Microarray analysis of fluoro-gold labeled rat dopamine neurons harvested by laser capture microdissection
Introduction
The central nervous system (CNS) is composed of a great variety of heterogeneous cell types. In addition to the major categories of neurons, astrocytes, oligodendrocytes and microglia, the neuronal population is further subdivided into a vast complex of phenotypes defined by differential gene expression, connectivity and function. Approaches that employ microarray methods for gene expression profiling of microdissected brain regions (Bunney et al., 2003, Mirnics et al., 2000) unavoidably sample a mixture of multiple cell types. Consequently, expression changes uniquely generated in a specific phenotypic component of the cellular mixture will be diluted by different responses in the other cells.
The ability to harvest individual cells by laser capture microdissection (LCM) (Cornea and Mungenast, 2002, Simone et al., 1998, Wittliff and Erlander, 2002) and to use the RNA extracted from these cells for gene expression profiling represents a major advance in the study of specific CNS neuronal phenotypes. However, effective use of LCM to harvest specific cellular phenotypes requires visual guidance by the distinctive anatomy, morphology or specific labeling of the target cells. Previous studies, by other groups, have used LCM to harvest specific neuronal phenotypes from brain for gene expression profiling.
In one approach, midbrain dopamine neurons were harvested by LCM following immunocytochemical identification of tyrosine hydroxylase (TH) immunopositive cells. RNA extracted from 200 TH-positive cells was amplified, radiolabeled and then used to probe a custom cDNA array of approximately one hundred genes (Backes and Hemby, 2003, Fasulo and Hemby, 2003). The paraformaldehyde tissue fixation required for immunostaining would be expected to cause extensive cross-linking of RNA and the absence of validation of the array results by in situ hybridization or quantitative RT-PCR (QRT-PCR) make it difficult to assess the utility of this approach.
In another gene profiling study, 100 cells from each of seven different brain nuclei were harvested by LCM guided by rapid Nissl staining to identify neurons and the known anatomical locations of the target nuclei (Bonaventure et al., 2002). Amplified RNA was then applied to a custom microarray of a few thousand cDNAs and comparisons were made between the different nuclei. For each nucleus, expression of one or two known signature genes was enriched. Further, expression differences between nuclei for four, randomly selected genes were shown to roughly correspond to expression levels determined independently by QRT-PCR and the novel finding of unexpectedly equal expression of a particular single gene across all nuclei was verified by in situ hybridization.
A related study used LCM to harvest relatively large subregions of brain and applied the amplified and labeled RNA derived from these sites to Affymetrix mouse chips for global gene profiling (Zirlinger, 2003). Results from this study found general agreement in gene expression between hand dissected material which did not require RNA amplification and the LCM-derived material which did, indicating that the amplification method preserved the overall transcriptome representation of the tissues. Moreover, in situ hybridization was shown to validate the expression patterns determined by microarray analysis of roughly 60% of 18 selected genes. Other studies have used LCM and gene profiling to analyze gene expression in single cells with promising results (Kamme et al., 2003, Tietjen et al., 2003).
The present study used a neuronal tracing technique to fluorescently label a well-characterized neuronal phenotype, the dopamine neurons of the substantia nigra (SN). Labeling was performed using fluoro-gold, a fluorescent dye taken up by synaptic endings or cut axons and retrogradely transported to neuronal cell bodies (Naumann et al., 2000). A single injection of the dye into the rat striatum has been shown to label cortical neurons in mid-cortical layers via the corticostriatal pathway and dopamine neurons of the substantia nigra (SN), zona compacta via the nigrostriatal pathway (McNeill et al., 1999). Pre-labeling the specific target cell population could direct LCM on frozen sections without subjecting the tissue RNA to destructive fixation protocols. The purpose of the study was to determine whether the labeled cells would effectively guide LCM harvesting and if the RNA extracted and then amplified from these cells would maintain transcriptional representation. The latter was evaluated by performing differential two-color gene expression analysis in which gene expression in fluorescently-labeled cortical neurons was compared to gene expression in fluorescently-labeled dopamine neurons. Gene expression in the dopamine neurons had been previously well-characterized and therefore these cells presented an array of positive control genes to test the validity of the approach. Further, microarray results on selected genes were further evaluated by quantitative RT-PCR.
Section snippets
Animals and stereotaxic injection
Male Sprague–Dawley rats 250–275 g (Harlan, Indianapolis, IN) were anesthetized with ketamine (75 mg/kg)/xylazine (10 mg/kg) and prepared for stereotaxic surgery according to procedures approved by the Wayne State University Animal Investigation Committee. The rats were placed in a Kopf (Tujunga, CA) stereotaxic instrument and 1 μl of a 5% fluoro-gold (Fluorochrome, Denver, CO) solution was bilaterally delivered to the striatum at 0.2 μl/min, using a CMA microinjection pump (North Chelmsford, MA)
Co-localization of fluoro-gold and tyrosine hydroxylase in SN neurons
Fig. 1 illustrates the distribution of fluoro-gold labeled neurons in rat SN 5 days after injection of the tracer into the striatum. The right panel of Fig. 1 indicates tyrosine hydroxylase immunoreactivity of the same section. These results showed that virtually all (>98%) of the fluoro-gold labeled cells also expressed tyrosine hydroxylase and were thus dopamine neurons. These results indicated that fluoro-gold in SN cells would serve as a reliable visual guide for the LCM harvesting of
Discussion
The present study shows that specific neuronal subtypes in the brain can be labeled and harvested by LCM, and that the RNA extracted from these cells can be amplified and used for informative microarray analyses. By labeling a well-characterized nucleus of midbrain dopamine neurons, results of the microarray directly reflect the representational quality of the amplified RNA. The genes exhibiting the greatest enrichment in the samples obtained from SN dopamine neurons were precisely those
Acknowledgements
This work was supported by NIH grant R41 DA016406 from the National Institute on Drug Abuse. Microarray analysis was supported by the Microarray and Bioinformatics Facility Core NIEHS Center grant P30 ES06639.
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