Expression And Biological Significance Of HCN2-protein In The Enteric Nervous System Of The Mouse Gastrointestinal Tracts

Hyperpolarization-activated cyclic nucleotide-gated cation channel is a kind of pace-origin channel, which widely distributes in the cells or neurons in charge of rhythmic electric activity in sinoatrial node, atrio-ventricular node,Purkinje fibers,central nervous system and peripheral nerveous system. It is widely accepted that these ion channels are associated with generation of spontaneous rhythmic electric activities. Classical neurotransmitter such as norepinephrine and Ach can affect the spontaneous rhythmic electric activities by changing the biophysical features of HCN. Inward current generated by this channel opening, called If, which plays a critical role in the onset of inward current by autorhythm generating cells. There are four members in the family of HCN channels, HCN1-HCN4 with distinctive electrophysiologic features, such as relative rates of action, voltage dependence and effects by cAMP regulation. In the enteric nervous system, If was found in AH neurons, which is a kind of sensory neurons in gastrointestinal (GI) tract. Researches indicated that HCN2 channel immunoreactivity occurred in the majority of enteric neurons in the mouse . However, the patterns of localization of this isoforms and its biologic significance in gastrointestinal tract have not been elucidated, therefore analysis of the patterns of localization of this isoform may provide us with related information about morphology and help us to understand the function of HCN and its physiological significance in the nerveous system of gastrointestinal tract.Interstitial cells of Cajal (ICCs) are a special kind of interstitial cells that distribute throughout the gastrointestinal tract and form a cellular network. They play a very important role in the regulation of gastrointestinal motility by generating and propagating spontaneous electric slow-wave activity and mediating excitatory and inhibitory neurotransmission. As the nature of the GI spontaneous rhythm generation is similar to that of heart, so it is very interesting to find out the relationship between them. So we wonder whether HCN plays an important role in spontaneous rhythm activity of GI tract. As we know, there have been not any reports on these questions in the world up to now, and the mechanisms of the GI tract spontaneous rhythm generation are unknown. Therefore, it makes sense to investigate the relationship between ICCs and HCN, and explore the role of HCN in the process of GI spontaneous rhythm generation.Previous studies have shown that the level of HCN changes along with the development of neural activity in the central nervous system.so, We wonder whether the change of HCN level during the development of ENS is similar to its change in the central nervous system. So far, there have not any reports on this question in the world. Therefore,it is critical for us to understand the changes of the HCN level during the development of ENS and explore the role of HCN in the development of peripheral nervous system. The primary objective of this study is to investigate the expression of HCN in the GI tract of mature mice and the development pattern of HCN2 in the small intestine of nascent mice as well as the potiential role of HCN2 in the GI nervous system by using morphology techniques such as immunohistochemistry and immunofluorescentce staining.The results show that:1. In the different segments of GI tract, the surface area of HCN2-immunoreactive nerve ganglion and the average density of HCN2-immunoreactive cells show the same variation trend, however, the average density of HCN2-immunoreactive neurons in the nerve ganglions remains the same. HCN2 is likely to adapt or modulate the functional changes of each segment of GI tract by the change of its expression level in different segments of GI tract.2. HCN2 channels express on cholinergic neurons. HCN2-immunoreactive neurons and cholinergic neurons are Dogiel IΙtype-neurons.So, HCN channels are endowed with high plasticity of function so that they are fit for the various functions of cholinergic nerves.3. Both HCN2-immunoreactive neurons and ICCs form independent and complete celluar network. No colocalization of HCN2-immunoreactive neurons and ICCs was observed. However, the processes of ICCs were close to the HCN2-immunoreactive neurons. ICCs might involve in the transmission of nervous signals from HCN2-immunoreactive nerves and then modulate GI motility. 4. With the development of ENS, the average density of HCN2-immunoreactive neurons in the wall of small intestine of nascent mice increases prominently. The increase of HCN2 may be related to the establishment of ENS neural network.5. The morphology of HCN2-immunoreactive neurons in the small intestine of nascent mice changes during the development, the number of Dogiel III type-neurons decreases gradually and the Dogiel IΙtype-neurons become the major neurons in the mature ENS. The transformation may be related to the chages of the subcelluar distribution and functional changes of neurons.In conclusion, the HCN2-immunoreactive nerves in the GI tract of mature mice show various distributions in different segments, which might involve in the spontaneous rhythm activity of GI tract. HCN channels are endowed with high plasticity of function, which might be the molecular basis for the various functions of cholinergic nerves. With the development of small intestine, the level of HCN2 and the distribution of HCN change.