Distribution, Colocalization And Neuron Type Identification Of P2x₆ Receptor And Oxytocin In Enteric Nervous System

The enteric nervous system (ENS) is a branch of autonomic nervous system which controls the function of digestion. The enteric nervous system includes extrinsic neurons from parasympathetic dorsal motor nucleus of vagus nerve, sacral parasympathetic nucleus and prevertebral ganglia and intrinsic neurons from intestinal tract. They form an inte-grated enteric nervous system. The ENS is called independent nervous system because they can mediate reflex activity in the absence of input from the brain or spinal cord. Ref-lex activity is mediated by neurons within the wall of gut. Several kinds of neurons exist in the gastrointestinal tract including intrinsic primary afferent neuron (as sensory neuron), Interneuron and motor neuron. These neurons mainly exist in two nerve plexuses, myen-teric nerve plexus and submucosal nervous plexus. These neurons connect with each other though synapses and chemical transmitter forming an integrated body of structure and function. Among them, neurotransmitter and neuropeptide take a great part in maintaining function of gastrointestinal tract such as ATP and oxytocin. But we know little about their function. So, characterization of their ditribution and function will expand our understand-ing about enteric nervous system.The studies conduncted on the level of molecular, cell and system has confirmed extracellular ATP as an important signal can act on purinergic receptor to take part in many important physiological and pathological process. P2X receptor is ATP-gated purinergic receptor. P2X₂, P2X₃, P2X5 and P2X₇ receptor exist in myenteric nerve plexus and P2X₂, P2X₃, and P2X₇ receptor exist in the submucosal nervous plexus. These purinergic recep-tors may exist in the intrinsic primary afferent neurons, interneurons and motor neurons to regulate sense of gastrointestinal tract, nerve conduction and gastrointestinal peristalsis. With double-labeling fluorescence immunohistochemistry, the expression of P2X₄ and P2X₆ receptor subunits in the gastrointestinal tract of the rat was studied. The results showed that P2X₆ receptors were expressed widely in the submucosal and myenteric plex-uses. In the myenteric plexus, P2X₆ receptors were expressed mainly in the large size neu-rons, the shapes of which resembled Dogiel type II neurons. These P2X₆ receptor immu-noreactive (ir) neurons also expressed calbindin-28K, calretinin and NeuN (neu-ronal nuc-lei), proteins that are markers of intrinsic sensory neurons. In the submucosal plexus all the calbindin-28K, calretinin and NeuN-ir cells were immunoreactive for P2X₆ receptors. P2X₆ receptors do not form homomultimers, but rather heteromultimers with either P2X₂ or P2X₄ receptors. P2X₄ receptors were not expressed in neurons, but were expressed in macrophages of the rat gastrointestinal tract. These data indicate that P2X₆ receptors are mainly expressed on intrinsic sensory neurons and that ATP, via P2X₆ re-ceptors probably in heteromeric combination with P2X₂ receptors, may be involved in regulating the physi-ological functions of these neurons.Early study revealed that oxytocin not only possess function of milk ejection reflex and parturition, still it can stimulate or inhibit function of intestinal tract. Previous data of oxytocin has given us some hint about its function, but the systemic morphological cha-racter is still poorly understood.In the second part of our work, we also systematically studied the distribution pattern and neurochemical density of oxytocin-immunoreactive (-ir) neurons in the digestive tract of the guinea pig. The present data demonstrates that oxytocin-immunoreactivity is distri-buted widely in the guinea pig gastrointestinal tract. 3%, 13%, 17%, 15% and 10% of gan-glion neurons were immunoreactive for oxytocin in the myenteric plexuses of gastric cor-pus, jejunum, ileum, proximal colon and distal colon, respectively. 36%, 40%, 52% and 56% of ganglion neurons were immunoreactive for oxytocin in the submucosal plexuses of jejunum, ileum, proximal and distal colon, respectively. In the myenteric plexus oxytocin was expressed exclusively in the intrinsic enteric afferent neurons, as identified by calbin-din 28K. In the submucosal plexuses, oxytocin was expressed in non-cholinergic secreto-motor neurons, as identified by vasoactive intestinal polypeptide. Oxytocin-ir nerve fibers in the inner circular muscle layer may arise from the myenteric oxytocin-ir neurons and oxytocin-ir nerve fibers in the mucosa may arise from both the myenteric and submucosal oxytocin-ir neurons. These data indicate that oxytocin in the digestive tract may be in-volved in gastrointestinal tract motility mainly via regulating the inner circular muscle and the balance of absorption and secretion of water and electrolytes.The further investigation revealed that P2X₆ receptor and OT may colocalize in some neurons. This result indicates the neurotransmitter and the neuropeptide may interact with each other. ATP may act on P2X₆ receptor to regulate secretion of OT and further influence the function of gastrointestinal tract. In the present study, the distribution patterns and morphological characteristics of P2X₆ receptor-ir neurons and OT-ir neurons have been studied systematically in all the major regions of the gastrointestinal tract of rat and guinea pig, providing data for further functional study. In the gastrointestinal tract of rat, ATP via P2X₆ receptors probably in he-teromeric combination with P2X₂ receptors, may be involved in regulating the physiologi-cal functions of these neurons. In the gastrointestinal tract of guinea pig, OT may act on the inner circular muscle layer to regulate motility of gastrointestinal tract and keep balance of absorption and secretion of water and electrolytes.