The role of neural activity in the migration and differentiation of enteric neuron precursors

Abstract

Background As they migrate through the developing gut, a sub-population of enteric neural crest-derived cells (ENCCs) begins to differentiate into neurons. The early appearance of neurons raises the possibility that electrical activity and neurotransmitter release could influence the migration or differentiation of ENNCs. Methods The appearance of neuronal sub-types in the gut of embryonic mice was examined using immunohistochemistry. The effects of blocking various forms of neural activity on ENCC migration and neuronal differentiation were examined using explants of cultured embryonic gut. Key Results Nerve fibers were present in close apposition to many ENCCs. Commencing at E11.5, neuronal nitric oxide synthase (nNOS), calbindin and IK Ca channel immunoreactivities were shown by sub-populations of enteric neurons. In cultured explants of embryonic gut, tetrodotoxin (TTX, an inhibitor of action potential generation), nitro-L-arginine (NOLA, an inhibitor of nitric oxide synthesis) and clotrimazole (an IK Ca channel blocker) did not affect the rate of ENCC migration, but tetanus toxin (an inhibitor of SNAREmediated vesicle fusion) significantly impaired ENCC migration as previously reported. In explants of E11.5 and E12.5 hindgut grown in the presence of TTX or tetanus toxin there was a decrease in the number nNOS+ neurons close to the migratory wavefront, but no significant difference in the proportion of all ENCC that expressed the pan-neuronal marker, Hu. Conclusions & Inferences (i) Some enteric neuron sub-types are present very early during the development of the enteric nervous system. (ii) The rate of differentiation of some sub-types of enteric neurons appears to be influenced by TTX-and tetanus toxin-sensitive mechanisms.

Early Emergence of Neural Activity in the Developing Mouse Enteric Nervous System

“…Along with the spinal cord, hindbrain and dorsal root ganglia (Spitzer and Baccaglini, 1976;Gust et al, 2003; Lech- ner et al, 2009), the ENS appears to be one of the earliest parts of the nervous system to exhibit electrical activity. Endogenous neural activity has been shown to influence early events in ENS development including ENCC migration and differentiation (Vohra et al, 2006;Hao et al, 2010;Li et al, 2010). Our study raises the possibility that neural activity also guides later developmental processes, such as axon pathfinding and synaptogenesis, and the development of functioning neural circuits in the ENS.…”

“…Combined, these data are consistent with a role for endogenous neurotransmitter signaling in ENS development. However, not all neurotransmitters appear to be involved, as inhibition of nitric oxide synthase activity did not affect ENCC migration or differentiation (Hao et al, 2010).…”

“…Previous studies found that inhibition of synaptic release using clostridial toxins also perturbs ENCC migration (Vohra et al, 2006;Hao et al, 2010). Also, mutant mice lacking the norepinephrine transporter (Li et al, 2010) or neuronal serotonin (Li et al, 2011) have reduced numbers of enteric neurons, indicating roles for these neurotransmitters in enteric neuron differentiation and/or survival.…”

“…Spontaneous activity in PGP9.5-positive neurons probably influences differentiation of their neurotransmitter phenotype. We have previously shown that inhibition of endogenous activity using TTX delays differentiation of nitrergic neurons in cultured explants of E11.5 gut (Hao et al, 2010). Similarly, in the developing spinal cord of Xenopus laevis, inhibition of [Ca 2ϩ ] i spikes in neurons retards differentiation of inhibitory neurons (Borodinsky et al, 2004).…”

“…In the developing CNS, electrical activity and neurotransmitter release are present during embryonic development and influence many aspects of neuron circuitry formation in partnership with genetic programs (Moody and Bosma, 2005;Spitzer, 2006;Ben-Ari, 2008;Ben-Ari and Spitzer, 2010). Similar mechanisms also operate within the developing ENS as inhibition of activity (Vohra et al, 2006;Hao et al, 2010) or genetic disruptions to neurotransmitter synthesis or uptake (Li et al, 2010(Li et al, , 2011 can perturb the migration and differentiation of ENCCs. However, several key questions remain unanswered: when do neurons in the embryonic gut become electrically active?…”