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The gastrointestinal (GI) tract is innervated by intrinsic enteric neurons and by extrinsic efferent and afferent nerves. The enteric (intrinsic) nervous system (ENS) in most regions of the gut consists of two main ganglionated layers; myenteric and submucosal ganglia, containing numerous types of enteric neurons and glial cells. Axons arising from the ENS and from extrinsic neurons innervate most layers of the gut wall and regulate many gut functions. The majority of ENS cells are derived from vagal neural crest cells (NCCs), which proliferate, colonize the entire gut, and first populate the myenteric region. After gut colonization by vagal NCCs, the extrinsic nerve fibers reach the GI tract, and Schwann cell precursors (SCPs) enter the gut along the extrinsic nerves. Furthermore, a subpopulation of cells in myenteric ganglia undergoes a radial (inward) migration to form the submucosal plexus, and the intrinsic and extrinsic innervation to the mucosal region develops. Here, we focus on recent progress in understanding the developmental processes that occur after the gut is colonized by vagal ENS precursors, and provide an up-to-date overview of molecular mechanisms regulating the development of the intrinsic and extrinsic innervation of the GI tract., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Insect neurons are individually identifiable and have been used successfully to study principles of the formation and function of neuronal circuits. In the fruitfly Drosophila, studies on identifiable neurons can be combined with efficient genetic approaches. However, to capitalise on this potential for studies of circuit formation in the CNS of Drosophila embryos or larvae, we need to identify pre- and postsynaptic elements of such circuits and describe the neuropilar territories they occupy. Here, we present a strategy for neurite mapping, using a set of evenly distributed landmarks labelled by commercially available anti-Fasciclin2 antibodies which remain comparatively constant between specimens and over developmental time. By applying this procedure to neurites labelled by three Gal4 lines, we show that neuritic territories are established in the embryo and maintained throughout larval life, although the complexity of neuritic arborisations increases during this period. Using additional immunostainings or dye fills, we can assign Gal4-targeted neurites to individual neurons and characterise them further as a reference for future experiments on circuit formation. Using the Fasciclin2-based mapping procedure as a standard (e.g., in a common database) would facilitate studies on the functional architecture of the neuropile and the identification of candiate circuit elements.

The inner ear (vestibular and cochlear) efferent neurons are a group of atypical motor-like hindbrain neurons which innervate inner ear hair cells and their sensory afferents. They are born in the fourth rhombomere, in close association with facial branchial motor neurons, from which they subsequently part through a specific migration route. Here, we demonstrate that the inner ear efferents depend on Phox2b for their differentiation, behaving in that respect like hindbrain visceral and branchial motor neurons. We also show that the vestibular efferent nucleus is no longer present at its usual site in mice inactivated for the bHLH transcription factor Mash 1. The concomitant appearance of an ectopic branchial-like nucleus at the location where both inner ear efferents and facial branchial motor neurons are born suggests that Mash1 is required for the migration of a subpopulation of rhombomere 4-derived efferents.

Vasoconstrictor responses to electrical field stimulation (EFS, 0.2-32 Hz, 0.1 ms, 12 V, for 1 min) were measured in endothelium-denuded segments of guinea-pig mesenteric vein and compared to responses in mesenteric artery. The distribution of both tyrosine-hydroxylase-like immunoreactivity (TH-LI) and neuropeptide Y-like immunoreactivity (NPY-LI) was also studied using anti-TH and anti-NPY antibodies. The effect of exogenous NPY (10 nM) on EFS (8 Hz, 0.3 ms, 12 V, for 1 min)-evoked overflow of noradrenaline (NA) was also studied using an HPLC technique with electrochemical detection. Veins responded with contractions at lower frequencies of stimulation than arteries. Prazosin (0.1 microM) abolished the EFS-evoked contractions in artery at 0.5-32 Hz and in vein at 0.2-1 Hz of stimulation. However, in vein, the contractile responses to EFS at 2-32 Hz of stimulation were only reduced by prazosin. Phentolamine (1 microM) abolished the responses to 0.5-4 Hz and reduced the responses to 8-32 Hz of EFS in artery. In vein, phentolamine (1 microM) abolished the responses to 0.2-1 Hz and facilitated the contractions elicited by 16-32 Hz. The NPY-receptor antagonist BIBP3226 (1 microM), in combination with phentolamine, abolished contractions in vein. Yohimbine (0.1 microM) abolished the responses to lower frequencies of stimulation in both artery (0.5-2 Hz) and vein (0.2-1 Hz). The responses to greater frequency stimulation were not affected by yohimbine in artery, and were facilitated in vein. Pre-treatment of animals for 24 h with reserpine abolished contractile responses to EFS in artery, whereas in vein, responses to 0.2-2 Hz were abolished while responses to 4-32 Hz were unchanged. Suramin (100 microM) or alpha,beta-methylene ATP (alpha,beta MeATP; 10-100 microM) treatment did not affect the contractile responses to EFS in either artery or vein. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS; 30 microM), even potentiated the responses to 2-16 Hz in vein. However, following resperine-treatment, both PPADS and suramin reduced the nerve-evoked contractions of vein. Either BIBP3226 (1 microM) alone or BIBP3226 in combination with PPADS or suramin abolished the contractile response to EFS in reserpine-treated veins. NPY (100 nM) produced significantly more contraction in vein than in artery (i.e., 93 +/- 2.5 versus 7 +/- 4% of the response to 70 mM KCl, respectively). NPY (10 nM) significantly reduced the NA overflow evoked by EFS at 8 Hz. Flat mount preparations and cryostat sections of both mesenteric artery and vein revealed that TH-LI and NPY-LI were co-localized in a dense network of fibers within the adventitial layer. In conclusion, NA exclusively mediates the contractile response to sympathetic nerve stimulation in guinea-pig mesenteric artery, whereas at least three neurotransmitters [i.e., NA, adenosine 5'-triphosphate (ATP) and NPY] are involved in the neural response of mesenteric vein.

