Neuropeptides immunocytochemistry

Fig. 2 Increased synthesis, content and release of vasopressin (AVP) in the PVN of high-anxiety (HAB) vs low-anxiety (LAB) rats under basal circumstances. Above left in situ hybridization. Above right immunocytochemistry (courtesy of Dr. N. Singewald, University of Innsbruck). Middle and below intra-PVN release of AVP and oxytocin (OXT) measured by in vivo microdialysis under basal conditions and in response to hypertonic stimulation to reveal the releasable neuropeptide pool. p<0.05, p<0.01 vs LAB. (Adapted from Wigger et al. 2004)...

Davis N. T., Homberg U., Teal P. E. A., Altstein M., Agricola H.-J. and Hildebrand J. G. (1996) Neuroanatomy and immunocytochemistry of the median neuroendocrine cells of the subesophageal ganglion of the tobacco hawkmoth, Manduca sexta immunoreactivities to PBAN and other neuropeptides. Microscopy Res. Techniq. 35, 201-229. 

Recently, the neuropeptide, corticotropin releasing factor (CRF), has been demonstrated in mitral and some tufted cells using both immunocytochemistry (Fig. 1 IB) and in situ hybridization in the rat (Imaki et al. 1989). CRF fibers were also observed in the molecular layer of the piriform cortex. This finding is consistent with CRF being a releasable neural peptide in mitral cells since mitral cells synaptically terminate in the molecular layer of the piriform cortex. A similar localization of CRF has been reported in the squirrel monkey suggesting that this peptide may be a conserved transmitter/ modulator in the mitral/tufted cells of many mammals (Bassett et al. 1992). Finally, calretinin, a calcium binding protein, has been shown by immunohistochemistry to be localized in mitral cells (Jacobowitz and Winsky, 1991). Transmitter candidates for mitral and tufted cells are discussed further in 2.5.3., Projections to olfactory cortex. 

Information on the primary structures enabled comparative endocrinologists to arrange peptides into structurally homologous peptide families one could also generate very specific antisera for localization of the neuropeptides in cells, tissues and organs with the aid of immunocytochemistry, and for peptide quantification (titre... 

Neuropeptides constitute the largest and most diverse class of signaling substances known in metazoans. Over the last 20 yr it has become apparent that neuropeptides have important roles as neurohormones, neuromodulators, cytokines, morphogenetic factors, and possibly in some cases, as true neurotransmitters. Each neuropeptide may even be multifimctional and exist in several isoforms in a given animal species. In the search for functions of neuropeptides, it has been critical to be able to localize sites of synthesis and release. Immunocytochemistry (ICC) has been instrumental in the accurate mapping of the cellular and subcellular distribution of neuropeptides in tissue. Other immunological assays, such as radioimmunoassay (RIA) and immxmo-enzymatic assay (ELISA) provide powerful complements for quantification of neuropeptides. Several important discoveries related to neuropeptides have relied on ICC, for example Different neuropeptides have very specific distributions in small populations of neurons (1—3), neuropeptides are commonly colocalized with low-mol-wt neurotransmitters or other neuropeptides (4), the chemical diversity of neurons is far greater than previously suspected (2,3), and neuropeptide synthesis and release can be episodic (5). 

Tibbetts, M. F. and Nichols, R (1993). Immunocytochemistry of sequence-related neuropeptides m Drosophila. Neuropeptides 24,321—325. 

The diversity of neuropeptide transmitters is fairly large, as compared to other, classical neurotransmitters, such as acetylcholine and glutamate. Currently, immunocytochemistry and in situ hybridization are the methods of choice to map neuropeptide expression patterns in the nervous system. A disadvantage of these methods, however, is that they give limited clues to the structural identities of processed and biologically active peptides contained in the cells. Moreover, these techniques do not reveal previously undescribed cotransmitters. 

Nassel DR, Ekstrom P (1997) Detection of neuropeptides by immunocytochemistry. Methods Mol Biol 72 71-101... 

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