Josep Tomàs

Muscarinic autoreceptors modulate transmitter release through protein kinase C and protein kinase A in the rat motor nerve terminal

We have used intracellular recording to investigate the existence of a functional link between muscarinic presynaptic acetylcholine (ACh) autoreceptors, the intracellular serine‐threonine kinases‐mediated transduction pathways and transmitter release in the motor nerve terminals of adult rats. We found the following. (1) Transmitter release was reduced by the M1 muscarinic acetylcholine receptor (mAChR) blocker pirenzepine and enhanced by the M2 blocker methoctramine. The unselective mAChR blocker atropine increased ACh release, which suggests the unmasking of another parallel release‐potentiating mechanism. There are therefore two opposite, though finely balanced, M1–M2 mAChR‐operated mechanisms that tonically modulate transmitter release. (2) Both M1 and M2 mechanisms were altered when protein kinase C (PKC), protein kinase A (PKA) or the P/Q‐type calcium channel were blocked. (3) Both PKC and PKA potentiated release when they were specifically stimulated [with phorbol 12‐myristate 13‐actetate (PMA) and Sp‐8‐Br cAMPs, respectively], and both needed the P/Q channel. (4) In normal conditions PKC seemed not to be directly involved in transmitter release (the PKC blocker calphostin C did not reduce release), whereas PKA was coupled to potentiate release (the PKA blocker H‐89 reduced release). However, when an imbalance of the M1–M2 mAChRs function was experimentally produced with selective blockers, an inversion of the kinase function occurred and PKC could then stimulate transmitter release, whereas PKA was uncoupled. (5) The muscarinic function may be explained by the existence of an M1‐mediated increased PKC activity‐dependent potentiation of release and an M2‐mediated PKA decreased activity‐dependent release reduction.

Pertussis Toxin-Sensitive G-Protein and Protein Kinase C Activity are Involved in Normal Synapse Elimination in the Neonatal Rat Muscle

Individual skeletal muscle fibers in most new‐born rodents are innervated at a single endplate by several motor axons. During the first postnatal weeks, the polyneuronal innervation decreases in a process of synaptic elimination. Previous studies showed that the naturally occurring serine‐protease thrombin mediates the activity‐dependent synapse reduction at the neuromuscular junction (NMJ) in vitro and that thrombin‐receptor activation may modulate nerve terminal consolidation through a protein kinase mechanism. To test whether these mechanisms may be operating in vivo, we applied external thrombin and its inhibitor hirudin, and several substances affecting the G protein‐protein kinase C system (GP‐PKC) directly over the external surface of the neonatal rat Levator auris longus muscle. Muscles were processed for immunocytochemistry to simultaneously detect acetylcholine receptors (AChRs) and axons for counting the percentage of polyinnervated NMJ. We found that exogenous thrombin accelerated synapse loss and hirudin blocked axonal removal. Phorbol‐12‐myristate‐13‐acetate, a potent PKC activator, had a similar effect as thrombin, whereas the PKC inhibitors, calphostin C and staurosporine, prevented axonal removal. Pertussis toxin, an effective blocker of GP function, blocked synapse elimination. These findings suggest that the normal synapse elimination in the neonatal rat muscle may be modulated, at least in part, by the pertussis‐sensitive G‐protein and PKC activity and that thrombin could play a role in the postnatal synaptic maturation in vivo. J. Neurosci. Res. 63:330–340, 2001.

Pre- and postsynaptic maturation of the neuromuscular junction during neonatal synapse elimination depends on protein kinase C

The distribution of acetylcholine receptors (AChRs) within and around the neuromuscular junction changes dramatically during the first postnatal weeks, a period during which polyneuronal innervation is eliminated. We reported previously that protein kinase C (PKC) activation accelerates postnatal synapse loss. Because of the close relationship between axonal retraction and AChR cluster dispersal, we hypothesize that PKC can modulate morphological maturation changes of the AChR clusters in the postsynaptic membrane during neonatal axonal reduction. We applied substances affecting PKC activity to the neonatal rat levator auris longus muscle in vivo. Muscles were then stained immunohistochemically to detect both AChRs and axons. We found that, during the first postnatal days of normal development, substantial axonal loss preceded the formation of areas in synaptic sites that were free of AChRs, implying that axonal loss could occur independently of changes in AChR cluster organization. Nevertheless, there was a close relationship between axonal loss and AChR organization; PKC modulates both, although differently. Block of PKC activity with calphostin C prevented both AChR loss and axonal loss between postnatal days 4 and 6. PKC may act primarily to influence AChR clusters and not axons, insofar as phorbol ester activation of PKC accelerated changes in receptor aggregates but produced relatively little axon loss.