In the present study we aimed to discretely characterise ganglionic and neuroeffector transmission to the ferret lower oesophageal sphincter (LOS) using a novel preparation of LOS muscle with intact vagal innervation in conjunction with isolated LOS muscle strips. In this way we could compare vagally mediated LOS relaxation with that of enteric inhibitory motorneurones which were directly stimulated. Preparations of LOS muscle, with or without attached vagus nerves, were dissected from adult ferrets and maintained under preload in organ baths, where LOS muscle developed spontaneous tone. LOS relaxations in response to vagal stimulation (0.5-5 Hz, 30 V) were recorded, alone and following pretreatment with tetrodotoxin (TTX), hexamethonium (Hex), Hex and atropine and NG-nitro-L-arginine (L-NNA). Direct activation of enteric inhibitory motorneurones was performed via electrical field stimulation (EFS). Vagal stimulation elicited frequency-dependent relaxations of the LOS that were abolished by tetrodotoxin (1 microM) and markedly reduced following L-NNA pretreatment (100 microM), but unaltered following pretreatment with the selective VIP or PACAP receptor antagonists VIP (10-28) or PACAP (6-38), respectively (each at 5 microM). The potent NOS inhibitor S-methyl-L-thiocitrulline (100 microM) inhibited LOS relaxation to the same degree at 5 Hz. Hex alone (500 microM) reduced maximal relaxation by 50%; in combination with atropine (2 microM), relaxation was almost abolished. In isolated LOS muscle strips, neither VIP (10-28) nor PACAP (6-38) altered EFS-induced relaxation. Taken together, these results suggest ganglionic neurotransmission to the ferret LOS occurs mainly through a combination of nicotinic and muscarinic receptors and utilises nitroxidergic enteric inhibitory motorneurones to relax the LOS. Moreover, LOS relaxation due to direct activation of inhibitory motorneurones also utilises primarily nitric oxide and other as yet undefined neurotransmitters. Neither VIP nor PACAP are involved in vagally mediated or direct enteric neuronally stimulated LOS relaxation in the ferret.

It is unknown which neurotransmitter receptors are involved in the transfer of sensory information from the digestive tract to the brainstem. We examined the potential involvement of central glutamate, acetylcholine, and CGRP receptors in vagal pathways arising from gut chemo- and mechanosensitive afferents. Unitary recordings were made from 21 vagal preganglionic efferent neurones. Neuronal discharge showed either excitation or inhibition in response to oesophageal balloon distension (1-2 ml), gastric distension (40-60 ml in whole stomach or 20 ml in proximal stomach), cholecystokinin-8s (100 pmol close intra-arterially) and bradykinin (18 nmol close intra-arterially). Effects of glutamatergic non-NMDA, muscarinic M1, and CGRP1 receptor antagonism on efferent responses were investigated using CNQX (75-155 nmol i.c.v.), pirenzepine (2.5-5.0 micromol/kg i.v.), and hCGRP8-37 (3.2-6.4 nmol i.c.v.), respectively. CNQX, pirenzepine, and hCGRP8-37, respectively, altered efferent responses in 65%, 23% and 41% of neurones. When both CNQX and hCGRP8-37 were administered, a further 58% of responses were reduced. CNQX and hCGRP8-37 reduced a proportion of efferent responses to all stimuli whereas pirenzepine selectively reduced only efferent responses to gastric distension. We conclude that central CGRP1 and non-NMDA receptors are involved in mediating a range of upper gastrointestinal mechano- and chemo-sensitive afferent inputs onto vagal efferents. M1 receptors, on the other hand, are selectively involved in neurotransmission from gastric mechanoreceptors.