Location of gene expression in CNS using hybridization histochemistry.
In this chapter we have placed heavy emphasis on our own recent work to lay out a workable recipe for hybridization histochemistry. Only a trickle of papers followed the initial benchmark excursions into in situ labeling of tissue sections. Our own entry into this field was as late starters in 1978, but since then a confluence of important questions and technical advances has served to make hybridization histochemistry much more attractive and universally applicable as a research tool. Hybridization histochemistry allows the location of anatomical sites of gene expression and viral replication with unique specificity and is able to solve some problems for which there is no other suitable technique available in the central nervous system. For example, allowing that peptides may enter neurons by a variety of mechanisms and then be christened neuroendocrine peptides, it has become a compelling issue to know which cells are manufacturing the peptide. Thus, much can be learned by the approach elegantly demonstrated by Gee et al., of locating mRNA and its peptide product within the same neuron. The intracellular location of specific mRNA for a neuropeptide in a cell body indicates a very high probability that the peptide is secreted as a neurotransmitter or a neuromodulator from sites associated with the cell body. Our introduction of the use of whole mouse sections and large sections of brain of large animals in hybridization histochemistry has great potential in locating hormonal, enzymatic, and growth factor gene expression. The technique has been applied most elegantly by others to developmental studies and for the examination of viral infection. Resolution down to a single cell in heterogeneous tissue was beyond the original expectation of the capability of 32P-labeled probes and single cells in sections shown in Fig. 2 is probably the limit of resolution with this isotope. There is no reason why other isotopes, fluorescent labels, or labels suitable for EM should not take the resolution of the technique to intracellular. The horizon of application is widened enormously by the successful application of synthetic oligonucleotide probes, and at the same time unshackles the procedure from dependence upon a fully functional molecular biology laboratory. Although hybridization is a valuable research tool which we have applied to location of neuropeptides in the brain, it should soon find a niche in many fields and in a short time should become a key diagnostic tool.(ABSTRACT TRUNCATED AT 400 WORDS)
D000818:Animals; D001345:Autoradiography / Q000379:methods; D001921:Brain / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004247:DNA / Q000378:metabolism; D005796:Genes; D007202:Indicators and Reagents; D009419:Nerve Tissue Proteins / Q000235:genetics; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D014158:Transcription, Genetic
D007202:Indicators and Reagents; D009419:Nerve Tissue Proteins; D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D004247:DNA