Expression of the sodium channel Nav1.2 in chemically identified myenteric neurons in the guinea pig

Abstract

Our purpose was to identify Na(v)1.2-expressing myenteric neurons of the small and large intestine of the guinea pig by using antibodies directed against Na(v)1.2 and selected neurochemical markers. Na(v)1.2-like immunoreactivity (-li) co-localized with immunoreactivity for choline acetyltransferase in all regions, representing 45%-67% of Na(v)1.2-positive neurons. Na(v)1.2-li co-localized with immunoreactivity for the neural form of nitric oxide synthase more frequently in the colon (20% of neurons exhibiting Na(v)1.2-li) than in the ileum (8%). Co-localization of Na(v)1.2-li with immunoreactivity for a form of neurofilament (NF145) was infrequently observed in the ileum and colon. Enkephalin-immunoreactive cell bodies co-localized with Na(v)1.2-li in all regions. Few myenteric cell bodies immunoreactive for neuropeptide Y were observed in the ileum, but all co-localized with Na(v)1.2-li. This and our previous data suggest that Na(v)1.2 is widely expressed within the guinea pig enteric nervous system, including the three main classes of myenteric neurons (sensory, motor, and interneurons), and is involved in both excitatory and inhibitory pathways. Notable exceptions include the excitatory motor neurons to the longitudinal smooth muscle, the ascending interneurons of the ileum, and the myenteric neurons immunoreactive for NF145, few of which are immunoreactive for Na(v)1.2.

Nav1.7 and Nav1.3 are the only tetrodotoxin‐sensitive sodium channels expressed by the adult guinea pig enteric nervous system

“…The in situ hybridization method fulfils the criterion of high selectivity, because specific antisense and sense oligonucleotide probes were designed from the coding region of the guinea pig cDNA sequences of the Na v 1.1, Na v 1.2, Na v 1.3, Na v 1.6, and Na v 1.7 α subunits. Unexpectedly, we found that, among the mRNAs of these five α subunits, only the Na v 1.3 and Na v 1.7 mRNAs could be detected in the ENS, although we could replicate the immunolabeling of ENS structures for Na v 1.2, Na v 1.3, and Na v 1.6 described by Bartoo et al (2005,2006a,b) with the antibodies they used. By using converging immunohistochemistry, Western blot, and molecular biology approaches, we determined the origin of the discrepancies between the immunohistochemistry and the in situ hybridization results and confirmed the validity of the in situ hybridization experiment results.…”

“…As indicated above, the Na v 1.2 α subunit has been previously immunolabeled using a polyclonal Ab raised against the 467–485 rat Na v 1.2 sequence (Westenbroek et al,1989; Planells‐Cases et al,2000; Bartoo et al,2005,2006a,b; Osorio et al,2005). Thirteen of the nineteen amino acids of this rat sequence are conserved in both the rat and the guinea pig Na v 1.3 orthologues.…”

“…In addition to relatively few neurons, LMMP preparations include various cell types (including gut and blood vessel muscle cells, glial cells, and fibroblasts), some of which are present in high numbers and all of which express different types of Na v 1 channels (Black et al,1994; Oh et al,1994; Schaller et al,1995; Jo et al,2004; Platoshyn et al,2005; Saleh et al,2005), so the RT‐PCR results obtained from these preparations cannot provide definitive information concerning the Na v 1 channels expressed by the ENS. On the contrary, from their immunohistochemistry data, Bartoo et al (2005,2006a,b) concluded that Na v 1.2, Na v 1.3, and Na v 1.6 are expressed in identified neuron subpopulations. Whether Na v 1.7 was expressed could not be determined, because no commercial antibody reveals this subunit in the guinea pig ENS (Bartoo et al,2005).…”

“…In the absence of Na v 1.8, the voltage‐sensitive sodium channels responsible for the rising phase of the action potential belong exclusively to the group of TTX‐sensitive Na + channels. To determine which TTX‐sensitive Na + α subunit is expressed in the guinea pig ENS, Bartoo et al (2005,2006a,b) performed standard RT‐PCR using total mRNA extracted from longitudinal muscle/myenteric plexus (LMMP) preparations and immunohistochemistry experiments. In addition to relatively few neurons, LMMP preparations include various cell types (including gut and blood vessel muscle cells, glial cells, and fibroblasts), some of which are present in high numbers and all of which express different types of Na v 1 channels (Black et al,1994; Oh et al,1994; Schaller et al,1995; Jo et al,2004; Platoshyn et al,2005; Saleh et al,2005), so the RT‐PCR results obtained from these preparations cannot provide definitive information concerning the Na v 1 channels expressed by the ENS.…”