16 research outputs found
Fast Photoswitchable Molecular Prosthetics Control Neuronal Activity in the Cochlea
Altres ajuts: CERCA Programme/Generalitat de Catalunya, Fundaluce and "la Caixa" foundations (ID 100010434, agreement LCF/PR/HR19/52160010)Artificial control of neuronal activity enables the study of neural circuits and restoration of neural functions. Direct, rapid, and sustained photocontrol of intact neurons could overcome the limitations of established electrical stimulation such as poor selectivity. We have developed fast photoswitchable ligands of glutamate receptors (GluRs) to enable neuronal control in the auditory system. The new photoswitchable ligands induced photocurrents in untransfected neurons upon covalently tethering to endogenous GluRs and activating them reversibly with visible light pulses of a few milliseconds. As a proof of concept of these molecular prostheses, we applied them to the ultrafast synapses of auditory neurons of the cochlea that encode sound and provide auditory input to the brain. This drug-based method afforded the optical stimulation of auditory neurons of adult gerbils at hundreds of hertz without genetic manipulation that would be required for their optogenetic control. This indicates that the new photoswitchable ligands are also applicable to the spatiotemporal control of fast spiking interneurons in the brain
Synthetic Photoswitchable Neurotransmitters Based on Bridged Azobenzenes
Photoswitchable neurotransmitters of ionotropic kainate receptors were synthesized by tethering a glutamate moiety to disubstituted C2-bridged azobenzenes, which were prepared through a novel methodology that allows access to diazocines with higher yields and versatility. Because of the singular properties of these photochromes, photoisomerizable compounds were obtained with larger thermal stability for their inert cis isomer than for their biologically activity trans state. This enabled selective neuronal firing upon irradiation without background activity in the dark
Control of Brain State Transitions with a Photoswitchable Muscarinic Agonist
Reproducció del document publicat a:https://doi.org/10.1002/advs.202005027The ability to control neural activity is essential for research not only in basic neuroscience, as spatiotemporal control of activity is a fundamental experimental tool, but also in clinical neurology for therapeutic brain interventions. Transcranial-magnetic, ultrasound, and alternating/direct current (AC/DC) stimulation are some available means of spatiotemporal controlled neuromodulation. There is also light-mediated control, such as optogenetics, which has revolutionized neuroscience research, yet its clinical translation is hampered by the need for gene manipulation. As a drug-based light-mediated control, the effect of a photoswitchable muscarinic agonist (Phthalimide-Azo-Iper (PAI)) on a brain network is evaluated in this study. First, the conditions to manipulate M2 muscarinic receptors with light in the experimental setup are determined. Next, physiological synchronous emergent cortical activity consisting of slow oscillations—as in slow wave sleep—is transformed into a higher frequency pattern in the cerebral cortex, both in vitro and in vivo, as a consequence of PAI activation with light. These results open the way to study cholinergic neuromodulation and to control spatiotemporal patterns of activity in different brain states, their transitions, and their links to cognition and behavior. The approach can be applied to different organisms and does not require genetic manipulation, which would make it translational to humans
Optical Control of Cardiac Function with a Photoswitchable Muscarinic Agonist
Light-triggered reversible modulation of physiological functions offers the promise of enabling on-demand spatiotemporally controlled therapeutic interventions. Optogenetics has been successfully implemented in the heart, but significant barriers to its use in the clinic remain, such as the need for genetic transfection. Herein, we present a method to modulate cardiac function with light through a photoswitchable compound and without genetic manipulation. The molecule, named PAI, was designed by introduction of a photoswitch into the molecular structure of an M2 mAChR agonist. In vitro assays revealed that PAI enables light-dependent activation of M2 mAChRs. To validate the method, we show that PAI photoisomers display different cardiac effects in a mammalian animal model, and demonstrate reversible, real-time photocontrol of cardiac function in translucent wildtype tadpoles. PAI can also effectively activate M2 receptors using two-photon excitation with near-infrared light, which overcomes the scattering and low penetration of short-wave-length illumination, and offers new opportunities for intravital imaging and control of cardiac function
