Alan Thorpe

Glucose and amino acid-stimulated insulin release in vivo in the European silver eel (Anguilla anguilla L.).

Abstract Glucose and amino acid-stimulated insulin release was studied in vivo in cannulated European silver eels ( Anguilla anguilla ). Glucose-stimulated insulin release was dose dependent over a range of glucose loads (10–100 mg/kg) while higher doses (300 and 500 mg/kg) produced no greater increments than 100 mg/kg. Arginine and lysine injections of 10, 25 and 100 mg/kg caused greater significant increases in plasma insulin levels than glucose at the same dose levels. Histidine (10 and 25 mg/kg) caused a small but significant reduction in the plasma insulin level. Simultaneous injection of arginine (100 mg/kg) and glucose (100 mg/kg) caused an increase in plasma insulin which was sustained for 60 min and remained significantly above the controls over the 360 min sampling period. Total insulin secretion appeared to be significantly greater over the entire sampling period than when arginine and glucose were injected alone.

Immunochemical characterization of gastrin/cholecystokinin-like peptides in the brain of the blowfly, Calliphora vomitoria

Gastrin/cholecystokinin-like material in the brains of the blowfly, Calliphora vomitoria, was extracted with boiling water and purified by immunoaffinity adsorption to a C-terminal-specific gastrin antiserum. The material eluted in two peaks after separation by gel filtration on Sephadex G-50. Both peaks reacted in radioimmunoassays using antisera specific for the C terminus of gastrin and CCK and did not react with antisera specific for N terminus or intact heptadecapeptide gastrin. Estimates of the concentrations of immunoreactive material depended on the antiserum used; the highest estimate was 1.9 pmol/g of Calliphora heads. The immunochemical data indicate that the Calliphora peptides resemble, but are distinguishable from, C-terminal fragments of the mammalian brain-gut peptide cholecystokinin and the structurally related hormone gastrin. Evidently representatives of this group of molecules are widely distributed in nervous tissue in invertebrates as well as vertebrates.

Cobalt-immunocytochemical identification of peptidergic neurons in Calliphora innervating central and peripheral targets

SummaryCertain neurons of the blowfly,Calliphora erythrocephala, show immunoreactivity to anti-gastrin/cholecystokinin (CCK) COOH terminal specific antisera. However, as is common to immunocytochemical staining, much of the structure of the immunoreactive neurons escapes detection. We describe here whole-neuron identification by backfilling with Co2+ and subsequent silver reduction, combined with immunocytochemistry of the filled cells. Cobalt-silver filled neurons can be examined directly by fluorescence microscopy for the presence of a secondary, rhodamine-conjugated antibody linked to the primary one. Two peptide-containing pathways have been resolved, one leading out of the brain to the corpus cardiacum, the other innervating certain higher brain centres, such as the central body. Both arise from neurosecretory cells of the mid-brain. Immunoreactive peptidergic neurons leading, respectively, to the corpus cardiacum and to the central body have been matched to single nerve cells visualized by Golgi impregnation, cobalt backfilling or focal injection of cobalt into the brain.

Allatostatins and allatotropin in the corpus cardiacum/corpus allatum complex of larval and adult lepidopterans studied by confocal laser scanning microscopy: correlation to juvenile hormone biosynthesis

Peptidergic innervation of the corpus cardiacum/corpus allatum (CC/CA) retrocerebral complex, and neurosecretory areas of the brain of the lepidopterans Lacanobia oleracea, Heliothis virescens and Manduca sexta was studied by immunocytochemistry linked to confocal laser scanning microscopy. The patterns of immunostaining resulting from the simultaneous application of fluorochrome-conjugated antibodies against Manduca sexta allatostatin (Mas-AS), M. sexta allatotropin (Mas-AT), and a representative of the −Y/FXFGL-NH2 superfamily of allatostatins was correlated with the physiological effects of these putative allatoregulatory peptides on juvenile hormone (JH) biosynthesis by the corpora allata. Whereas the two types of allatostatin immunoreactivity are present in both larval and adult CA of the three species, allatotropin immunoreactivity occurs only in the adult gland. The conclusion that withdrawal of the stimulatory effect of allatotropin is unlikely to be involved in the downregulation of CA activity prior to the onset of metamorphosis, but that an inhibitory influence of at least Mas-AS is important, is borne out in physiological experiments on JH biosynthesis in M. sexta larvae (Mas-AS inhibitory, Mas-AT without effect). Immunoreactivity to the Y/FXFGL-NH2 allatostatins is present in both larval and adult CA and CC, frequently co-localised with Mas-AS. The function of this peptide family in the retrocerebral complex remains enigmatic since experiments on JH biosynthesis, either when the peptide is administered alone, or together with Mas-AS, show no effect on JH biosynthesis.

