Two-photon neuronal and astrocytic stimulation with azobenzene-based photoswitches

This is an open access article published under an ACS AuthorChoice License. See Standard ACS AuthorChoice/Editors' Choice Usage Agreement - https://pubs.acs.org/page/policy/authorchoice_termsofuse.htmlSynthetic photochromic compounds can be designed to control a variety of proteins and their biochemical functions in living cells, but the high spatiotemporal precision and tissue penetration of two-photon stimulation have never been investigated in these molecules. Here we demonstrate two-photon excitation of azobenzene-based protein switches and versatile strategies to enhance their photochemical responses. This enables new applications to control the activation of neurons and astrocytes with cellular and subcellular resolution

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

Effectiveness of an intervention for improving drug prescription in primary care patients with multimorbidity and polypharmacy:Study protocol of a cluster randomized clinical trial (Multi-PAP project)

This study was funded by the Fondo de Investigaciones Sanitarias ISCIII (Grant Numbers PI15/00276, PI15/00572, PI15/00996), REDISSEC (Project Numbers RD12/0001/0012, RD16/0001/0005), and the European Regional Development Fund ("A way to build Europe").Background: Multimorbidity is associated with negative effects both on people's health and on healthcare systems. A key problem linked to multimorbidity is polypharmacy, which in turn is associated with increased risk of partly preventable adverse effects, including mortality. The Ariadne principles describe a model of care based on a thorough assessment of diseases, treatments (and potential interactions), clinical status, context and preferences of patients with multimorbidity, with the aim of prioritizing and sharing realistic treatment goals that guide an individualized management. The aim of this study is to evaluate the effectiveness of a complex intervention that implements the Ariadne principles in a population of young-old patients with multimorbidity and polypharmacy. The intervention seeks to improve the appropriateness of prescribing in primary care (PC), as measured by the medication appropriateness index (MAI) score at 6 and 12months, as compared with usual care. Methods/Design: Design:pragmatic cluster randomized clinical trial. Unit of randomization: family physician (FP). Unit of analysis: patient. Scope: PC health centres in three autonomous communities: Aragon, Madrid, and Andalusia (Spain). Population: patients aged 65-74years with multimorbidity (≥3 chronic diseases) and polypharmacy (≥5 drugs prescribed in ≥3months). Sample size: n=400 (200 per study arm). Intervention: complex intervention based on the implementation of the Ariadne principles with two components: (1) FP training and (2) FP-patient interview. Outcomes: MAI score, health services use, quality of life (Euroqol 5D-5L), pharmacotherapy and adherence to treatment (Morisky-Green, Haynes-Sackett), and clinical and socio-demographic variables. Statistical analysis: primary outcome is the difference in MAI score between T0 and T1 and corresponding 95% confidence interval. Adjustment for confounding factors will be performed by multilevel analysis. All analyses will be carried out in accordance with the intention-to-treat principle. Discussion: It is essential to provide evidence concerning interventions on PC patients with polypharmacy and multimorbidity, conducted in the context of routine clinical practice, and involving young-old patients with significant potential for preventing negative health outcomes. Trial registration: Clinicaltrials.gov, NCT02866799Publisher PDFPeer reviewe

New azobenzene-based photoswitches for two-photon optical control of neuronal receptors

Aquest projecte persegueix el desenvolupament d’una sèrie de nous interruptors moleculars per a controlar el funcionament d’un canal iònic present en les cèl·lules del sistema nerviós central. Fins a l’actualitat els interruptors desenvolupats d’aquest tipus es basen en sistemes azobenzènics, fet que fa que presentin una limitació fonamental: la radiació UV-vis necessària per a interconvertir els dos estats del sistema és poc penetrant en teixits biològics i pot, fins i tot, provocar processos de fotodegradació d’aquests teixits. Aquest inconvenient es podria solucionar utilitzant llum del infraroig proper (NIR) per a dur a terme la fotoisomerització del interruptor mitjançant absorció multifotònica (a dos fotons). Per tal d’assolir aquest objectiu, es van plantejar dues estratègies: (i) la photoisomerització sensibilitzada a dos fotons del nucli azobenzènic; i (ii) la directa photoisomerització a dos fotons del nucli azobenzènic. La primera aproximació es podria aconseguir afegint una quarta unitat activa a l’estructura de l’interruptor original descrit per Gorostiza et al. Aquest nou fragment actuaria com a sensibilitzador, absorbint llum NIR mitjançant un procés multifotònic i transferint la corresponent energia d’excitació electrònica mitjançant un mecanisme de transferència d’energia ressonant (RET) a la unitat d’azobenzè. Per contra, la segona estratègia requeriria la introducció d’asimetria electrònica entre els dos anells, per tal de millorar la capacitat del sistema d’absorbir a dos fotons. Per validar aquestes dues aproximacions, dues families d’interruptors moleculars es van preparar i el seu comportament fotoquímic va ser evaluat tant en solució com en teixits biològics.The development of optical methods for remote and non-invasive control of biological functions has recently emerged as a promising area of research. To attain this goal, it has been proposed the design of molecular photoswitches capable of regulating cellular activity upon reversible light-induced interconversion between their two states. Gorostiza et al. have demonstrated that photoinduced trans-cis isomerisation of azobenzene derivatives using UV-vis light allows the ion channels in neurons to be gated on demand. In spite of their successful performance, operation of these systems under irradiation with NIR light would be highly desired, since it has more penetration depth in biological tissues as well as causes less biological damage than UV-vis light. In view of this situation, this project pursues the development of a new series of photoswitches allowing light-induced control with IR light of the neuronal ion channels regulated by ionotropic glutamate receptors (iGluR). With this aim, two different strategies have been explored: (i) two-photon sensitised photoisomerisation of their azobenzene switch; and (ii) direct two-photon photoisomerisation of their azobenzene switch. On the former, an additional unit has been incorporated to the original photoswitch structure reported by Gorostiza et al. This new fragment should act as a sensitiser, absorbing NIR light via a non-linear optical process and then transferring its electronic excitation energy to the azobenzene group, which should eventually undergo photoisomerisation. On the other hand, the second strategy requires push-pull substitution of the azoaromatic group to enhance the intrinsic two-photon absorption cross-section of the system. To validate these approaches, two types of molecular photoswitches have been prepared and their photochemical activities have been tested both in solution and in biological tissues