Rationally designed azobenzene photoswitches for efficient two-photon neuronal excitation
Manipulation of neuronal activity using two-photon excitation of azobenzene photoswitches with near-infrared light has been recently demonstrated, but their practical use in neuronal tissue to photostimulate individual neurons with three-dimensional precision has been hampered by firstly, the low efficacy and reliability of NIR-induced azobenzene photoisomerization compared to one-photon excitation, and secondly, the short cis state lifetime of the two-photon responsive azo switches. Here we report the rational design based on theoretical calculations and the synthesis of azobenzene photoswitches endowed with both high two-photon absorption cross section and slow thermal back-isomerization. These compounds provide optimized and sustained two-photon neuronal stimulation both in light-scattering brain tissue and in Caenorhabditis elegans nematodes, displaying photoresponse intensities that are comparable to those achieved under one-photon excitation. This finding opens the way to use both genetically targeted and pharmacologically selective azobenzene photoswitches to dissect intact neuronal circuits in three dimensions
Optical control of endogenous receptors and cellular excitability with light
[eng] Light-controlled manipulation of neuronal activity has transformed the field of neurobiology. Light provides temporal and spatial resolution over activation or inhibition of targeted populations of neurons, one single neuron or single synapses. Such precision could be achieved with optogenetics, which is based on the over-expression of light-sensitive proteins, but it requires gene therapy and often alters cellular physiology. An alternative to optogenetics is offered by photopharmacology (the development of light-regulated drugs or photoswitches), which can operate on endogenous receptors without genetic manipulation. Several new photoswitches are described in this thesis to improve their pharmacological and optical properties.
In photopharmacology, azobenzene is the most commonly used light switch. Photoswitchable tethered ligands (PTLs) are tri-modular molecules able to anchor to target receptors and alter their function by switching the azobenzene group. In the first chapter we demonstrate the ability to target endogenous receptors of hippocampal neurons and olfactory bulb neurons from Xenopus larvae. The chemical strategy used was to introduce a highly reactive anchoring group to a PTL with similar structure to the reported MAG compound used in the light regulated glutamate receptor, LiGluR. These new kind of PTLs were called “Targeted Covalent Photoswitches” (TCPs).
The promiscuity of the reactive group of TCP limits the possibility to spatially confine the drug conjugation. Thus, we designed a photolabile TCP that can be conjugated to its target with a specific wavelength, and then be photoswitched at another wavelength.
Despite the advantages of all molecules described above, they share a common limitation: the activation wavelength of azobenzene is in the UV-violet range. Increasing the push-pull effect at the azobenzenic core by adding different substituents, we achieved a TCP derivative activatable at the visible range and ultrafast relaxing. In this way a single wavelength of stimulation can be used. Both features make them great candidates to control ultrafast neurotransmission processes such as the ones implicated in sound coding in the cochlea. We demonstrated in a gerbil animal model the capacity of this blue-activated TCP to photosensitize native receptors of adult gerbil cochlea. This first proof of concept opens new possibilities to develop optical cochlear implants for the treatment of hearing loss avoiding gene therapy.
Chemical substitutions can also be rationally designed to enhance two-photon (2P) absorptivity without modifying the dynamics of photoswitches. In the third chapter we described two new 2P enhanced MAG derivatives to photocontrol LiGluR. We validated their properties with an all-optical approach, by recording calcium induced neuronal responses in organotypic hippocampal slices, and in vivo in Caenorhabditis elegans. The combination of slow relaxation lifetime and enhanced 2P absorptivity is translated into an accumulation of the active isomer of the photoswitch that successfully enhances its functional effect even at low illumination power.
Another way to spectrally modify the characteristics of a chromophore while avoiding mutation screening and new synthetic processes is by using light-harvesting fluorophores. Spectral overlapping of fluorophore emission and chromophore absorption broadens its activation spectrum. By means of chemical protection and deprotection, we orthogonally control the conjugation of fluorophores and photoswitches. This is the first demonstration of light-harvesting strategy for optogenetics and photopharmacology.
