Axonal injuries can result in paralysis and profound degenerative alterations to skeletal muscle leading to muscle atrophy, force deficits, even after repair employing micro-surgical techniques. Beyond surgical repair of the nerve, there are currently no treatment options for these patients. Understanding the basic mechanisms of neural regeneration after injury is a pre-requisite for developing appropriate treatments.
Neuronal activity has long been known to modulate many processes during development. Moreover, it has also been seen that neuronal activity can lead to regeneration in the peripheral nervous system (PNS). The manipulation of neuronal activity with light is currently the state of the art approach to investigate the nervous system, due to its specificity and capacity to induce more physiological neural activation. In this proposal we plan to use optogenetics (genetic manipulation to express light-sensitive proteins) and photophamacology (the development of light-regulated drugs) as tools to control neuronal activity. We aim to enhance the intrinsic capacity of peripheral nerves to regenerate by modulating neuronal activity. The laboratory of José A. del Río has developed an axotomy microfluidic device system that allows for simultaneous 3D co-culture and compartmentalization of mouse embryonic ventral spinal cord explants and skeletal muscle [1, 2]. This platform capitalizes on microfluidic designs for 3D cultures and the presence of an axotomy channel for the study of neuromuscular junction (NMJ) injuries and regeneration.
Preliminary results pointed out the importance of the modulation of neuronal activity to induce axonal regeneration after axotomy. Additionally, the system enables the culture of functional NMJs and myofibers, the evaluation of muscle cell contractility, the observation of axonal regeneration after axotomy, and the formation of functional NMJs, proving it to be useful as a tool to investigate NMJ mechanisms and treatments in health and disease, including axotomy.
We will combine the characterization of neuronal regeneration using our axotomy microfluidic device and advanced imaging techniques, with spatiotemporally and pharmacologically selective photostimulation of neuronal activity. In particular, we will use (1) transfection of muscle cells and postsynaptic neurons with excitatory optogenetic constructs (Channelrhodopsin-2), and (2) light-regulated ligands of postsynaptic receptors (acetylcholine, glutamate, and gamma-aminobutyric acid (GABA) receptors) to control the activity of NMJs and neuron-neuron synapses with patterns and pulses of illumination. The laboratory of Pau Gorostiza has developed several light-regulated agonists and antagonists of ionotropic  and metabotropic  glutamate receptors, and is currently working on ionotropic GABA receptors , muscarinic  and nicotinic receptors including muscle- and brain-subtypes. These compounds allow manipulating the activity of endogenous synaptic receptors without genetic manipulations and thus offer a powerful research tool and an interesting therapeutic opportunity for activity-dependent regeneration.
This multidisciplinary proposal encompasses the objective of engineering neuronal regeneration with a great diversity of experimental methods, ranging from microfluidics, 3D cell culture, and fluorescnce imaging, to advanced photomanipulation techniques like optogenetics and photopharmacology. Therefore, the project offers unique opportunities to train students and to develop novel neuroregeneration therapies.
 Tong Z, Seira O, Casas C, Reginensi D, Homs-Corbera A, Samitier J, Del Río JA (2014) Engineering a functional neuro-muscular junction model in a chip. RSC Advances 4 (97): 54788-54797.
 Tong Z, Segura-Feliu M, Seira O, Homs-Corbera A, Del Río JA, Samitier J (2015). A microfluidic neuronal platform for neuron axotomy and controlled regenerative studies. RSC Advances. 90 (20): 73457-73466.
 Izquierdo-Serra M, Bautista-Barrufet A, Trapero A, Garrido-Charles A, Díaz-Tahoces A, Camarero N, Pittolo S, Valbuena S, Pérez-Jiménez A, Gay M, García-Moll A, Rodríguez-Escrich C, Lerma J, de la Villa P, Fernández E, Pericàs MÀ, Llebaria A, Gorostiza P. Nat Commun. 2016 Jul 20;7:12221. doi: 10.1038/ncomms12221.
 Pittolo S, Gómez-Santacana X, Eckelt K, Rovira X, Dalton J, Goudet C, Pin JP, Llobet A, Giraldo J, Llebaria A, Gorostiza P. Nat Chem Biol. 2014 Oct;10(10):813-5. doi: 10.1038/nchembio.1612.
 Maleeva G, Wutz D, Rustler K, Nin-Hill A, Alfonso-Prieto M, Petukhova E, Bautista-Barrufet A, Gomila-Juaneda A, Scholze P, Peiretti F, Rovira C, König B, GorostizaP, Bregestovski P. Under review (2018).
 Riefolo, F; Matera, C; Garrido-Charles, A; Gomila-Juaneda, A; Agnetta, L; Claro, E; Masgrau, R; Holzgrabe, U; Batlle, M; Decker, M; Guasch, E; Gorostiza, P. Under review (2018).