Applications of In Situ Hybridization and Immunocytochemistry for Localization and Quantification of Peptide Gene Expression — A Lesson From Islet Amyloid Polypeptide

Abstract

The identification of the messenger phenotype of neuroendocrine cells is crucial for the understanding of neuroendocrine mechanisms. Neuroendocrine cells generally express several neurohormonal peptide messengers—sometimes together with non-peptide messengers—and this expression may be subject to alterations during ontogeny and under pathological conditions. In addition, the level of messenger expression is regulated in response to physiological stimuli. At present, several techniques can be used to assess messenger expression in neuroendocrine cells. Immunocytochemistry, using antibodies directed at these messengers and applied as single, double or triple immunostaining, reveals the localization and co-localization of neurohormonal peptides. In situ hybridization, using probes designed to hybridize with a target mRNA in tissue sections, demonstrates the cellular localization of peptide mRNA; in situ hybridization can be combined with immunocytochemistry and, thus, simultaneously demonstrate the site of expression of both mRNA and peptide. Furthermore, in situ hybridization can, by the aid of computerized image analysis, be used to quantitate levels of mRNA in tissue sections and thereby monitor alterations in gene expression under different conditions. Collectively, these techniques have greatly expanded the present-day knowledge of the neuroendocrine system. In this review, we focus on the recently discovered islet hormone candidate—islet amyloid polypeptide (IAPP or amylin)—to illustrate the use and relevance of these techniques. Protocols for in situ hybridization are given and discussed from a practical standpoint. IAPP is structurally related to calcitonin gene-related peptide (CGRP) and adrenomedullin and is predominantly co-expressed with insulin in pancreatic islets; in some species, such as rat, IAPP is also expressed in islet somatostatin cells. Moreover, IAPP is expressed in gastrointestinal neuroendocrine cells, most of which co-express somatostatin, and in a population of CGRP-containing sensory neurons. Under diabetes-like experimental conditions induced by dexamethasone or streptozotocin, the islet expression of IAPP and insulin dissociate, in that IAPP is over-expressed relative to insulin. The implications of the dissociated expression of IAPP and insulin remain unclear but may be relevant to the pathogenesis and course of diabetes, due to the metabolic and paracrine effects of IAPP-restraining insulin action and release. The role of IAPP in the gastrointestinal tract and in sensory neurons remains to be clarified.