Síntesi i caracterització de compostos azobenzènics per la preparació de nous interruptors moleculars

Aquest treball de recerca té com a objectiu la síntesi i caracterització de nous interruptors moleculars basats en cromòfors azobenzènics i amb aplicació a nivell biològic. Més concretament, es planteja el desenvolupament d’interruptors moleculars basats en el sistema MAG per a controlar el funcionament d’un canal iònic de les cèl·lules del sistema nerviós central governat pel receptor iGluR. Tot i que ja s’han descrit interruptors moleculars tipus MAG que permeten el control d’aquest canal, en aquest treball es pretenen optimitzar dues de les seves propietats: augmentar el rendiment quàntic de fotoisomerització entre els estats trans i cis de l’interruptor i augmentar la constant tèrmica d’isomerització cis-&trans. Aquestes dues propietats depenen de les característiques estructurals del cromòfor azobenzènic central del sistema MAG

New azobenzene-based photoswitches for two-photon optical control of neuronal receptors

Aquest projecte persegueix el desenvolupament d'una sèrie de nous interruptors moleculars per a controlar el funcionament d'un canal iònic present en les cèl·lules del sistema nerviós central. Fins a l'actualitat els interruptors desenvolupats d'aquest tipus es basen en sistemes azobenzènics, fet que fa que presentin una limitació fonamental: la radiació UV-vis necessària per a interconvertir els dos estats del sistema és poc penetrant en teixits biològics i pot, fins i tot, provocar processos de fotodegradació d'aquests teixits. Aquest inconvenient es podria solucionar utilitzant llum del infraroig proper (NIR) per a dur a terme la fotoisomerització del interruptor mitjançant absorció multifotònica (a dos fotons). Per tal d'assolir aquest objectiu, es van plantejar dues estratègies: (i) la photoisomerització sensibilitzada a dos fotons del nucli azobenzènic; i (ii) la directa photoisomerització a dos fotons del nucli azobenzènic. La primera aproximació es podria aconseguir afegint una quarta unitat activa a l'estructura de l'interruptor original descrit per Gorostiza et al. Aquest nou fragment actuaria com a sensibilitzador, absorbint llum NIR mitjançant un procés multifotònic i transferint la corresponent energia d'excitació electrònica mitjançant un mecanisme de transferència d'energia ressonant (RET) a la unitat d'azobenzè. Per contra, la segona estratègia requeriria la introducció d'asimetria electrònica entre els dos anells, per tal de millorar la capacitat del sistema d'absorbir a dos fotons. Per validar aquestes dues aproximacions, dues families d'interruptors moleculars es van preparar i el seu comportament fotoquímic va ser evaluat tant en solució com en teixits biològics.The development of optical methods for remote and non-invasive control of biological functions has recently emerged as a promising area of research. To attain this goal, it has been proposed the design of molecular photoswitches capable of regulating cellular activity upon reversible light-induced interconversion between their two states. Gorostiza et al. have demonstrated that photoinduced trans-cis isomerisation of azobenzene derivatives using UV-vis light allows the ion channels in neurons to be gated on demand. In spite of their successful performance, operation of these systems under irradiation with NIR light would be highly desired, since it has more penetration depth in biological tissues as well as causes less biological damage than UV-vis light. In view of this situation, this project pursues the development of a new series of photoswitches allowing light-induced control with IR light of the neuronal ion channels regulated by ionotropic glutamate receptors (iGluR). With this aim, two different strategies have been explored: (i) two-photon sensitised photoisomerisation of their azobenzene switch; and (ii) direct two-photon photoisomerisation of their azobenzene switch. On the former, an additional unit has been incorporated to the original photoswitch structure reported by Gorostiza et al. This new fragment should act as a sensitiser, absorbing NIR light via a non-linear optical process and then transferring its electronic excitation energy to the azobenzene group, which should eventually undergo photoisomerisation. On the other hand, the second strategy requires push-pull substitution of the azoaromatic group to enhance the intrinsic two-photon absorption cross-section of the system. To validate these approaches, two types of molecular photoswitches have been prepared and their photochemical activities have been tested both in solution and in biological tissues

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

Two-photon neuronal and astrocytic stimulation with azobenzene-based photoswitches

This is an open access article published under an ACS AuthorChoice License. See Standard ACS AuthorChoice/Editors' Choice Usage Agreement - https://pubs.acs.org/page/policy/authorchoice_termsofuse.htmlSynthetic photochromic compounds can be designed to control a variety of proteins and their biochemical functions in living cells, but the high spatiotemporal precision and tissue penetration of two-photon stimulation have never been investigated in these molecules. Here we demonstrate two-photon excitation of azobenzene-based protein switches and versatile strategies to enhance their photochemical responses. This enables new applications to control the activation of neurons and astrocytes with cellular and subcellular resolution