Proceedings of The Physiological Society

University of Central Lancashire (2002) J Physiol 543P, S228


Neurochemical coding of gastric vago-vagal pathways in the ferret

Richard L. Young*, Nicole J. Cooper* and L. Ashley Blackshaw*†

*Nerve-Gut Research Laboratory, Royal Adelaide Hospital and †Department of Medicine, Adelaide University, SA 5000, Australia

Anatomical and functional subdivisions of the somatic and enteric nervous systems have been distinguished according to unique neurochemical content. In the ferret, a widely used model of the gut-brain axis, gastric vagal afferents exist as two broad modality-signalling populations (Page & Blackshaw, 1998) but no histochemical correlate has been evaluated. We assessed histochemistry of calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4), selective markers of nociceptive afferents in the rat, as potential neurochemical markers of gastric vagal afferents in the ferret. Histochemistry of the nodose ganglia and dorsal medulla was performed with retrograde tracing of vagal pathways from the proximal stomach with a tracer cocktail containing cholera toxin subunit-B-conjugated FITC (5 %) and wheatgerm agglutinin-conjugated Alexa Fluor 488 (AF488, 2.5 %). Six ferrets were anaesthetized with halothane (2.5 % in O2), a laparotomy performed and tracer (10 X 5 µl) injected into the ventral and dorsal subserosa of the proximal stomach, 10 mm from the gastro-oesophageal junction. Four days later ferrets were anaesthetized with urethane (1.25 g kg-1 I.P.), humanely killed and perfuse-fixed. The left nodose ganglia and dorsal medulla were then removed, cryoprotected and sectioned (20 µm). CGRP immunoreactivity was detected using a rabbit anti-rat CGRP primary and an AF350-conjugated secondary. IB4 labelling was detected using IB4-conjugated biotin and streptavidin-AF546. Nodose neurons were counted in six transverse sections representing the rostrocaudal extent of each ganglion; total cell counts were obtained using differential interference contrast. 16 % of left-nodose neurons (afferents) innervated the ferret proximal stomach; 69 % of these contained CGRP, 53 % were positive for IB4 labelling, 46 % showed dual labelling (CGRP/IB4) and 23 % showed no label. Centrally, CGRP and IB4 labelled fibres in the subnucleus gelatinosus, lateral medial subnucleus of the NTS and at the dorsal border of the dorsal motor vagal subnucleus (at obex); IB4 also selectively labelled medial regions of the medial subnucleus of the NTS. In rostral sections (obex +1.5 mm) CGRP selectively labelled the interstitial subnucleus. In the dorsal motor vagal nucleus, gastric vagal efferent soma were unlabelled, whereas CGRP, IB4 and dual labelled fibres were seen in close apposition with these pre-ganglionic motor neurons, where they may make functional connections. In conclusion, neurochemical coding of gastric vagal afferents in the ferret can be defined according to CGRP and IB4 labelling. Differential labelling of these markers in central subnuclei of the gastric circuit may be relevant to processing in these vago-vagal pathways. Triple immunohistochemical studies are underway to assess these marker combinations, and others, to identify functional classes of gastric vagal afferents.

This work was funded by AstraZeneca.

Experiments were carried out with the approval and under the guidelines of the Ethics Committee of the IMVS, Adelaide.

Where applicable, experiments conform with Society ethical requirements