Galanin immunoreactivity in the blowfly nervous system: localization and chromatographic analysis

Abstract

In this study chromatographic, immunochemical, and immunocytochemical methods provide evidence of a galanin-like peptide(s) in an invertebrate, the blowfly Phormia terraenovae. The major portion of the galanin-like immunoreactivity (GAL-LI) in fly heads was extractable in acetic acid but not in boiling water, which suggests that the peptide(s) may be highly basic in nature. GAL-LI was present both in the head and body portion of the blowfly in roughly the same amounts. Initial gel filtration data, using a G-50 Sephadex column and a weak phosphate-buffer (pH 6.5) as eluent, suggested that a fly GAL-LI peptide(s) from fly heads, eluting as an apparent single peak, was smaller than porcine GAL(1-29) and GAL(1-15). However, concomitant analysis using a G-25 Sephadex column and acetic acid (0.2 M) as eluent, spread the immunoreactive material over a great portion of the chromatogram, although the main portion of the material eluted in the same size range as porcine GAL(1-29). Taken together, the gel filtration data thus suggest that fly GAL-LI peptide(s) may be highly basic but presumably similar in size to vertebrate GAL(1-29). However, the hydrophobic properties of the fly GAL-LI peptide(s) differ from that of porcine GAL as demonstrated by the presence of several immunoreactive components eluting both early as well as late in the chromatogram when using reverse-phase high performance liquid chromatography (HPLC); early peaks may represent highly basic and/or possibly smaller GAL-immunoreactive peptide(s), whereas later peaks may represent less basic and possibly elongated forms. Immunocytochemistry indicated that GAL-LI was present in the nervous system of the blowfly. About 160 GAL-immunoreactive neurons were found in the brain and subesophageal ganglion, 26 in the fused thoracic ganglion and 30 in the fused abdominal ganglion. In the brain, GAL-immunoreactive fibers supply specific subdivisions of the central body, optic lobe, superior protocerebrum, and tritocerebrum as well as neuropil in the subesophageal ganglia. In the thoracico-abdominal ganglia, GAL-immunoreactive neuron processes are found inside synaptic neuropil as well as in the neural sheath of the ganglia and several of the dorsal nerve roots. Many of the GAL-immunoreactive neurons react also with an antiserum against porcine galanin message associated peptide, a peptide present in the preprogalanin protein. Immunocytochemical double-labeling indicated that some GAL-immunoreactive neurons also reacted with antisera against the molluscan peptides FMRFamide and SCPB, whereas no evidence could be found for colabeling with antisera against tyrosine hydroxylase, substance P and physalaemin.(ABSTRACT TRUNCATED AT 400 WORDS)