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

Certain 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.