Finally, we describe the use of novel PCLs containing chromophores other than linear azobenzene for the light-controlled activation of hippocampal neurons. Cyclic azobenzenes reverse isomer thermal stability and consequently also the activity of the photoswitch. On the other hand, stilbenes allow the irreversible but non-photo-destructive activation of the original molecule, thus avoiding the creation of photo-products.
In conclusion, this thesis puts forward several advances in the photochromism and pharmacology of photoswitches for the control of neurotransmission without need of genetic manipulation.[cat] L'estudi electrofisiològic i d’imatge de l’activitat neuronal en temps real requereix una gran resoluciĂł espacial i temporal. AmbdĂłs trets es poden aconseguir amb la precisiĂł, velocitat i control remot que permet la llum. En aquesta tesi es descriu la caracteritzaciĂł de nous fotocommutadors dirigits a fotosensibilitzar receptors endògens de glutamat i estratègies per la millora de les seves caracterĂstiques òptiques.
Els primers fotoconmutadors descrits capaços de controlar amb llum l’activitat de canals endògens de glutamat son eficaços en altres aproximacions experimentals com ara en la inducciĂł amb llum de potencials d’acciĂł en neurones en cultiu, en cultius organotĂpics d’hipocamp de rata o in vivo en larves de Xenopus electroporades, a nivell d’un grup neuronal, d’una Ăşnica neurona o d’una Ăşnica espina sinĂ ptica. Modificant la part reactiva del fotocommutador, som podem controlar espacialment la conjugaciĂł mitjançant patrons d’il·luminaciĂł.
Canviant l’estructura quĂmica s’aconsegueix un fotocommutador actiu en el rang visible de llum i alhora extremadament rĂ pid. Fet que el fa ideal pel control de sinapsis ultra rĂ pides com ara les encarregades de codificar el so en la còclea. El disseny racional d’altres modificacions quĂmiques en fotocommutadors de receptors de glutamat (LiGluRs) obre les portes al disseny de molècules sensibles a l’activaciĂł per 2 fotons. L’estimulaciĂł de 2 fotons amb lĂ sers polsats de llum infraroja comporta importants avantatges com ara l’augment de la capacitat de penetraciĂł en teixit i una disminuciĂł de possibles danys degut a una sobreexposiciĂł a la llum. CaracterĂstiques molt Ăştils per estudis in vivo. Modificacions quĂmiques dels fotocommutadors comporten altres modificacions en les seves caracterĂstiques fotodinĂ miques. Per aquest motiu, hem desenvolupat una estratègia de conjugaciĂł de fluoròfors col·lectors de llum que mitjançant RET transfereixen la seva energia al cromòfor (fotocommutador), modificant-ne les caracterĂstiques espectrals.
Es demostra que cromòfors no conjugables permeten controlar l’activitat neuronal. Amb diversos avantatges respecte l’azobenzè clĂ ssic: els azobenzens cĂclics reverteix l’estabilitat i en conseqüència l’activitat de la molècula; i els estilbens permeten l’activaciĂł irreversible però no foto-destructiva de la molècula original, evitant aixĂ la creaciĂł de foto-productes.
En conclusió, en aquesta tesi es presenten noves molècules i estratègies de disseny per al control de la neurotransmissió sense necessitat de modificacions genètiques
Optical control of endogenous receptors and cellular excitability with light
Light-controlled manipulation of neuronal activity has transformed the field of neurobiology. Light provides temporal and spatial resolution over activation or inhibition of targeted populations of neurons, one single neuron or single synapses. Such precision could be achieved with optogenetics, which is based on the over-expression of light-sensitive proteins, but it requires gene therapy and often alters cellular physiology. An alternative to optogenetics is offered by photopharmacology (the development of light-regulated drugs or photoswitches), which can operate on endogenous receptors without genetic manipulation. Several new photoswitches are described in this thesis to improve their pharmacological and optical properties.
In photopharmacology, azobenzene is the most commonly used light switch. Photoswitchable tethered ligands (PTLs) are tri-modular molecules able to anchor to target receptors and alter their function by switching the azobenzene group. In the first chapter we demonstrate the ability to target endogenous receptors of hippocampal neurons and olfactory bulb neurons from Xenopus larvae. The chemical strategy used was to introduce a highly reactive anchoring group to a PTL with similar structure to the reported MAG compound used in the light regulated glutamate receptor, LiGluR. These new kind of PTLs were called “Targeted Covalent Photoswitches” (TCPs).
