Hybridization approaches to the study of neuropeptides

During the course of evolution, species have increased in complexity, and their nervous systems have evolved correspondingly with an increase in the diversity of their capabilities to respond. Part of that diversity has resulted from an increase in cell types and numbers and their interconnections. In addition, much of it comes from the panoply of neurotransmitters available, of which the neuropeptides represent a major portion. The application of the techniques of molecular biology to the nervous system has led to an appreciation of some of the genetic means by which such diversity can be generated. The cloning and sequencing of peptide precursor genes has shown the existence of gene families, genes with duplications of internal sequences, and genes evolutionarily related to one another, suggesting that one response to the increasing complexity of the organism has been a genetic diversification of the precursor population for peptides. As the precursor genes evolved and thereby provided increasing numbers of peptides, the receptor genes may have evolved simultaneously to provide diversification in the responses to these peptides (for example, the opioid peptide precursors) (Comb et al 1983). The precursor sequences obtained have led not only to the predictions of new peptides but also to the discovery of alternative methods of generating diversity from a single gene. At one extreme, the gene is translated into a polyprotein containing several peptides, which are produced in and released from the same cell. At the other extreme, the nuclear transcript of the gene is differentially spliced such that one peptide is expressed in one tissue and another in a different tissue (Calcitonin-CGRP), or one peptide may be expressed with or without a second peptide in different cells (substance P-substance K). The net result is either one neuron producing a multiplicity of responses to several co-released peptides derived from a polyprotein (POMC or PE) or a tissue- or cell-specificity in terms of which peptide is produced and released. Numerous applications have been made utilizing the cDNA probes generated from the cloning of neuropeptide precursors. Hybridization analyses, including in vitro transcription run-off, have demonstrated that the transcription of neuropeptide genes is regulated by transsynaptic activation of transmitter receptors located in the neuronal membrane, or by hormones, or by as yet unveiled mechanisms. Hybridization techniques have allowed assessment of the dynamic state of neuropeptides functioning as neuromodulators.(ABSTRACT TRUNCATED AT 400 WORDS)