Neuromuscular junction (NMJ) is the functional contact (synapse) between an axon of motor neuron and muscle fiber. It is generally accepted to consider this contact only as a specialized morpho-functional structure, where chemical transmission (via release of the acetylcholine (ACh)) of electrical signal from motor neuron to muscle fiber occurs, ultimately causing the muscle to contract. This synaptic contact is probably one of the most studied synapses since it has relatively large size and easy accessibility for various experimental manipulations. A great body of data is received on the development, molecular organization, morphology, and physiology of both pre- and postsynaptic regions of the NMJ. It's not so long ago that it seemed that practically all was known about the NMJ. However, due to the significant progress in the improvement and application of electrophysiological, genetic, pharmacological, biochemical and immunohistochemical methods a number of previously unknown aspects of neuron and muscle interaction were revealed. So, according to numerous studies, not only ACh (which by the way does not always lead to a contraction of the muscle fiber) is released in the vertebrate neuromuscular synapse, but also a number of other synaptically active molecules. And these molecules can be released from both nerve terminal (anterograde signal), and from muscle fiber (retrograde signal).
Before starting the consideration of the facts relating to the yet poorly studied non-cholinergic signaling, it should be recalled main points of the structure and functioning of the NMJ.
2. Neuromuscular junction organization: Brief overview
Motor neurons in the ventral region of the spinal cord send axons out toward the periphery (Fig. 1). In mammals and many higher vertebrates, each muscle fiber typically has a single synaptic site innervated by a single motor axon branch. In front of the contact, the motor axon loses its myelin sheath and forms nerve terminal branches. Several non-myelinating Schwann cells are located over these nerve terminal branches and make processes that are closely covered to them. Terminal Schwann cells, motor nerve terminal branches and the postsynaptic specializations of sarcolemma (also known as a motor end plate) together form the neuromuscular junction (or myoneural junction).
The motor nerve ending contains a large number of small synaptic vesicles which store molecules of the neurotransmitter ACh. The latter is synthesized in nerve terminals from choline and acetyl coenzyme A by the cytoplasmic enzyme choline acetyltransferase (ChAT) and transferred by a vesicular ACh transporter (VAChT) into synaptic vesicles. The transmitter contained in a single vesicle (in vertebrate NMJ it is about 5000 – 10000 molecules of ACh) is often referred to as a ‘quantum’, because during vesicle exocytosis relatively stable portion of chemical substance is released.
ACh diffuses across the synaptic cleft (50 – 100 nm) to be received by ACh receptors on the postsynaptic sarcolemma. One of the most striking structural features of this region is the deep infolding of sarcolemma. The crests of the folds contain a high density of ACh receptors whereas in the depths of the folds a density of voltage-gated sodium channels are presents. The binding of ACh to receptors causes the opening of cation-selective ion channels and allows a net flux of positive charge into the skeletal muscle. When rising depolarization is adequate to open voltage-gated sodium channels, the threshold for action potential generation is reached. Then action potential sweeps across the muscle fiber membrane and the muscle fiber contracts. The neurotransmitter action is terminated by localized in synaptic cleft enzyme acetylcholinesterase (AChE) hydrolyzing ACh to choline and acetate. Choline is recycled into the motor nerve terminal by a high-affinity uptake system, making it available for the resynthesis of ACh.
3. Neurotransmission in neuromuscular junction
First of all it must be recalled, that ACh release from the motor nerve ending does not always leads to muscle fiber contraction, and motor neuron not only induce a contractile activity in the muscle, but also control of a number of morphological and functional properties of muscle fibers. This latter influence is usually referred to as neurotrophic and it often involve the control of gene expressions in the muscle [8-13]. At the NMJ have revealed the presence of several distinct types of ACh release: spontaneous quantal, nerve impulse evoked quantal and non-quantal release. Molecular mechanisms, features and functional significance of these secretion types are described in detail and systematized in reviews [6,14-17]. Here we will briefly consider these processes.
Although molecular mechanisms of action of spontaneously released mediator are not yet fully established, the majority of data indicate that tonic neurotransmitter release is one of the neurotrophic control factors whereas the physiological role of the evoked quantal ACh release is to ensure clear transmission of the electric impulse from the motor nerve to the muscle fiber [15,17]. At the same time obtained experimental results suggest a possible trophic role of ACh released by quantal manner in response to the nerve action potential [8,22]. However, until recent time, the fact that other signaling molecules can be released together with the ACh from motor nerve endings and participate in the neuromuscular transmission was ignored.
