Photoswitchable glutamate receptors to control neurotransmission with light

Abstract

Optical tools to control neuronal activity include synthetic photoswitchable ligands of receptors and ion channels. Photoswitches can act either as soluble molecules (photochromic ligands, PCLs) or tethered to the protein (photoswitchable tethered ligands, PTLs), and they have been used to photocontrol many ion channels and receptors such as voltage-gated potassium channels, acetylcholine or glutamate receptors. Recognizing both the need for new optical tools in neuroscience and the opportunities offered by photoswitches, this work is focused on the use of light gated glutamate receptors to control neuronal activity and neurotransmission. In the first chapter of results of the thesis, we demonstrate that the Ca2+-permeable LiGluR can be used as a tool to reversibly control neurosecretion by directly affecting the intracellular [Ca2+]. To achieve this goal, LiGluR was expressed in cultured bovine chromaffin cells and cultured hippocampal neurons. We measured secretion in chromaffin cells using two techniques, amperometry and membrane capacitance, and current-clamp recordings to assess neurotransmission in cultured neurons. The results indicated that the magnitude of LiGluR-mediated Ca2+ influx is sufficiently large to trigger regulated exocytosis in chromaffin cells and neurons. In addition, LiGluR induced secretion can be modulated with the wavelength of illumination. This new application of LiGluR opens the possibility to reversibly control the activity of individual synapses, which might help to understand the computational properties of neurons and to unravel how brain circuits work. To use LiGluR as an effective method to interrogate the neuronal function it should support high-spatial 3D resolution and tissue penetration. Multiphoton excitation with near-infrared light enables stimulation in intact tissue with cellular and subcellular resolution, and it has been extensively applied to optical actuators such as caged compounds and more recently to optogenetics. However, two-photon stimulation of synthetic photoswitches had not been explored before. In the second section of the results, the two-photon stimulation of LiGluR is investigated. Two new photoswitches were designed (MAG2p and MAGA2p) based on the structure of the original photoswitch (MAG) and intended to enhance the two-photon absorption ability of the azobenzene switch. The three PTLs, including MAG, successfully activate LiGluR under two-photon stimulation, suggesting that multiphoton excitation can be applied to other azobenzene-based molecules. Interestingly, the rationally designed photoswitches were more efficient in opening LiGluR as lower power and shorter simulation time were required. Finally we validated MAG2p and MAG as new tools to control the activation of neurons and astrocytes with cellular and subcellular resolution. In the last chapter, a new method based on the affinity labeling approach is presented in order to confer light sensitivity to endogenous receptors. Glutamate-azobenzene-reactive PTLs with different lengths and reactive groups were tested on kainate receptors. These reactive PTLs successfully and functionally conjugate to the ionotropic glutamate receptor subunit GluK1, thus enabling to photoswitch its activity, as evidenced from photocurrent recordings of mammalian cells overexpressing the non-mutated receptor. These results are also supported by the photocontrol of GluK1 currents in dorsal root ganglion neurons, where GluK1 is the main glutamate subunit that is endogenously expressed. The new strategy proposed is versatile and we suggest that it can be extended to label other endogenous receptors, giving rise to novel optpharmacological therapies. The new methods here developed improve the LiGluR performance, and address photoswitches to use endogenous neuronal receptors to optically control neuronal activity, being able to stimulate them in small volumes corresponding to the neuronal functional unit (i.e. synaptic terminals). In this way, light would emerge as a unique tool to dissect neuronal physiology and to understand the function of neuronal circuits.

L’estudi de la neurotransmissió requereix noves eines moleculars, i els fotocommutadors ofereixen grans possibilitats. Aquesta tesi està centrada en l’ús de receptors de glutamat activables per llum (LiGluRs) pel control de l’activitat neuronal i dels processos de neurosecreció. Al primer bloc de resultats, la permeabilitat a Ca2+ dels receptors de glutamat s’aprofita per manipular de manera directa -independentment del voltatge de membrana- i reversible la concentració intracel•lular de Ca2+ amb llum. Així, és possible desencadenar els processos de secreció en cèl•lules cromafins i de neurotransmissió en neurones hipocampals. A la segona part dels resultats de la tesi, s’investiga l’estimulacio multifotó del receptor de glutamat modificat químicament amb fotocommutadors basats en l’azobenzè. Els resultats mostren l’estimulació per dos fotons del LiGluR, incloent-hi dos fotocommutadors nous, que milloren l’absorció multifotó del commutador azobenzè. Finalment, s’aplica aquesta tècnica per estimular neurones i astròcits amb una resolució a l’escala d’una cèl•lula o de compartiment subcel•lular. Al tercer capítol dels resultats, es descriu un nou mètode per aconseguir el fotocontrol de receptors neuronals endògens, utilitzant lligands covalents. Amb l’aplicació d’aquest mètode es pot fotocommutar l’activitat del receptor de kainat subtipus 1 (GluK1) no mutant, quan se sobreexpressa el receptor en cèl•lules de mamífer. Aquesta estratègia també permet obtenir fotocorrents en cultius de neurones dels ganglis de l’arrel dorsal, on GluK1 és la subunitat de glutamat endògenament més expressada. Els nous mètodes desenvolupats en aquesta tesi milloren la utilització dels LiGluRs, encaminant l’ús dels fotocommutadors cap al control òptic dels receptors endògens, amb la possibilitat d’estimular cèl•lules individuals o estructures subneuronals, fet que situaria els fotocomutados com a eines indispensables per l’estudi del cervell, des de la fisiologia fins als circuits neuronals.