Immunolocalisation of AMPA receptor subunits within the enteric nervous system of the mouse colon and the effect of their activation on spontaneous colonic contractions Short running title: AMPA receptors in the mouse colon Authors:

Background: The appropriate expression of specific neurotransmitter receptors within the cellular networks which compose the enteric nervous system (ENS) is central to the regulation of gastrointestinal (GI) functions. Whilst the ENS expression patterns of the neurotransmitter glutamate have been well documented, the localisation of its receptors on ENS neurons remains to be fully characterised. We investigated the expression patterns of glutamate receptor AMPA subunits within ENS neurons of the mouse colon and the consequences of their pharmacological activation on spontaneous colonic contractility. Methods: RT-PCR was used to detect individual AMPA receptor (GluR 1-4) subunit expression at the mRNA level in mouse colon tissue. Immunohistochemistry and confocal microscopy was used to localise the expression of the GluR1, 4 subunits in colon tissue. Brain tissue was used as a positive control. Organ bath preparations were used to determine the effect of AMPA receptors activation on the force and frequency of colonic longitudinal smooth muscle spontaneous contractions. Key Results: GluR1, 3, 4 mRNA was detected in the mouse colon. Immunoreactivity for GluR1 and 4 subunits was detected on the somatic and dendritic surfaces of subpopulations of neurochemically defined ENS neurons. The pharmacological activation of AMPA receptors increased the force but not frequency of spontaneous colonic contractions. Conclusions & Inferences: Molecularly distinct AMPA receptor subtypes are differentially expressed within the neural networks of the mouse colon and have a direct role in motility. These data provide the rationale for the development of AMPAselective ligands for the therapeutic delivery to the GIT in motility disorders.