Immunohistochemical Identification of Purinergic Neurons in the Enteric Nervous System

Propulsive gastrointestinal (GI) motility patterns depend on coordinated contraction and relaxation of GI smooth muscles. The enteric nervous system (ENS) controls GI motility. Currently, it is accepted that smooth muscle relaxation is mainly mediated by the co‐release of ATP and nitric oxide (NO) from one class of inhibitory motor neuron (IMN). ATP and NO mediate fast and slow inhibitory junction potentials, respectively. Neuronal ATP and NO release depend on activation of voltage‐gated calcium channels (VGCC) and different VGCC may drive ATP and NO release independently. Functional data also suggest that descending interneurons communicate with other interneurons and IMNs via ATP release. However, direct identification of purinergic nerves in the ENS is lacking. Our study aim was to directly identify purinergic nerves in the ENS using an antibody against the vesicular nucleotide transporter (VNUT, SLC17A9), a purinergic nerve marker to distinguish the purinergic and non‐purinergic neurons in mice and guinea pig colon and ileal myenteric plexus. We used immunohistochemical methods to localize nitric oxide synthase (NOS), VNUT, choline acetyl transferase (ChAT) and the a1E subunit of the R‐type Ca 2+ channels. . In addition, longitudinal muscle myenteric plexus (LMMP) preps were placed in tissue culture with colchicine for 48 h to facilitate staining of purinergic and non‐purinergic nerve cell bodies. Our results show VNUT+/NOS‐‐, NOS+/VNUT‐, ChAT+/VNUT‐, and ChAT‐/VNUT+ nerve fibers supplying the muscle layers. Moreover, multiple populations of cell bodies were identified (VNUT+ neurons, NOS+ neurons, and NOS‐/VNUT+ neurons), NOS+/VNUT+ neurons being the smallest population observed. In addition, VNUT and ChAT did not co‐localize in myenteric neurons. Finally NOS and the a1E subunit localize in the same axon bundles, but not in the same axons, that innervate the smooth muscle. These results suggest that there may be two types of inhibitory motorneuron supplying the muscle layers in the gut (purinergic and nitrergic). There may also be two types of excitatory interneurons: cholinergic and puringergic. Further studies of neuromuscular and synaptic transmission are required to establish the function of the puringeric, nitrergic and cholinergic neurons. (Supported by 1R01DK094932) Support or Funding Information Supported by 1R01DK094932