Immunocytochemical studies on peptidergic neurons in the Colorado potato beetle and some other insect species

This thesis describes the distribution, numbers, and morphology of peptidergic neurons and neurosecretory cells in the Colorado potato beetle, as detected with immunocytochemistry with antisera to various regulatory peptides from vertebrates, as well as to the molluscan cardioexcitatory peptide FMRFamide, and to the insect neuropeptide proctolin.<p/>In chapter one, a description is given of peptidergic neurons in the Colorado potato beetle which are immunoreactive with antisera to FMRFamide and bovine pancreatic polypeptide. Results from cross-adsorption experiments suggest that the antisera to FMRFamide and bovine pancreatic polypeptide cross-react and reveal the same substance(s) in the Colorado potato beetle. Some of the immunoreactive neurons branch extensively in the neuropile, suggesting that the immunoreactive substance is used as a transmitter or modulator. The corpus cardiacum contains numerous immunoreactive axon terminals from neurosecretory cells located in the suboesophageal ganglion. These cells probably use the peptide as a hormone.<p/>In chapter two, ten different antisera were used to differentiate between neurons in the neuroendocrine system of the Colorado potato beetle. Four antisera to vasopressin, three to oxytocin and one to vasotocin, neurophysin-1 and neurophysin-2 each were used. All antisera revealed immunoreactive cells, except for the two neurophysin antisera. The antisera to vasopressin all gave different results. Whereas one antiserurn revealed only a single neuron pair, the other three antisera revealed additional groups of immunoreactive cells. The three oxytocin antisera also gave different results. It was further found that the fixation procedure also greatly influenced the immunocytochemical results. It was concluded that several vasopressin and oxytocin immunoreactive peptides are present in the Colorado potato beetle, all with a different degree of resemblance to vasopressin and oxytocin.<p/>In chapter three, it was investigated whether immunocytochemical techniques could be used for the demonstration of homologous neurosecretory cells in the suboesophageal ganglion of the Colorado potato beetle and of the migratory locust. The antisera were anti-FMRFamide, anti-bovine pancreatic polypeptide, two antisera to vasopressin, which all had been used before in the work described in chapters 1 and 2, and an antiserurn to α-MSH. It is shown that neurosecretory cells are present in the suboesophageal ganglion of both species and that they occur in similar locations and have the same immunoreactivity. This suggests that the two species contain homologous neurosecretory cells. It is speculated that they also have similar functions.<p/>In chapter four, the concept of homologous neurons is further developed and ten different insect species were examined for the presence of neurons immunoreactive with a specific antiserurn to gastrin-releasing peptide. Immunoreactive neurons were revealed in seven species, belonging to seven orders. The concentration of antiserurn required to demonstrate immunoreactive cells was much higher in insects than in chicken proventriculus, known to contain gastrin-releasing peptide. Some immunoreactive neurons were found in the same numbers and approximately the same locations within the nervous system of different insect species. This suggests that the substances in these neurons have been relatively stable during evolution and that these neurons are homologous.<p/>In chapter five, a large number of antisera to various regulatory vertebrate peptides was tested on the Colorado potato beetle, to reveal peptidergic neurons and neurosecretory cells and to differentiate between these cells. Immunoreactive cells were revealed by antisera to ACTH, gastrin, cholecystokinin, α-endorphin, β-endorphin, γl-MSH, insulin, human calcitonin, motilin, growth hormone, somatostatin, corticotropin-releasing factor, ovine prolactin and rat prolactin. Some of these immunoreactive cells seem to function as neurons, whereas others function as neurosecretory cells. It is postulated that if the part of the substance recognized by the antiserurn is of physiological importance to the insect, that part is retained in several species. The antiserurn should then reveal homologous neurons in different insect species. Given the fact that insect species differ widely in their immunocytochemical responses, the criterion of staining of probably homologous neurons offers some help in separating relevant from irrelevant immunoreactions. The immunocytochemical data are evaluated according to this concept.<p/>In chapter six, neurons in the central and peripheral nervous system of the Colorado potato beetle were described which contain substances immunoreactive with antiserurn to the insect neuropeptide proctolin. This peptide was originally isolated from cockroaches, in which it stimulates contractions of the gut musculature. Immunoreactive axon terminals were also found on the muscles of the fore- and hindgut, and abdominal segments, as well as in the vas deferens of the testis. Furthermore, the nervous system was extracted to investigate whether proctolin-like bioactivity could be demonstrated. A proctolin-like bioactive peptide was demonstrated, that behaved chromatographically like proctolin. This suggests that at least some of the immunocytochemically demonstrated proctolin is identical with or at least similar to proctolin.