Activity-Induced Expression of Common Reference Genes in Individual CNS Neurons

Abstract

T he quantitative study of gene expression in neurons under physiological and pathophysiological conditions has gained considerable importance. Consequently, a variety of different techniques to quantify gene expression have evolved. Generally, such approaches can be subdivided into strategies that provide absolute versus relative quantitation of a target transcript. Quantitative procedures such as quantitative Northern blots or conventional quantitative reverse transcription polymerase chain reaction (RT-PCR) methods have several disadvantages. For instance, Northern blots require substantial amounts of RNA, rendering investigation of mRNA levels from single cells impossible. Comparability of results is extremely dependent upon identical amounts of starting mRNA used in different samples, a condition that cannot usually be achieved with small cell numbers or minute amounts of tissue. To circumvent these obstacles, various techniques have been developed to quantify gene expression relative to a reference gene (Fink et al, 1998; Waha et al, 1998). This obviates the need to perform an accurate determination of initial mRNA concentrations. On the other hand, a suitable reference gene is essential as an internal control for relative mRNA quantitation. This internal control is crucial for a quantitative comparison of gene expression between different tissue types, at various developmental stages, and between control and disease tissue. Ideally, internal control transcripts should show constant, highlevel expression independent of experimental conditions. The genes for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and b-actin have been commonly used as reference genes. In recent years, cyclophilin A and hypoxanthine phosphoribosyl-transferase (HPRT) have also been adopted for this purpose. However, several classical reference genes may exhibit considerable changes in expression in cell types or under disease conditions (Suzuki et al, 2000). Here, we have analyzed several of these genes as reference transcripts for activity-dependent chronic changes in central nervous system (CNS) gene expression. Pilocarpine-induced epilepsy in rats was employed as experimental paradigm. We find that GAPDH is not suitable for this purpose, whereas the neuron-specific gene synaptophysin appears remarkably stable following intense neuronal activity. To study activity-dependent alterations in reference gene expression, seizure activity was induced in vivo by applying a single dose of the convulsant pilocarpine (male Wistar rats 150–200 g, 300–380 mg/kg, ip) after prior administration of methyl-scopolamine (1 mg/kg, sc, 30 minutes before pilocarpine, ip). Seizures were terminated after 2 hours by application of diazepam (0.1 mg/kg, sc). Age-matched rats, injected with saline served only as controls. Thirty days after pilocarpine/saline injection, the brains of pilocarpinetreated and control animals (n 5 5 each) were removed. One hippocampus was immediately frozen in liquid nitrogen for in situ hybridization, whereas the contralateral hippocampus was used for preparation of 400-mm transverse vibratome slices. From these slices, single CA1 neurons were harvested with a glass pipette in less than 10 ml of extracellular solution (Beck et al, 1999). mRNA was isolated using the Dynabead mRNA Direct Micro Kit (Dynal, Hamburg, Germany) according to the manufacturer’s protocol. For real-time RT-PCR fluorescent hybridization, oligonucleotides and primers were chosen for the five putative reference genes: GAPDH, b-actin, cyclophilin A, HPRT, and synaptophysin (Eurogentec, Seraing, Received March 9, 2001. This work is supported by the Deutsche Forschungsgemeinschaft (SFB 400), the BONFOR program of the University of Bonn Medical Center, and the German-Israel Program of the Bundesministerium fur Bildung und Fors and Ministry of Science. Address reprint requests to: Dr. Albert J. Becker, Department of Neuropathology, University of Bonn Medical Center, Sigmund-Freud Str. 25, 53105 Bonn, Germany. E-mail: albert_becker@uni-bonn.de 0023-6837/01/8106-913$03.00/0 LABORATORY INVESTIGATION Vol. 81, No. 6, p. 913, 2001 Copyright © 2001 by The United States and Canadian Academy of Pathology, Inc. Printed in U.S.A.