Characterization of the innervation of the gallbladder mucosa in the Australian brush-tailed possum

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Intrinsic and extrinsic nerves innervate the gallbladder epithelium and modulate its functions. Some neurochemicals of this innervation are known in some species, however, the intrinsic and extrinsic origin of this innervation is unclear. Aim: To characterize the neurons projecting to the gallbladder epithelium in the Australian Brush-tailed possum using in vitro retrograde tracing, extrinsic denervation in culture and immunohistochemistry. Methods: Gallbladders were harvested from 9 anesthetized possums. In 6 gallbladders, a fluorescent neural tracer was focally applied to the epithelial surface to identify intrinsic neurons innervating the epithelium. Following organotypic culture, the preparations were dissected and an antiserum against nitric oxide synthase (NOS) was applied. Retrogradely traced and antibody labeled neurons were visualized using fluorescence microscopy and their positions recorded. A further 3 gallbladders were cut into 4 longitudinal segments. Two segments from each gallbladder were fixed immediately and the others placed in organotypic culture for 5 days to achieve extrinsic penervatlon. Multiple-labeling immunohistochemistry for substance P (SP), vasoactive intestinal polypeptide (VlP), calcitonin gene related peptide (CGRP) and tyrosine hydroxylase (TH) were used to characterize the intrinsic and extrinsic innervation of the ganglionated and non-ganglionated sub-epithelial plexus of the gallbladder. Results: Retrogradely traced neurons were found within lOmm of the tracer application site, exclusively within the subepithelial plexus, with 63 _+ 9% being immunoreactive for NOS. The majority of neurons within the sub-epithelial plexus colocalized both NOS and MIP. Some SP immunoreactive neurons were seen. Varicose nerve fibres showing immunoreactivity for NOS, VIP, SP, CGRP and TH were seen within the sub-epithelial plexuses in freshly fixed tissue. Immunoreactivity for CGRP and TH were abolished post-culture, with some reduction in the SP and MIP innervation. Summary: NOS and VlP-colocalizing and SP-immunoreactive neurons of the ganglionated sub-epithelial plexus innervate the gallbladder epithelium, with no innervation from the muscular and serosal plexuses. This is the first demonstration of the neurochemistry and spatial organisation of the intrinsic innervation of the gallbladder epithelium. Sympathetic and extrinsic afferent neurons also directly innervate the epithelium and the ganglionated subepithelial plexus.