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

Abstract

Neurons of the enteric nervous system (ENS) arise from neural crest cells that migrate into and along the developing gastrointestinal tract. A subpopulation of these neural-crest derived cells express pan-neuronal markers early in development, shortly after they first enter the gut. However, it is unknown whether these early enteric "neurons" are electrically active. In this study we used live Ca 2ϩ imaging to examine the activity of enteric neurons from mice at embryonic day 11.5 (E11.5), E12.5, E15.5, and E18.5 that were dissociated and cultured overnight. PGP9.5-immunoreactive neurons from E11.5 gut cultures responded to electrical field stimulation with fast [Ca 2ϩ ] i transients that were sensitive to TTX and -conotoxin GVIA, suggesting roles for voltage-gated Na ϩ channels and N-type voltage-gated Ca 2ϩ channels. E11.5 neurons were also responsive to the nicotinic cholinergic agonist, dimethylphenylpiperazinium, and to ATP. In addition, spontaneous [Ca 2ϩ ] i transients were present. Similar responses were observed in neurons from older embryonic gut. Wholecell patch-clamp recordings performed on E12.5 enteric neurons after 2-10 h in culture revealed that these neurons fired both spontaneous and evoked action potentials. Together, our results show that enteric neurons exhibit mature forms of activity at early stages of ENS development. This is the first investigation to directly examine the presence of neural activity during enteric neuron development. Along with the spinal cord and hindbrain, the ENS appears to be one of the earliest parts of the nervous system to exhibit electrical activity.

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies

“…This is important as developing enteric neurons (Hao et al, 2012) and adult neural stem cells (Moe et al, 2005) can express neuronal markers without being electrically active. These assays could be performed using a variety of electrophysiological and functional imaging approaches including sharp electrode intracellular recordings, patch clamping (Hao et al, 2012), calcium (Hao et al, 2011) or voltage-sensitive imaging.…”

Enteric nervous system development: migration, differentiation, and disease

“…Serotonergic neurons, in turn, require the norepinephrine transporter for proper development, which suggests that norepinephrine uptake may shape neuronal differentiation. Recent work has also demonstrated that nascent enteric neurons are electrically active very early in the colonization process (87) and that inhibition of this activity reduces the number of early-born nitrergic neurons close to the ENCDC wave front (88), suggesting that the interdependence of different types of enteric neuron is not limited to the late-born populations. Finally, neuronal activity may be important for ENCDC colonization of the bowel, since tetanus and botulinum neurotoxins slow neurite extension and ENCDC migration (201).…”

Optical Tools to Investigate Cellular Activity in the Intestinal Wall

“…To examine the development of ENS circuitry, activity of enteric neurons has been imaged using Fluo-4 101 and Wnt1-Cre;R26R-GCaMP3 mice. 102 Initially, Fluo-4 Ca 2+ imaging was performed on ENCCs isolated from different embryonic ages to examine [Ca 2+ ] i responses to electrical field stimulation.…”

“…102 Initially, Fluo-4 Ca 2+ imaging was performed on ENCCs isolated from different embryonic ages to examine [Ca 2+ ] i responses to electrical field stimulation. 101 More recently, the availability of a conditional GCaMP3-expressing mouse line has allowed Ca 2+ imaging to be performed on intact explants of embryonic gut, thereby preserving the native cell-cell connections of the developing ENS. 102 Using the Wnt1-Cre transgene to induce expression of GCaMP3 in all neural crest derivatives, the contribution of different subunits of nicotinic receptors to cholinergic neurotransmission throughout ENS development has been characterized.…”