The promiscuity of the reactive group of TCP limits the possibility to spatially confine the drug conjugation. Thus, we designed a photolabile TCP that can be conjugated to its target with a specific wavelength, and then be photoswitched at another wavelength.
Despite the advantages of all molecules described above, they share a common limitation: the activation wavelength of azobenzene is in the UV-violet range. Increasing the push-pull effect at the azobenzenic core by adding different substituents, we achieved a TCP derivative activatable at the visible range and ultrafast relaxing. In this way a single wavelength of stimulation can be used. Both features make them great candidates to control ultrafast neurotransmission processes such as the ones implicated in sound coding in the cochlea. We demonstrated in a gerbil animal model the capacity of this blue-activated TCP to photosensitize native receptors of adult gerbil cochlea. This first proof of concept opens new possibilities to develop optical cochlear implants for the treatment of hearing loss avoiding gene therapy.
Chemical substitutions can also be rationally designed to enhance two-photon (2P) absorptivity without modifying the dynamics of photoswitches. In the third chapter we described two new 2P enhanced MAG derivatives to photocontrol LiGluR. We validated their properties with an all-optical approach, by recording calcium induced neuronal responses in organotypic hippocampal slices, and in vivo in Caenorhabditis elegans. The combination of slow relaxation lifetime and enhanced 2P absorptivity is translated into an accumulation of the active isomer of the photoswitch that successfully enhances its functional effect even at low illumination power.
Another way to spectrally modify the characteristics of a chromophore while avoiding mutation screening and new synthetic processes is by using light-harvesting fluorophores. Spectral overlapping of fluorophore emission and chromophore absorption broadens its activation spectrum. By means of chemical protection and deprotection, we orthogonally control the conjugation of fluorophores and photoswitches. This is the first demonstration of light-harvesting strategy for optogenetics and photopharmacology.
Finally, we describe the use of novel PCLs containing chromophores other than linear azobenzene for the light-controlled activation of hippocampal neurons. Cyclic azobenzenes reverse isomer thermal stability and consequently also the activity of the photoswitch. On the other hand, stilbenes allow the irreversible but non-photo-destructive activation of the original molecule, thus avoiding the creation of photo-products.
In conclusion, this thesis puts forward several advances in the photochromism and pharmacology of photoswitches for the control of neurotransmission without need of genetic manipulation.L'estudi electrofisiològic i d’imatge de l’activitat neuronal en temps real requereix una gran resoluciĂł espacial i temporal. AmbdĂłs trets es poden aconseguir amb la precisiĂł, velocitat i control remot que permet la llum. En aquesta tesi es descriu la caracteritzaciĂł de nous fotocommutadors dirigits a fotosensibilitzar receptors endògens de glutamat i estratègies per la millora de les seves caracterĂstiques òptiques.
Els primers fotoconmutadors descrits capaços de controlar amb llum l’activitat de canals endògens de glutamat son eficaços en altres aproximacions experimentals com ara en la inducciĂł amb llum de potencials d’acciĂł en neurones en cultiu, en cultius organotĂpics d’hipocamp de rata o in vivo en larves de Xenopus electroporades, a nivell d’un grup neuronal, d’una Ăşnica neurona o d’una Ăşnica espina sinĂ ptica. Modificant la part reactiva del fotocommutador, som podem controlar espacialment la conjugaciĂł mitjançant patrons d’il·luminaciĂł.