4. Сotransmission and neuromodulation
In neurobiology for decades the ‘Dale’s principle’ dominated, according to which, one neuron synthesizes, stores, and releases a single transmitter liberated from all own’s axon terminals. In this regard, vertebrate motoneuron for a long time considered as a cell capable to release ACh only. However by the early 90’s a large amount of experimental data was obtained, the analysis of which led to the formation of the modern theory of ‘cotransmission’ [23-28]. According to this theory, one or several types of synaptically active molecules – cotransmitters (coexisting transmitters) are released from the neuron together with basic mediator. These cotransmitters are capable of exerting its own effects in the target cell, regulating the release of primary neurotransmitter (presynaptic modulation) or modulating the physiological response in the postsynaptic cell (postsynaptic modulation). At present, it can be stated that the phenomenon of corelease of several neurotransmitters from the nerve endings is the rule rather than the exception for the entire nervous system, including peripheral part [24,25,27,28].
Some signaling molecules that do not meet the definition of ‘cotransmitters’ are involved in the functioning of the synaptic apparatus too. They are released from either neuron, but independently of the primary neurotransmitter, or have a glial origin or they are released from the postsynaptic cell and, along with cotransmitters, exert their modulating and/or neurotrophic effects.
5. Purinergic signaling
Like many signaling molecules, ATP released from the cell is metabolized in the extracellular space. ATP is broken down to ADP and AMP by extracellular ATPases . Further, as shown directly in the rat NMJ, AMP was either dephosphorylated into adenosine by ecto-5’-nucleotidase or deaminated into inosine monophosphate by ecto-AMP deaminase . Inosine is an inactive metabolite , but adenosine is a signaling molecule that activates its own receptors . Formed adenosine is removed from the synaptic cleft of the NMJ by dipyridamole-sensitive adenosine uptake system, and there are reasons to believe that adenosine uptake is more important than adenosine deamination in the regulation of extracellular adenosine concentrations .
Pharmacological evidence of the presynaptic localization of adenosine (P1) receptors were obtained on preparations of NMJ, both in amphibians  and mammals [45,46]. In the latter case the presence of A1 and A2A receptor subtypes on the nerve ending was defined. Subsequently, confirmation of exclusively presynaptic localization of A2A receptors in the the NMJ of mouse was obtained by the means of immunohistochemistry . At the same time, on the plasma membrane of human skeletal fiber adenosine A2A and A2B receptors were revealed by means of immunohistochemistry .
As for P2 receptors, the following is known at present time. P2X7 receptor subunits were found on presynaptic motor nerve terminals of mouse, but there is no evidence for P2X1, P2X2, P2X3, P2X4, P2X5 or P2X6 receptor subunits . According to a number of electrophysiological studies metabotropic P2Y receptors are also localized on the motor nerve endings of both amphibian and mammals NMJ [44,50-52]. However, P2Y receptors were found on the postsynaptic membrane of skeletal muscle fiber. Moreover, the presence of P2Y1 and P2Y2 receptors on the plasma membrane was precisely established [53,54]. Developing mammalian skeletal muscle fibers are able to express 4 subtypes of metabotropic purine receptors (P2Y1, P2Y2, P2Y4 and P2Y11) and, what is interesting, all types of P2X receptors which, apparently, are absent on the mature innervated muscle fibers [29,55-57].
In addition to the role of ATP in the process of synaptogenesis a lot of data is obtained about the modulator effects of purines on the processes of ACh release in the mature vertebrate NMJ. So, it was found that ATP and adenosine significantly reduced the intensity of both evoked and spontaneous quantal release of ACh, activating presynaptic purine receptors [44,50,51,61,62]. However adenosine can also facilitate the quantal release of ACh what, apparently, depends on the pattern of motor nerve stimulation . Extracellular ATP induces presynaptic inhibition of ACh release via its own P2Y receptors, which modulate voltage-gated Ca2+ channels [50,51]. Adenosine also inhibits quantal release of ACh, acting through P1 receptors and its mechanism of action does not affect the operation of calcium channels [50,51]. As for the influence of purines on the non-quantal release of ACh it is established that its intensity remains unchanged in the presence of adenosine, but it decreases via activation of P2Y receptors by the ATP molecules and this mechanism is not coupled to presynaptic voltage-dependent Ca2+ channels [52,63].