The Interaction between Tropomyosin-Related Kinase B Receptors and Presynaptic Muscarinic Receptors Modulates Transmitter Release in Adult Rodent Motor Nerve Terminals

The neurotrophin brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4) and the receptors tropomyosin-related kinase B (trkB) and p75NTR are present in the nerve terminals on the neuromuscular junctions (NMJs) of the levator auris longus muscle of the adult mouse. Exogenously added BDNF or NT-4 increased evoked ACh release after 3 h. This presynaptic effect (the size of the spontaneous potentials is not affected) is specific because it is not produced by neurotrophin-3 (NT-3) and is prevented by preincubation with trkB-IgG chimera or by pharmacological block of trkB [K-252a (C27H21N3O5)] or p75NTR [Pep5 (C86H111N25O19S2)] signaling. The effect of BDNF depends on the M1 and M2 muscarinic acetylcholine autoreceptors (mAChRs) because it is prevented by atropine, pirenzepine and methoctramine. We found that K-252a incubation reduces ACh release (∼50%) in a short time (1 h), but the p75NTR signaling inhibitor Pep5 does not have this effect. The specificity of the K-252a blocking effect on trkB was confirmed with the anti-trkB antibody 47/trkB, which reduces evoked ACh release, like K-252a, whereas the nonpermeant tyrosine kinase blocker K-252b does not. Neither does incubation with the fusion protein trkB-IgG (to chelate endogenous BDNF/NT-4), anti-BDNF or anti-NT-4 change ACh release. Thus, the trkB receptor normally seems to be coupled to ACh release when there is no short-term local effect of neurotrophins at the NMJ. The normal function of the mAChR mechanism is a permissive prerequisite for the trkB pathway to couple to ACh release. Reciprocally, the normal function of trkB modulates M1- and M2-subtype muscarinic pathways.

Localization of brain-derived neurotrophic factor, neurotrophin-4, tropomyosin-related kinase b receptor, and p75NTR receptor by high-resolution immunohistochemistry on the adult mouse neuromuscular junction

Neurotrophins and their receptors, the trk receptor tyrosine kinases (trks) and p75NTR, are differentially expressed among the cell types that make up synapses. It is important to determine the precise location of these molecules involved in neurotransmission. Here we use immunostaining and Western blotting to study the localization and expression of neurotrophin brain‐derived neurotrophic factor (BDNF) and neurotrophin‐4 (NT‐4) and the receptors tropomyosin‐related kinase b (trkB) and p75NTR at the adult neuromuscular junction. Our confocal immunofluorescence results on the whole mounts of the mouse Levator auris longus muscle and on semithin cross‐sections showed that BDNF, NT‐4, trkB, and p75NTR were localized on the three cells in the neuromuscular synapse (motor axons, post‐synaptic muscle and Schwann cells).

Changes in the neuromuscular synapse induced by an antibody against gangliosides

In this study, we used a monoclonal IgM antibody from a patient with a pure motor chronic demyelinating polyneuropathy, which binds specifically to the complex gangliosides GM2, GalNAc‐GD1a, and GalNAc‐GM1b, which appear to have a common epitope of ‐[GalNAcβ1‐4Gal(3‐2αNeuAc)β1]. This was done for the following reasons: (1) to localize these gangliosides in specific cellular components of the neuromuscular junction (NMJ), and (2) to describe the anti–ganglioside antibody–induced structural and functional changes in the NMJs to gain insight into the role of gangliosides in the synaptic function. Using immunofluorescence techniques, we found that these gangliosides are located only in the presynaptic component of the motor end‐plates, both in nerve terminals and in Schwann cells. After 2 weeks of continued passive transfer of the IgM monoclonal antibody over the mouse levator auris longus muscle, electromyography showed an axonal or NMJ disorder. Morphology showed important nerve terminal growth and retraction changes. Using intracellular recording electrophysiology, we found neurotransmitter release alterations, including quantal content reduction and an immature expression of voltage‐dependent calcium channels similar to what occurred during NMJ development and regeneration. These changes were complement independent. The results showed that these gangliosides were involved in the reciprocal Schwann cell–nerve terminal interactions, including structural stability and neurotransmission. Ann Neurol 2005;57:396–407

Presynaptic membrane receptors in acetylcholine release modulation in the neuromuscular synapse

Over the past few years, we have studied, in the mammalian neuromuscular junction (NMJ), the local involvement in transmitter release of the presynaptic muscarinic ACh autoreceptors (mAChRs), purinergic adenosine autoreceptors (P1Rs), and trophic factor receptors (TFRs; for neurotrophins and trophic cytokines) during development and in the adult. At any given moment, the way in which a synapse works is largely the logical outcome of the confluence of these (and other) metabotropic signalling pathways on intracellular kinases, which phosphorylate protein targets and materialize adaptive changes. We propose an integrated interpretation of the complementary function of these receptors in the adult NMJ. The activity of a given receptor group can modulate a given combination of spontaneous, evoked, and activity‐dependent release characteristics. For instance, P1Rs can conserve resources by limiting spontaneous quantal leak of ACh (an A1R action) and protect synapse function, because stimulation with adenosine reduces the magnitude of depression during repetitive activity. The overall outcome of the mAChRs seems to contribute to upkeep of spontaneous quantal output of ACh, save synapse function by decreasing the extent of evoked release (mainly an M2 action), and reduce depression. We have also identified several links among P1Rs, mAChRs, and TFRs. We found a close dependence between mAChR and some TFRs and observed that the muscarinic group has to operate correctly if the tropomyosin‐related kinase B receptor (trkB) is also to operate correctly, and vice versa. Likewise, the functional integrity of mAChRs depends on P1Rs operating normally.

