Participació dels receptors muscarínics presinàptics (mAChRs) en l'eliminació de les connexions sinàptiques redundants durant el desenvolupament neuromuscular

Identifier:  TDX:609

Handle: https://hdl.handle.net/20.500.11797/TDX609

Authors:  Salón Cabré, Maria Isabel

Abstract:
AntecedentsDuring first postnatal developing days the motor endplates are multiinnervated, this multiinnervation decrease at the next weeks until the adult age, when the motor endplates are monoinnervated. Is in this axonal losing or synaptic elimination where there are implicated presynaptic and postsynaptic mechanisms related with neuromuscular activity levels. In this context, muscarinic autoreceptors (mAChRs) can be involved. In the cholinergic neuromuscular junctions there are presynaptic muscarinic autoreceptors(mAChRs) with sensibility to neurotransmitter released by themselves. ObjectivesIn this work we investigate the participation of different subtypes of mAChRs in the synaptic elimination period in the newborn rat neuromuscular junction with electrophysiological recording techniques. We want previously determining, the presence of different subtypes, the functional behaviour and the voltage dependent calcium channels (VDCC)-dependence of the mAChRs in the adult motor nerve terminals.MethodsWe investigate presynaptic muscarinic autoreceptors involved in the modulation of neurotransmitter release and their VDCC-dependence, in the Levator Auris Longus muscle neuromuscular junction of newborn and adult rats. We use intracellular recording techniques to study who different muscarinic antagonists affect the endplate potentials (EPP) in the adult and newborn neuromuscular junctions.We previously incubated the muscle with VDCC blockers (Nitrendipine, -Conotoxin- GVIA, -Agatoxin-IVA) before determining the muscarinic response, to study the possible relation to mAChRs with VDCC.ResultsIn the adult, the M1 antagonist Pirenzepine (10 µM) decreases the evoked release. On the other hand, the M2 antagonist Methoctramine (1 µM) increases the evoked neurotransmitter release. Both mechanisms, depends on the calcium influx from the external media via P/Q calcium channels. We found no indices of M3 (4-DAMP-sensitive) or M4 (Tropicamide-sensitive) muscarinic receptors in the adult animals muscles.In the newborn, in dually innervated fibers, a second EPP can appear after the first when the intensity of the electrical stimulus is raised. The lowest and highest EPP amplitudes are designed as 'the small- EPP' and 'the large- EPP', respectively.The muscarinic agents response in the nerve terminal of monoinnervated synapses is similar to the large EPP response in dually innervated junctions. This response involved an ACh release inhibition by M1 muscarinic antagonist Pirenzepine and by M2 muscarinic antagonist Methoctramine. However, M1 antagonist Pirenzepine and M4 antagonist Tropicamide increase the small EPP.In relation to VDCC-dependence we observe a gradual change in the small EPP, from the P/Q, N, and L VDCC implication in all responses where the M1, M2 and M4 muscarinics are involved, to the implication of a lonely channel, the P/Q, in the M1 and M2 responses in the monoinnervated endplates.This indicates the existence of a progressive shutoff in parallel with maturation and specialization of the adult type P/Q channel.Main conclusionIn summary, muscarinic autoreceptors can directly modulate both, large-EPP generating ending potentiation, and small-EPP generating ending depression by their association with VDCC in developing neuromuscular junction.