Postsynaptic modulator effects of purines in the mature neuromuscular synapse were also established. So it was found that ATP can increase ACh receptor activity [64-66] and inhibit chloride channels in mammalian skeletal muscle .
6. Glutamatergic signaling
In experiments on the culture of spinal neurons and skeletal muscle fibers of
7. Peptidergic signaling
High concentrations of NAAG have been found in spinal cord motoneurons and motor components of cranial nerve nuclei [98-100]. Moreover, this dipeptide was found in sciatic nerve [98,101] and phrenic nerve terminals . NAAG can be involved in neurotransmission as: (i) direct agonist of glutamate ionotropic NMDA receptors and metabotropic GluR3 receptors and (ii) as a glutamate precursor, which is formed directly in the extracellular space during hydrolysis by the enzyme glutamate carboxypeptidase II (GCP II), also known as N-acetylated α-linked acidic dipeptidase (NAALADase) .This peptidase is a membrane-bound protein which was detected in non-myelinating presynaptic Schwann cells surrounding motor nerve terminals [87,103].
Experiments on rat NMJ showed that NAAG is able to depress non-quantal ACh release . The mechanism of neuropeptide action is realized through its extracellular hydrolysis by the GCP II with the formation of glutamate molecules, which, as was shown earlier , activate glutamate postsynaptic NMDA receptors and thereby trigger the NO-mediated mechanism of reducing the intensity of the non-quantal ACh release .
The presence of substance P in frog motor nerve endings was shown by immunohistochemistry . Later, data demonstrating the neuropeptide release during the stimulation of the motor nerve was obtained . NK-1 receptors, localized in perisynaptic Schwann cells NMJ were found by the same authors. Substance P was not found by immunohistochemistry in the motor nerve endings of rodents [108,109], however, it was found in the muscle fibers. Soleus muscle had a significantly higher content (0.61 ng/g) than the extensor digitorum longus (0.22 ng/g) .
In studying the signaling function of substance P in the frog NMJ its influence on all compartments NMJ was revealed: on motor nerve terminal, on postsynaptic membrane and on Schwann cell. So, following effects were shown: (i) facilitating effect of neuropeptide (at a concentration till 1 µM) on spontaneous and evoked quantal release of ACh ; (ii) reduction of the sensitivity of the postsynaptic membrane to ACh at the concentration peptide above than 1 µM [111,112]; and (iii) induction of Ca2+ release from internal stores in Schwann cells . In the mammalian NMJ also was noted presynaptic facilitatory action of substance P. Neuropeptide facilitated the indirect twitch responses of the rat diaphragm and increased amount of ACh released into the bathing medium in response to tetanic stimulation of the phrenic nerve .
Frog motor neurons express CGRP-like immunoreactivity and this immunoreactivity in motor nerve terminals is confined within so called ‘large dense-core vesicles’ . CGRP-like immunoreactivity was found in the mouse and rat motor nerve terminals [116,117]. In rat hind limb CGRP-like immunoreactivity is heterogeneously present in the endplates and, apparently, correlates with the muscle fibers phenotype . Motoneurons of small and slow-twitch motor units in general have lower levels than motoneurons of large and fast-twitch motor units . It is established that the CGRP is released by nerve impulse activity . Calcrl mRNA and CALCRL protein were found directly in postsynaptic region of rats muscle fibers . The CGRP receptor, and its two associated components (RAMP1 and RCP), are highly concentrated at the adult avian NMJ where they co-localize with AChE and ACh receptors .
Physiological role of CGRP was revealed not only at establishment and development NMJ, but also in the process of its functioning. Thus, on cultured chick myotubes it was shown that the CGRP stimulates the turnover of phosphoinositides and the accumulation of inositol phosphates  and also increases the number of surface ACh receptors . In 1-day-old
In experiments on mature rodent neuromuscular synapse it was shown that CGRP enhances muscle contraction during stimulation of the nerve fibers or direct stimulation of the muscle . The ability of neuropeptide to enhance the intensity of spontaneous quantal ACh release was revealed . The effect of the CGRP facilitating the secretion of ACh was also described in the frog neuromuscular synapse .