Plastic-embedded semithin cross-sections as a tool for high-resolution immunofluorescence analysis of the neuromuscular junction molecules: Specific cellular location of protease-activated receptor-1.

In the neuromuscular junction (NMJ), three cellular elements (nerve ending, postsynaptic muscle component, and teloglial Schwann cell) are closely juxtaposed and functionally interdependent. It is important to determine the precise location of the relevant molecules involved in structural stability and neurotransmission at the three cellular components of this synapse in order to understand the molecular mechanisms underlying NMJ formation, maintenance, and functionality. In this paper, we show that plastic‐embedded 0.5‐μm semithin cross‐sections from whole‐mount multiple‐immunofluorescence‐stained muscles provide a simple and sensitive high‐resolution procedure for analyzing the cellular and subcellular distribution of molecules at the NMJ. We have used this procedure to resolve the location of protease‐activated receptor 1 (PAR‐1). Previously, by immunohistochemistry we had detected PAR‐1 in muscle fibers concentrated in the synaptic area but could not determine whether PAR‐1 is expressed only in the muscle fiber at the NMJ. Our present results demonstrate that PAR‐1 is concentrated in the postsynaptic region but not in the presynaptic terminal and that the labelling pattern for PAR‐1 overlapped with Schwann cell staining.

Involvement of brain-derived neurotrophic factor (BDNF) in the functional elimination of synaptic contacts at polyinnervated neuromuscular synapses during development.

We use immunohistochemistry to describe the localization of brain‐derived neurotrophic factor (BDNF) and its receptors trkB and p75NTR in the neuromuscular synapses of postnatal rats (P6–P7) during the synapse elimination period. The receptor protein p75NTR is present in the nerve terminal, muscle cell and glial Schwann cell whereas BDNF and trkB proteins can be detected mainly in the pre‐ and postsynaptic elements. Exogenously applied BDNF (10 nM for 3 hr or 50 nM for 1 hr) increases ACh release from singly and dually innervated synapses. This effect may be specific for BDNF because the neurotrophin NT‐4 (2–8 nM) does not modulate release at P6–P7. Blocking the receptors trkB and p75NTR (with K‐252a and anti‐p75‐192‐IgG, respectively) completely abolishes the potentiating effect of exogenous BDNF. In addition, exogenous BDNF transiently recruits functionally depressed silent terminals, and this effect seems to be mediated by trkB. Calcium ions, the L‐type voltage‐dependent calcium channels and protein kinase C are involved in BDNF‐mediated nerve ending recruitment. Blocking experiments suggest that endogenous BDNF could operate through p75NTR receptors coupled to potentiate ACh release in all nerve terminals because the anti‐p75‐192‐IgG reduces release. However, blocking the trkB receptor (K‐252a) or neutralizing endogenous BDNF with the trkB‐IgG fusion protein reveals a trkB‐mediated release inhibition on almost mature strong endings in dual junctions. Taken together these results suggest that a BDNF‐induced p75NTR‐mediated ACh release potentiating mechanism and a BDNF‐induced trkB‐mediated release inhibitory mechanism may contribute to developmental synapse disconnection.

Adenosine A1 and A2A receptor-mediated modulation of acetylcholine release in the mice neuromuscular junction

Immunocytochemistry shows that purinergic receptors (P1Rs) type A1 and A2A (A1R and A2AR, respectively) are present in the nerve endings at the P6 and P30 Levator auris longus (LAL) mouse neuromuscular junctions (NMJs). As described elsewhere, 25 μm adenosine reduces (50%) acetylcholine release in high Mg2+ or d‐tubocurarine paralysed muscle. We hypothesize that in more preserved neurotransmission machinery conditions (blocking the voltage‐dependent sodium channel of the muscle cells with μ‐conotoxin GIIIB) the physiological role of the P1Rs in the NMJ must be better observed. We found that the presence of a non‐selective P1R agonist (adenosine) or antagonist (8‐SPT) or selective modulators of A1R or A2AR subtypes (CCPA and DPCPX, or CGS‐21680 and SCH‐58261, respectively) does not result in any changes in the evoked release. However, P1Rs seem to be involved in spontaneous release (miniature endplate potentials MEPPs) because MEPP frequency is increased by non‐selective block but decreased by non‐selective stimulation, with A1Rs playing the main role. We assayed the role of P1Rs in presynaptic short‐term plasticity during imposed synaptic activity (40 Hz for 2 min of supramaximal stimuli). Depression is reduced by micromolar adenosine but increased by blocking P1Rs with 8‐SPT. Synaptic depression is not affected by the presence of selective A1R and A2AR modulators, which suggests that both receptors need to collaborate. Thus, A1R and A2AR might have no real effect on neuromuscular transmission in resting conditions. However, these receptors can conserve resources by limiting spontaneous quantal leak of acetylcholine and may protect synaptic function by reducing the magnitude of depression during repetitive activity.