Presynaptic Membrane Receptors Modulate ACh Release, Axonal Competition and Synapse Elimination during Neuromuscular Junction Development

Abstract

During the histogenesis of the nervous system a lush production of neurons, which establish an excessive number of synapses, is followed by a drop in both neurons and synaptic contacts as maturation proceeds. Hebbian competition between axons with different activities leads to the loss of roughly half of the neurons initially produced so connectivity is refined and specificity gained. The skeletal muscle fibers in the newborn neuromuscular junction (NMJ) are polyinnervated but by the end of the competition, 2 weeks later, the NMJ are innervated by only one axon. This peripheral synapse has long been used as a convenient model for synapse development. In the last few years, we have studied transmitter release and the local involvement of the presynaptic muscarinic acetylcholine autoreceptors (mAChR), adenosine autoreceptors (AR) and trophic factor receptors (TFR, for neurotrophins and trophic cytokines) during the development of NMJ and in the adult. This review article brings together previously published data and proposes a molecular background for developmental axonal competition and loss. At the end of the first week postnatal, these receptors modulate transmitter release in the various nerve terminals on polyinnervated NMJ and contribute to axonal competition and synapse elimination.

Keywords: PKC; TrkB; acetylcholine release; axonal competition; muscarinic acetylcholine receptors; postnatal synapse elimination; protein kinases; voltage-dependent calcium channels.

Figure 1

Confocal immunofluorescence images. The pictures show representative confocal fluorescence images of monoinnervated and polyinnervated synapses from C57BL/6J P7 control mice. The levator auris longus (LAL) neuromuscular junctions (NMJs) show the axons stained by 200-kD neurofilament antibody in green and the postsynaptic nicotinic acetylcholine receptor (nAChR) clusters stained in red with TRITC-α- BTX. Scale bar: 10 μm.

Figure 2

Diagrams showing an overall representation of the data. The diagrams show the effect of several substances on acetylcholine (ACh) release (evoke endplate potential, EPP size represented by circles: increase [green], decrease [red] and no change [black] with respect to untreated controls) in developing (P7) single axons on monoinnervated junctions [M], the strong [S] and weak [W] synaptic contacts on dual junctions and in adult (P30) nerve endings [A]. Silent synaptic contacts [R] can be observed in some NMJs in treated muscles after recovering ACh release. Here, R shows the effect of some substances on the polyinnervation index (PI, the mean number of axons per synapse) of these treated muscles (green = increased PI, black = no change). The axonal loss rate (represented with squares in the outermost concentric layer in A and B) is quantified by direct axonal counts in confocal LAL preparations from B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice. In these histological preparations we counted the percentage of singly-, dually- and triply- (or more) innervated synapses at P7 after 2 days of subcutaneous applications of several muscarinic and TrkB substances. Delayed axon loss in red squares, accelerated loss in green and no change in black. (A) shows the effect of several subtype-selective muscarinicsubstances. The M₂ receptor is selectively blocked with methoctramine (MET) or AFX-116. The M₁ receptor is selectively blocked with pirenzepine (PIR) or muscarinic toxin 7 (MT7). The M₃ subtype is blocked with 4-DAMP and the M₄ subtype is blocked with tropicamide (TRO) or muscarínic toxin 3 (MT3). (B) shows the effect of neurotrophins and trophic cytokines (Brain-derived neurotrophic factor, BDNF, NT4, NT3, GDNF and CNTF) and related substances (TrkB-Fc chimera and k-252a). (C) shows the effect of voltage-dependent calcium channels (VDCC) blockers (nitrendipine [L type], ω-AGA [P type] and ω-CON [N type]), ion concentration change (0.5 mM magnesium) and PKC blockers (calphostin C (CaC) and chelerythrine (Che)). In all cases, significance is at P < 0.05.

Figure 3

Changes in polyneuronal innervation of the NMJ after inhibiting the mAChR and blocking the TrKB signaling. The figure shows the percentage of singly-, doubly- and triply (or more) innervated NMJs in untreated YFP control mice (exposed to PBS applications) and after two (P7 in A) and four (P9 in B) applications (one application each day after P5) of the mAChR antagonists PIR, MET and MT3. The figure also shows the effect of the TrkB blocking pathway agent TrkB-Fc. ***p < 0.001,**p < 0.01, *p < 0.05. This figure has been adapted and redrawn from the Figures 3, 4 in the original article “[Presynaptic muscarinic ACh autoreceptors (M1, M2 and M4 subtypes), adenosine receptors (A₁ and A^2A) and tropomyosin-related kinase B receptor (TrkB) modulate the developmental synapse elimination process at the NMJ]” by Nadal et al. (2016). The original article is an open access article distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/2.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

Figure 4

The figure is a plausible interpretation of the role of mAChR (M₁, M₂ and M₄) and the BDNF-TrkB pathways in the process of eliminating the weakest endings around P7. The explanation can be found in the conclusion to the main text. Green arrows indicate activation or stimulation, and red arrows inhibition.