Caratterizzazione elettrofisiologica e morfologica di sinapsi glutammatergiche nel muscolo scheletrico di mammifero

Each of the behavioral tasks performed by the nervous system, from the perception of sensory input and the control of motor output to cognitive functions such as learning and memory, depends on precise interconnections of many millions of neurons. These connections are developed during embryonic and post natal development. The study of the mechanisms that underlie synaptic formation has always interested a great number of scientists. In the last ten years, microscopy and molecular biology techniques have supplied a great number of knowledges about the mechanisms of synaptic formation. Despite the amount of information about synaptogenesis there are still many unknown processes behind the development of a synapse, such as the mechanisms that induce the correct expression of neurotransmitters (and their receptors). It is well known that acetylcholine is the only neurotransmitter in mammalian neuromuscular junction, but recently it has been demonstrated that glutamatergic neurons are able to form functional synapses with mammalian muscle under particular conditions. At the University of Brescia it has been observed that a new glutamatergic innervation of skeletal muscle replaces the original cholinergic one when the transversus abdominal muscle is denervated and a distal nerve stump is inserted in the white lateral matter of spinal cord in rat. Moreover, Xenopus muscle cells express several classes of transmitter receptors in addition to those for acetylcholine in early embryonic development. During normal differentiation and innervation of muscle, the other classes of receptors disappear. Changing the expression of transmitters by altering calcium spike activity leads to retention of the classes of cognate, noncholinergic receptors. This indicates that the expression of appropriate receptors is due to the neurotransmitter The aim of this work was to study the mechanisms that underlie the formation of glutamatergic muscular synapses observed at the University of Brescia. Muscles, to which descending glutamatergic fibres had been diverted from the spinal cord by means of a peripheral nerve graft, were studied by intracellular electrophysiological techniques in the first part of the project. Surgical procedures were performed at Brescia University. Unfortunately, all analyzed muscles showed cholinergic innervation. In the second part of the project, murine co-cultures with cortical neurons and skeletal muscle cells were developed. Electrophysiological, biochemical and morphological analysis showed that, under these conditions, cortical neurons develop functional glutamatergic synapses with muscle cells. Immunofluorescence studies also have demonstrated the presence of AMPA receptors (AMPARs) on muscular cells in the cocultures. Calcium imaging studies showed that electrical stimulation of cortical neurites, reaching myotubes, induced an increase of intracellular calcium concentration in the muscular cells. Electrical stimulation of neurites also produced contraction of myotubes. The contractions were resistant to curare, a common blocker of acetylcholine receptors, but they were sensitive to the glutamate AMPAR antagonist (GYKI 52466). Finally, coimmunoprecipitation and immunoblotting analysis demonstrated that only myotubes cocultured with cortical neurons express AMPARs and moreover it was seen that muscle cells express proteins of central post synaptic density (PSD). These data show, for the first time, that it is possible to induce the formation of a glutamatergic synapse on mammalian muscle cell in vitro.