The distributions of PHI and VIP in porcine gut and their co-localisation to a proportion of intrinsic ganglion cells

VIP and PHI share sequence homology and certain biological actions. Immunocytochemistry and radioimmunoassay were used to see if the two peptides also have similar distributions in the gut of the pig. PHI-immunoreactive fibres were found, like those containing VIP, in all layers of the bowel wall but in lesser numbers. Unlike VIP-immunoreactive nerves, however, which are ubiquitous in the gastrointestinal tract, PHI-containing neurons were numerous in all areas except the fundus, where only few fibres and no ganglion cells were found to be reactive to PHI antibodies. PHI and VIP immunoreactive materials were also quantified by specific radioimmunoassay of tissue extracts. The concentrations of PHI and VIP were similar in all regions of the gut, except in the fundus where the quantities of VIP-immunoreactivity far exceeded those of PHI. The presence of both VIP- and PHI-immunoreactivities in ganglion cells of the sub-mucous plexus allowed investigation of the co-localisation of the peptides. Serial sections through ganglion cells revealed that a major proportion contain both PHI- and VIP-immunoreactivity. Some cells contained VIP alone, or VIP and weak, equivocal immunostaining of PHI, and a sub-population contained no peptide-immunoreactivity. The presence of both VIP- and PHI-immunoreactivities in the same ganglion cell supports the recent reports of the isolation and characterisation, using genetic technology, of their common precursor molecule. The finding of VIP and not PHI in the fundic region suggests the differential expression of the two peptides.     

Chemical coding of neurons in the myenteric plexus and external muscle of the small and large intestine of the mouse

Immunohistochemical techniques were used to examine the presence and co-localisation of a range of putative neurotransmitters and other neuronal markers in the myenteric plexus of the small and large intestine of the mouse. Distinct sub-populations of myenteric neurons were identified, based on the combinations of substances they contained and the distribution of their fibres. In the small intestine, there were two major classes of circular muscle motor neurons; one class was characterised by the presence of nitric oxide synthase, vasoactive intestinal peptide plus neuropeptide Y (NOS/VIP/NPY), and the second class contained calretinin plus substance P (CalR/SP). There were seven classes of neurons that innervated myenteric ganglia; these contained NOS, VIP, NOS/VIP, NPY, CalR/calbindin (CalB), SP or 5-HT. In the large intestine, there were five major classes of motor neurons that contained NOS, NOS/VIP, GABA, SP, or CalR/SP, and seven major classes of neurons that innervated myenteric ganglia and contained NOS, VIP, CalR/CalB, CalR, SP, GABA or 5-HT. Although some aspects of the patterns of co-localisation are similar to those in other species, this study re-inforces recent analyses that indicate significant species differences in neurochemical patterns in the enteric neurons of different species. Received: 28 August 1995 / Accepted: 30 November 1995     

Localisation of three host-protective oncospheral antigens of Taenia ovis

Immunohistochemistry, confocal immunofluorescence and immunogold labelling were used to determine the localisation of the host-protective antigens To16, To18 and To45W in Taenia ovis oncospheres. During maturation of the adult tapeworm the antigens were initially seen as diffuse staining in the developing oncospheres but in mature oncospheres four distinct cells stained positively for the antigens. Confocal fluorescence microscopy using different fluorophores revealed that each of the antigens co-localises within the same cells in the oncosphere. No surface localisation was seen in non-activated or recently activated parasites. Immunogold labelling of non-activated oncosphere sections viewed in transmission electron microscopy revealed labelling of bilateral cells, however the identities of these cells was unclear due to deficiencies in the current level of understanding of oncosphere ultrastructure. Localisation of all the antigens changed dramatically after oncospheres were activated in vitro with each of the antigens being dispersed more generally throughout the parasite parenchyma. During development of the parasites in in vitro culture, surface localisation of the proteins was seen in parasites after 3 or more days in culture. All three antigens were found to be completely absent in parasites by 15 days of culture. The location of the host-protective antigens suggests that initially the invading oncospheres are not susceptible to vaccine-induced antibody and complement mediated attack, but that as the parasites mature, the host-protective antigens come to be associated with the parasite’s surface, rendering them susceptible to immune attack.     

Detecting fluorescent protein expression and co-localisation on single secretory vesicles with linear spectral unmixing

Many questions in cell biology and biophysics involve the quantitation of co-localisation and the interaction of proteins tagged with different fluorophores. However, the incomplete separation of the different colour channels due to the presence of autofluorescence, along with cross-excitation and emission "bleed-through" of one colour channel into the other, all combine to render the interpretation of multi-band images ambiguous. Here we introduce a new live-cell epifluorescence spectral imaging and linear unmixing technique for classifying resolution-limited point objects containing multiple fluorophores. We demonstrate the performance of our technique by detecting, at the single-vesicle level, the co-expression of the vesicle-associated membrane protein, VAMP-2 (also called synaptobrevin-2), linked to either enhanced green fluorescent protein (EGFP) or citrine [a less pH-sensitive variant of enhanced yellow fluorescent protein (EYFP)], in mouse cortical astrocytes. In contrast, the co-expression of VAMP-2-citrine and the lysosomal transporter sialine fused to EGFP resulted in little overlap. Spectral imaging and linear unmixing permit us to fingerprint the expression of spectrally overlapping fluorescent proteins on single secretory organelles in the presence of a spectrally broad autofluorescence. Our technique provides a robust alternative to error-prone dual- or triple colour co-localisation studies.     

Immunogold staining procedure for the localisation of regulatory peptides

The use of protein A- and IgG-conjugated colloidal gold staining methods for the immuno-localisation of peptide hormones and neurotransmitters at light- and electron microscope level are described and discussed. Bright-field and dark-ground illumination modes have been used to visualise the gold-labelled antigenic sites at the light microscope level. Immunogold staining procedures at the ultrastructural level using region-specific antisera have been adopted to localise specific molecular forms of peptides including gastrin (G17 and G34), glucagon and pro-glucagon, insulin and pro-insulin, in normal tissue and in tumours of the gastroenteropancreatic system. Similar methods have been used to demonstrate the heterogeneity of p-type nerves in the enteric nervous system. Vasoactive intestinal polypeptide (VIP) has been localised to granular sites (mean +/- S.D. granule diameter = 98 +/- 19 nm) in nerve terminals of the enteric plexuses and in tumour cells of diarrhoeogenic VIP-producing neoplasias (mean +/- S.D. granule diameter = 126 +/- 37 nm) using immunogold procedures applied to ultraviolet-cured ultrathin sections. Co-localisation of amines and peptides in carotid body type I cells and in chromaffin cells of normal adrenal medulla and phaeochromocytomas has also been demonstrated. Advantages of the immunogold procedures over alternative immunocytochemical techniques are discussed.