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Optopharmacological brain mapping of autism mouse models

Periodic Reporting for period 1 - OPTOFRAX (Optopharmacological brain mapping of autism mouse models)

Reporting period: 2015-07-01 to 2017-06-30

The OPTOFRAX project aims at developing the seeds for a totally new therapeutic method for neurological disorders. Current pharmacological treatments suffer from known limitations: when a patient takes a drug, it distributes throughout the whole organism, including sick regions and healthy regions of the body, and usually at the same concentration everywhere. If one could deliver a drug only to the sick tissue and could adjust its concentration locally, many side effects would be avoided. Optopharmacology is a new promising technology that could achieve this goal. It is based on the development of photo-regulated drugs: molecules that can be activated or inactivated with light, so that its action can be finely controlled in space and time. These drugs could be activated only in the sick tissue with a flash of light, and only during the desired amount of time.

In this line, our experiments pretended to test novel optopharmacological tools to control neuronal physiology with light. The first objective was to test modulators of glutamate receptors in brain slices and to demonstrate that we can control neuronal activity with rapid flashes of light. The second objective was to use this technique to map the known therapeutic effect of a photoswitchable inhibitor of the metabotropic glutamate receptors in an animal model of one autism spectrum disorder, the Fragile X syndrome. We wanted to test these drugs at different levels of resolution of the neuronal system: at a macroscale resolution (whole-brain level) and at microscale resolution (single-synapse level). Overall, this project was a proof-of-concept test for pre-clinical studies on the use of light-regulated drugs for neurological disorders.
The first step towards the achievement of these goals was to establish the experimental approaches that we designed to modulate neuronal activity using these new photoswitchable molecules. We set up protocols to prepare acute slices from rodent brains, or slices that can be maintained in a culture for days or weeks. A transfection method to introduce fluorescent genes into those neurons was also established. At the same time, an in vivo approach using whole-brain Xenopus tropicalis tadpoles was also established. In both cases, neuronal activity was monitored using fluorescent calcium sensors (genetic or chemical). Several photoswitches that target the family of glutamate receptors were tested. Our results shows that, using covalently-targeted photoswitches, neuronal activity could be successfully activated or silenced by means of shining light (Fig. 1). UV light activated neurons and green light silenced them. The ability to control not only neurons (Fig. 1C-D) but also single synapses (Fig. 1E-H) was also tested and effectively achieved. The fine control of one single synapse was possible at unprecedented time resolution (1 sec) and space resolution (1 micron) using these covalently attached photo-sensitive molecules (Fig. 1E-H). We determined that these photoswitches preferentially targeted receptors of the AMPA and KA type, but not NMDA or metabotropic type (mGluR). Another photoswitchable molecule that selectively targeted only mGluRs were used to trigger morphological plasticity of dendritic spines (Fig. 1G-H). Spine morphology is altered in several animal models of autism spectrum disorders, including Fragile X syndrome. We had found that, while mGluR dependent plasticity is intact, NMDAR-dependent plasticity is altered in neurons from these sick animals. Our results showed that there must be an interaction between mGluR and NMDAR, since mGluR agonists can induce NMDAR-dependent plasticity. This plasticity can, therefore, be controlled with light using photo-sensitive mGluR modulators. We now aim at testing whether these photoswiches can revert this phenotype at the level of single dendritic spines before attempting to revert brain-wide neurological phenotypes. In this line, we tested the same compounds at the macroscale level of whole-brain in Xenopus tadpoles. We successfully used targeted covalent photoswitches to activate and inactive endogenous receptors in neurons of several brain regions of these animals (Fig. 1A-B). The next step is now testing if the same responses can be obtained in the genetic or chemical models of autism disorders.
The results obtained so far in this project demonstrate that we have successfully pushed the boundaries of the existing state of the art technologies. We have implemented a novel combination of techniques that allowed us to conquer a previously unexplored area of neuroscience. The combination of our newly-designed light-regulated drugs (optopharmacological tools) with the most advance light imaging techniques (2-photon and confocal microscopy) and their application as therapeutic mapping and physiological control of neural tissue, neurons and synapses, represent a clear progress in the development of innovative technologies towards the achievement of future phototherapies for neurological disorders. Our results show that we can control neuronal activity using photoswitches that target different specific subtypes of glutamate receptors. Some of these receptors are essential for the normal physiology of neural circuits (AMPAR, KAR), while some others are modulatory receptors implicated in neurological diseases (mGluR). These results, therefore, have important implications for the development of preclinical studies for phototherapies for these brain diseases, including autism spectrum disorders. Furthermore, we demonstrate that we can regulate neuronal activity and plasticity at multiple levels of resolution in the brain: from the macroscopic level of a whole brain in an intact organism (Fig 1A-B), to the mesoscopic level of single neurons (Fig 1C-D), down the microscopic level of single synapses (Fig 1E-H), which is the minimum and essential basis of neuronal transmission. These experiments are technically demanding and, to successfully obtain the results, we had to overcome the existing limits of several technologies. Therefore, there is no doubt these results will have an imminent impact in the scientific community, i.e. with a potential high number of citations. It will also have a mid-term high impact in the medical field, as this is the first preclinical data of the application of these optopharmacological tools for autism spectrum disorders. It will also have a mid-term impact on the industry as some commercial applications of similar drugs are already on the way from our lab (i.e. there is the clear possibility to patent these new drugs as well as the therapeutic mapping technique itself). Finally, these results will definitely have a clear impact on the society. Optopharmacology is still a largely unknown technology for non-scientific lay people. The dissemination of these results and the introduction of this new technology to the general audience, therefore, is sure to create a great social impact. Thus, our current efforts to disseminate these results will undoubtedly payoff for the achievement of our scientific goals, as it will attract the attention of members of the scientific, commercial, medical and cultural communities.