and PV-negative tissues. There was no expression of either GFAP or Cd11b in PV-ir cells compared with a positive band of GFAP in PV-negative tissue. F, although calcium/calmodulin-dependent protein kinase II alpha (αCaMKII), which only expressed in cortical pyramidal neurons (Liu and Jones, 1996), was observed in PV-negative control tissue, all of other three genes, including calbindin (CB), calretinin (CR), and αCaMKII, were not expressed in PV-ir interneurons. Scale bar in B = 50 μm for A–D.

Figure 5

Comparison between the concentration of the RNA isolated from LMD-picked cells and the concentration of cDNA synthesized and amplified from the RNA. The DNA concentration was increased about 400 times from 3.20 ng/μl to 1258.95 ng/μl. * indicates the significant difference between two concentrations (p<0.001).

Figure 6

Real-time PCR response curves. A and B, amplification curves of GAPDH from different dilutions (1:1, 1:4,1:16, 1:64, 1:256), in which threshold cycle (Ct), the numbers of cycles required to reach threshold, was determined (A). The “single” peak (at ~ 85°C) exhibited in melting curves (B) suggests a single and specific product. Inset in B, standard curve of the GAPDH derived from (A) showing a correlation coefficient of 0.965 (R² = 0.9306), slope = −3.35 and PCR efficiency of 98.9%. Efficiency is relevant to slope and the mathematical algorithm is: (1+Efficiency) ^−slope = 10. Figures C to F were derived from same LMD sample for PV gene. C and D, response curves (C) and melting curves (D) of 40-cycle reactions using preamplified cDNA of PV as template (same concentration with 6 repetitions). Ct values determined in C ranged from 29.8 to 32.9, and all peaks were located around 85°C in the melting curves (D), indicating the aRNA amplification was successful and specific. E and F, 50-cycle reactions using reverse transcripted cDNA of PV without aRNA amplification. In contrast, there was no amplification and peak in this sample, suggesting that aRNA amplification is an essential step for real-time PCR for LMD sample.

Figure 7

Relative mRNA expression of NMDA receptor subunits and PV in PV-ir interneurons in normal adult rat PFC versus treatment with MK-801. A, it is clear that the mRNA expression of NR2A subunit is significantly higher than that of NR2B (p<0.05) in the PV-ir interneurons of adult rat PFC (postnatal day 90). Note the high expression of PV and undetectable level of NR2D subunit in PV-ir cells. B, subchronic treatment with MK-801 significantly downregulated the mRNA expression of PV to an almost undetectable level (337.2-fold) and all subunits of NMDA receptors in PV-ir interneurons, i.e., 6.13-fold for NR1, 17.7-fold for NR2A, and 21.0-fold for NR2B (p<0.05).