Locustatachykinin immunoreactivity in the blowfly central nervous system and intestine

Abstract

An antiserum raised against locustatachykinin I, one of four myotropic peptides that have been isolated from the locust brain and corpora cardiaca, was characterized by enzyme-linked immunosorbent assay (ELISA) and used for immunocytochemical detection of neurons and endocrine cells in the nervous system and intestine of the blowfly Calliphora vomitoria. The ELISA characterization indicated that the antiserum recognizes the common C-terminus sequence of the locustatachykinins I-III. Hence, the cross reaction with locustatachykinin IV is less, and in competitive ELISAs no cross reaction was detected with a series of vertebrate tachykinins tested. It was also shown that the antiserum recognized material in extracts of blowfly heads, as measured in ELISA. In high-performance liquid chromatography the extracted locustatachykinin-like immunoreactive (LomTK-LI) material eluted in two different ranges. A fairly large number of LomTK-LI neurons was detected in the blowfly brain and thoracicoabdominal ganglion. A total of about 160 LomTK-LI neurons was seen in the proto-, deuto-, and tritocerebrum and subesophageal ganglion. Immunoreactive processes from these neurons could be traced in many neuropil regions of the brain: superior and dorsomedian protocerebrum, optic tubercle, fan-shaped body and ventral bodies of the central complex, all the glomeruli of the antennal lobes, and tritocerebral and subesophageal neuropil. No immunoreactivity was seen in the mushroom bodies or the optic lobes. In the fused thoracicoabdominal ganglion, 46 LomTK-LI neurons could be resolved. The less evolved larval nervous system was also investigated to obtain additional information on the morphology and projections of immunoreactive neurons. In neither the larval nor the adult nervous systems could we identify any efferent or afferent immunoreactive axons or neurosecretory cells. The widespread distribution of LomTK-LI material in interneurons suggests an important role of the native peptide(s) as a neurotransmitter or neuromodulator within the central nervous system. Additionally a regulatory function in the intestine is indicated by the presence of immunoreactivity in endocrine cells of the midgut.

Expression and Functional Characterization of aDrosophila Neuropeptide Precursor with Homology to Mammalian Preprotachykinin A

“…The others appear to be local interneurons. By comparison, the blowfly displays a larger number of LTK-LI neurons both in the brain and ventral nerve cord (30). The distribution of LTK-LI cells in the larval and adult midgut of Drosophila is very similar to that revealed in the blowfly Calliphora (47).…”

“…Tissues were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer for 3-4 h. Central nervous systems and intestines were used for whole-mount immunocytochemistry as described (30) with an antiserum raised against the locust TRP LomTK-I (31) at a dilution of 1:1000. Either the peroxidase/ anti-peroxidase technique or immunofluorescence with a secondary antiserum tagged with Cy3 was used.…”

“…Preabsorption controls were made with antiserum (at 1:1000) incubated with 50 nmol of LomTK-I. The specificity of the antiserum has been established previously (30,31).…”

Insect tachykinin-related neuropeptides: Developmental changes in expression of callitachykinin isoforms in the central nervous system and intestine of the blowfly,Calliphora vomitoria

“…LomTK-like immunoreactivity (LomTK-LI) was demon-*Abbreviations used: BSA = bovine serum albumin; CavTK = callitachykinin; CNS = central nervous system; ELISA = enzyme-linked immunosorbent assay; HPLC = high performance liquid chromatography; LomTK = locustatachykinin; RIA = radioimmunoassay; TFA = trifluoroacetic acid; TK-LI = tachykinin-like immunoreactivity; TRP = tachykinin-related peptide. strated in interneurons in the central nervous system (CNS) of both larvae and imagos of C. vomitoria (Lundquist et al, 1994b). More recently, we analyzed extracts of larval and adult CNS by a combination of high performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) utilizing a LomTK antiserum to reveal whether both CavTKs were present in the tissues .…”

“…In the blowfly Calliphora vomitoria, peptides from the family of insect tachykinin-related peptides (TRPs) (Schoofs et al, 1990a(Schoofs et al, ,b, 1993 would be suitable for such an analysis. In C. vomitoria only two tachykinin-related peptides, callitachykinins I and II (CavTK* I and II), have been isolated and sequenced so far (Lundquist et al, 1994b). These peptides were isolated from an extract of whole insects, but it was possible to determine the cellular localization with an antiserum raised against the locust peptide locustatachykinin I (LomTK I), which crossreacts with all known insect TRPs (Lundquist et al, 1994a,b;Muren and Nässel, in press).…”