8. Nitric oxide signaling
Neuronal and endothelial NO-synthases are activated by calcium and calmodulin, whereas the inducible isoform binds irreversibly to the calmodulin right after the translation, so this enzyme produces NO independently of changes in intracellular calcium concentration . It is established that during muscle contraction the activity of NO-synthases increases by several times [137,138]. It is well explained by the increase of cytosolic calcium concentration, which facilitates the interaction of the enzyme with calmodulin. According to several authors skeletal muscle produces from 2 to 25 (average ~ 10) pmol min-1 mg-1 of nitric oxide [137,139,140].
It is interesting to note that, apparently, in amphibians the localization of NO-synthases is differ from mammals. So, in frog NMJ NO-synthase immunostaining was found at the membrane and occasionally in the cytoplasm of perisynaptic Schwann cells and was not detected in the nerve terminal or muscle .
NO-mediated signaling plays a certain role in the formation of the NMJ. In particular, the role of NO both in presynaptic and postsynaptic differentiation of NMJ was shown [142,143]. In mature neuromuscular synapse physiological significance of NO-mediated signaling was revealed in processes metabolism and contraction of muscle fiber, as well as in modulation of ACh release from the motor nerve ending.
It is shown that the NO-synthase activity can modulate mitochondrial respiration in skeletal muscle. So, inhibitory effect of NO on oxygen consumption of muscle tissue was revealed [144,130]. Modulatory influence of NO was demonstrated with respect to carbohydrate metabolism. It was shown that NO-synthases blocking inhibits the reuptake of 2-deoxyglucose, whereas exogenous NO molecules donor leads to its increase [138,139]. On the other hand, the possibility of NO to inhibit the activity of glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was revealed . Finally, data on the NO-mediated inhibition of the creatine kinase activity in skeletal muscle was obtained . It can lead to decrease in the synthesis of ATP from creatine phosphate.
The action of NO on contractile function of muscle fibers is complex.
Namely the fact of finding the post-synaptic localization of NO-synthase and modulating effect of NO molecules on the process of ACh release from motor nerve terminals allow us to declare that this signaling molecule acts as a retrograde synaptic mediator in the NMJ. NO reduces the intensity of both spontaneous and evoked quantal ACh release in the neuromuscular synapse of the frog [152,153]. The inhibitory action of nitric oxide on spontaneous and induced synaptic currents was shown also in the developing neuromuscular contacts
Until now, many people share the opinion that intercellular contact between motor neuron and skeletal muscle fiber is very well studied morpho-functional structure, which provide the one-way transmission of electrical impulse from the motor neuron to the muscle for the initiation of the contractile act. However, this opinion is totally wrong and one of the proofs for that is this review which describes a number of most studied signaling pathways mediated by molecules that previously were not considered in the aspect of the functioning of the NMJ. Experimental facts proving; (i) the formation of these molecules in the neuromuscular synapse; (ii) their release in the synaptic cleft; (iii) the interaction with specific receptor proteins; and (iv) the existence of a specific physiological effect for each of these signaling molecules are presented and analyzed here. It is necessary to emphasize that the author intentionally considered those signaling molecules (ATP, glutamate, NAAG, substance P and NO), which act as an individual neurotransmitter in the mature organism, but in synapses of other parts of the nervous system [27,29,69,105,156,157]. CGRP, in its turn, also plays its role in mature intercellular contact, acting as a cotransmitter in sensory-motor neurons .
A number of signaling molecules which are also participate in the signaling between motor neuron, Schwann cell and skeletal muscle fiber remained beyond the review. At least nerve growth factor (NGF), glial-cell-line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4) and transforming growth factor-beta 2 (TGF-β2) are among them. The main role in regulating of neuronal survival, plasticity, growth, and death is ascribed to them. However, it turned out that these proteins act also as regulators of the maintenance, function, and regeneration of skeletal muscle fibers . So, it was shown that BDNF, NT-3, NT-4 are expressed both in motor neurons and in muscle fibers. GDNF, in its turn, is expressed in Schwann cell and in muscle fiber. Activity-dependent synthesis and release of these factors in extracellular space have been reported. Receptors for all these factors were revealed in mature NMJ, their participation in the regulation of neuromuscular transmission was shown also at the expense of influence on the processes of ACh release [158-160].
Thus, NMJ is a rather complicated and flexible compartment for multicircuit intercellular communication between a motor neuron and muscle fiber, what provides the synaptic plasticity and reliability of synaptic transmission.
Preparation of this chapter was supported by grants from the RFBR (projects nos. 11-04-01188, 11-04-01471) and program of the President of the Russian Federation (Science School No. 2669.2012.7).
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