The effects of diabetogenic and hypoglycaemic agents in the northern pike, Esox lucius L.

Abstract 1. The effects of intra-arterial injection of mammalian diabetogenic and hypoglycaemic agents on three blood metabolites, and pancreatic islet and liver cytology were studied in cannulated pike. 2. Alloxan (50 and 100 mg/kg) produced a sustained hyperglycaemia with return to normal levels after 24 hr at the lower dose only. 3. Streptozotocin (100 mg/kg) effected a transient hyperglycaemia after 0·5 hr. 4. Anti-insulin serum (1ml/kg) produced a marked hyperglycaemia between 3 and 9 hr after injection 5. Tolbutamide (50 and 100 mg/kg) produced a marked hyperglycaemia after 0·5 hr. 6. No effects on plasma amino acid nitrogen or cholesterol were observed by these treatments. Similarly, neither alloxan nor streptozotocin affected liver or islet cytology. 7. With the possible exception of anti-insulin serum, these agents exhibit different properties in the pike as compared with mammals.

Immunocytochemical mapping of neuronal pathways from brain to corpora cardiaca/corpora allata in the cockroach Diploptera punctata with antisera against Met-enkephalin-Arg6-Gly7-Leu8

SummaryNeuronal circuits in the brain and retrocerebral complex of the cockroach Diploptera punctata have been mapped immunocytochemically with antisera directed against the extended enkephalin, Met-enkephalin-Arg6-Gly7-Leu8 (Met-8). The pathways link median and lateral neurosecretory cells with the corpus cardiacum/corpus allatum complex. In females, nerve fibres penetrate the corpora allata and varicosities or terminals, immunoreactive to Met-8, surround the glandular cells. Males differ in having almost no Met-8 immunoreactivity in the corpora allata. The corpora cardiaca of both males and females are richly supplied with Met-8 immunoreactive material, in particular in the ‘cap’ regions immediately adjacent to the corpora allata. A similarity in the amino-acid sequences of Met-8 and the C-terminus of the recently characterised allatostatins of D. punctata suggests that the pathways identified with the Met-8 antisera may be the same as those by which the allatostatins are transported from the brain to the corpus allatum. In comparative studies on the blowfly Calliphora vomitoria, similar neuronal pathways have been identified except that no sexual dimophism with respect to amounts of immunoreactive material within the corpus allatum has been observed. These results suggest a possible homology in the neuropeptide regulation of the gland.

Purification, Characterisation and Cellular Distribution of Insect Neuropeptides with Special Emphasis on their Relationship to Biologically Active Peptides of Vertebrates

Immunocytochemical techniques have been used on the insect nervous system to study the occurrence and distribution of neuropeptides related to the biologically active peptides of vertebrates.

Identification of Neurons Containing Vertebrate-Type Brain-Gut Peptides by Antibody and Cobalt Labelling

Different chemical compounds such as cobalt chloride, heme proteins and fluorescent dyes (e.g. Procion yellow and Lucifer yellow) have proved invaluable for tracing neurons. They do not, however, reveal the identity of the chemical contents, whereas immunocytochemistry does. Immunocytochemical methods are inferior, however, for revealing the complete dendritic domain and axonal pathway of a particular cell due to a variety of reasons. First, with the thin sections (5–10 µm) obligatory for this technique, a curved and branched axonal pathway cannot be followed very far since the plane of section does not usually coincide with the axonal trajectory. Second, there are considerable variations in the metabolic activities of peptidergic neurons that are reflected in a changing distribution of material in both the perikarya and the axons. Factors such as age, diet, sex and, possibly, behavioural history are very important in determining how much of a particular pathway is stainable at any particular time (see also Chap. 13).

Immunochemical Identification of Vertebrate-Type Brain-Gut Peptides in Insect Nerve Cells

Immunocytochemistry has been used with great success over the past decade in the identification of the sites of the so-called brain-gut peptides of vertebrates. Information resulting from the use of this technique has made it clear that bioactive peptides that were once thought to be strictly gut peptides or hormones, such as cholecystokinin (CCK) and vaso-active intestinal peptide (VIP), are also widespread in the central and peripheral nervous systems. Conversely, neuropeptides such as the enkephalins and substance P have been shown to occur in the gut (cf. Hokfelt et al. 1980 a, b). The list of these dual occurrence brain-gut peptides is constantly being extended.