Synergistic Action of Presynaptic Muscarinic Acetylcholine Receptors and Adenosine Receptors in Developmental Axonal Competition at the Neuromuscular Junction

Abstract

The development of the nervous system involves the initial overproduction of synapses, which promotes connectivity. Hebbian competition between axons with different activities leads to the loss of roughly half of the overproduced elements and this refines connectivity. We used quantitative immunohistochemistry to investigate, in the postnatal day 7 (P7) to P9 neuromuscular junctions, the involvement of muscarinic receptors (muscarinic acetylcholine autoreceptors and the M₁, M₂, and M₄ subtypes) and adenosine receptors (A₁ and A_2A subtypes) in the control of axonal elimination after the mouse levator auris longus muscle had been exposed to selective antagonists in vivo. In a previous study we analyzed the role of each of the individual receptors. Here we investigate the additive or occlusive effects of their inhibitors and thus the existence of synergistic activity between the receptors. The main results show that the A_2A, M₁, M₄, and A₁ receptors (in this order of ability) delayed axonal elimination at P7. M₄ produces some occlusion of the M₁ pathway and some addition to the A₁ pathway, which suggests that they cooperate. M₂ receptors may modulate (by allowing a permissive action) the other receptors, mainly M₄ and A₁. The continued action of these receptors (now including M₂ but not M₄) finally promotes axonal loss at P9. All 4 receptors (M₂, M₁, A₁, and A_2A, in this order of ability) are necessary. The M₄ receptor (which in itself does not affect axon loss) seems to modulate the other receptors. We found a synergistic action between the M₁, A₁, and A_2A receptors, which show an additive effect, whereas the potent M₂ effect is largely independent of the other receptors (though can be modulated by M₄). At P9, there is a full mutual dependence between the A₁ and A_2A receptors in regulating axon loss. In summary, postnatal axonal elimination is a regulated multireceptor mechanism that involves the cooperation of several muscarinic and adenosine receptor subtypes.

Membrane Receptor-Induced Changes of the Protein Kinases A and C Activity May Play a Leading Role in Promoting Developmental Synapse Elimination at the Neuromuscular Junction

“…We simultaneously applied two inhibitors (two selective antagonists from two different receptors) to reveal the possible additive or occlusive crosstalk effects between the corresponding pathways. The histograms in Figure 2 show the individual and the paired effects of these inhibitors on axonal loss at P9 (percentage of the monoinnervated synapses after exposure to blockers (data drawn from previous studies: Nadal et al, 2016a , b , 2017 ; Tomàs et al, 2017 ). The paired inhibition data of the AR and TrkB shown in histograms i and j from Figure 2A have not been previously published).…”

“…Membrane receptor signaling can play a role in axonal competition by allowing the various nerve endings to have an activity-dependent influence on one another directly or with the involvement of the postsynaptic cell and the associated glial cell/s (Keller-Peck et al, 2001 ; Tomàs et al, 2014 ). We observed that presynaptic muscarinic acetylcholine receptors (mAChR; subtypes M 1 , M 2 and M 4 ), adenosine receptors (AR; A 1 and A 2A ) and the neurotrophin receptor (NTR) tropomyosin-related kinase B receptor (TrkB) all cooperate in the developmental synapse elimination process at this synapse [NMJ from the Levator auris longus —LAL—muscle of the B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice (hereinafter YFP mice), and from C57BL/6J P7 mice] by favoring axonal competition and loss (Nadal et al, 2016a , b , 2017 ; Tomàs et al, 2017 ). Other receptors, for example glutamate receptors at the mice NMJ (Waerhaug and Ottersen, 1993 ) may collaborate because developmental synapse loss is slowed by reducing activation of the glutamate-NMDA receptor pathway (Personius et al, 2016 ).…”

“…The receptors (Figure 2A ) with the exception of the M 4 subtype (Figure 2B ), directly accelerate axon loss at P9 (when selectively blocked between P5 and P8, axonal elimination is reduced and this shows their tonic effect in normal conditions). All diagrams in Figure 3 (taken from previous articles, except some unpublished data in Figure 3D , see below; Nadal et al, 2016b , 2017 ), show the effect of the selective inhibitors in order of their ability to finally delay monoinnervation and keep a high percentage of synapses innervated by two or more axons (methoctramine (MET), M 2 inhibitor; PIR, M 1 inhibitor; 8-Cyclopentyl-1,3-IP3, inositol triphosphate (DPCPX), A 1 inhibitor; SCH58261, A 2A inhibitor; inhibitor recombinant human TrkB-Fc Chimera (TrkB-Fc), TrkB inhibitor). The red arrows show approximately how effective the selective blockers are at delaying axonal elimination (the thicker they are, the greater their effect, although their absolute pharmacological potency cannot be directly compared).…”

“… Cooperation between mAChR, AR and TrkB receptors. All diagrams (A–D ; redrawn from previous work, except some unpublished data in diagram (D) , Nadal et al, 2016b , 2017 ), show the effect of the selective inhibitors in order of their ability to finally delay monoinnervation and keep a high percentage of synapses innervated by two or more axons (MET, M 2 inhibitor; PIR, M 1 inhibitor; DPCPX, A 1 inhibitor; SCH58261, A 2A inhibitor; TrkB-Fc, TrkB inhibitor). The red arrows show how effective the blockers are at delaying elimination (the thicker they are, the greater their effect).…”

Research progress on adenosine in central nervous system diseases

“…Both A2AR antagonists and mGLu5 antagonists, when used alone or in combination, can significantly reduce the dyskinesia caused by 6‐hydroxydopamine (6‐OHDA) damage, and the combined use of the two could also increase the inhibition of D2 signals of GABA neurons in the globus pallidus . Adenosine A2AR has synergistic effects with the M receptor, showing the effect of superposition . That is, A2AR can stimulate the release of acetylcholine in striatal neurons, and the cholinergic nerve fiber endings can also regulate the release of acetylcholine by negative feedback from muscarinic (M) receptors in the presynaptic membrane.…”