Canviant l’estructura quĂmica s’aconsegueix un fotocommutador actiu en el rang visible de llum i alhora extremadament rĂ pid. Fet que el fa ideal pel control de sinapsis ultra rĂ pides com ara les encarregades de codificar el so en la còclea. El disseny racional d’altres modificacions quĂmiques en fotocommutadors de receptors de glutamat (LiGluRs) obre les portes al disseny de molècules sensibles a l’activaciĂł per 2 fotons. L’estimulaciĂł de 2 fotons amb lĂ sers polsats de llum infraroja comporta importants avantatges com ara l’augment de la capacitat de penetraciĂł en teixit i una disminuciĂł de possibles danys degut a una sobreexposiciĂł a la llum. CaracterĂstiques molt Ăştils per estudis in vivo. Modificacions quĂmiques dels fotocommutadors comporten altres modificacions en les seves caracterĂstiques fotodinĂ miques. Per aquest motiu, hem desenvolupat una estratègia de conjugaciĂł de fluoròfors col·lectors de llum que mitjançant RET transfereixen la seva energia al cromòfor (fotocommutador), modificant-ne les caracterĂstiques espectrals.
Es demostra que cromòfors no conjugables permeten controlar l’activitat neuronal. Amb diversos avantatges respecte l’azobenzè clĂ ssic: els azobenzens cĂclics reverteix l’estabilitat i en conseqüència l’activitat de la molècula; i els estilbens permeten l’activaciĂł irreversible però no foto-destructiva de la molècula original, evitant aixĂ la creaciĂł de foto-productes.
En conclusió, en aquesta tesi es presenten noves molècules i estratègies de disseny per al control de la neurotransmissió sense necessitat de modificacions genètiques
Nanoengineered Light-Harvested Proteins for Optogenetics and Photopharmacology
Chemical modification with nanometer
precision can be used to probe and to improve the function of complex molecular
entities, from organic materials to proteins and their assemblies. Using the
pigment arrangement in photosynthetic light-harvesting as inspiration, we show
that molecular photosensitizers can be located at well-defined distances from
photoisomerizable units in proteins in order to enhance and spectrally shift their
photoresponses. The approach is demonstrated in Channelrhodopsin-2 (ChR2) and
in the light-gated ionotropic glutamate receptor (LiGluR), two archetypical
actuators in optogenetics and photopharmacology that have been used both for
fundamental and therapeutic purposes. These proof-of-concept experiments together
with theoretical simulations predict that the photosensitivity can be increased
several orders of magnitude using these means, thus providing a unique
methodology to boost the performance of current optogenetic and
photopharmacological toolboxes.</p
Control of Cardiac Function in vivo with a Light-Regulated Drug
Remote
control of physiological functions with light offers the promise of unveiling
their complex spatiotemporal dynamics in vivo, and enabling highly focalized
therapeutic interventions with reduced systemic toxicity. Optogenetic methods
have been implemented in the heart, but the need of genetic manipulation
jeopardizes clinical applicability. This study aims at developing, testing and
validating the first light-regulated drug with cardiac effects, in order to
avoid the requirement of genetic manipulation offered by optogenetic methods. A
M2 muscarinic acetylcholine receptors (mAChRs) light-regulated drug (PAI) was
designed, synthesized and pharmacologically characterized. The design was based
on the orthosteric mAChRs agonist Iperoxo, an allosteric M2 ligand, and a
photoswitchable azobenzene linker. PAI can be reversibly photoisomerized
between cis and trans configurations under ultraviolet (UV) and visible light,
respectively, and it reversibly photoswitches the activity of M2 muscarinic
acetylcholine receptors. We have evaluated in
vitro photoresponses using a calcium imaging assay in genetically
unmodified receptors overexpressed in mammalian cells. Furthermore, using this new
chemical tool, we demonstrate for the first time photoregulation of cardiac
function in vivo in wildtype frog
tadpoles and in rats with a method that does not require genetic manipulation.
Such a new approach may enable enhanced spatial and temporal selectivity for
cardiovascular drugs.</p
Synthetic Photoswitchable Neurotransmitters Based on Bridged Azobenzenes
Photoswitchable neurotransmitters of ionotropic kainate receptors were synthesized by tethering a glutamate moiety to disubstituted C2-bridged azobenzenes, which were prepared through a novel methodology that allows access to diazocines with higher yields and versatility. Because of the singular properties of these photochromes, photoisomerizable compounds were obtained with larger thermal stability for their inert cis isomer than for their biologically activity trans state. This enabled selective neuronal firing upon irradiation without background activity in the dark