Single-photon optogenetics enables precise, cell-type-specific modulation of neuronal circuits, making it a crucial tool in neuroscience. Its miniaturization in the form of fully implantable wide-field stimulator arrays enables long-term interrogation of cortical circuits and bears promise for brain-machine interfaces for sensory and motor function restoration. However, achieving selective activation of functional cortical representations poses a challenge, as studies show that targeted optogenetic stimulation results in activity spread beyond one functional domain. While recurrent network mechanisms contribute to activity spread, here we demonstrate with detailed simulations of isolated pyramidal neurons from cats of unknown sex that already neuron morphology causes a complex spread of optogenetic activity at the scale of one cortical column. Since the shape of a neuron impacts its optogenetic response, we find that a single stimulator at the cortical surface recruits a complex spatial distribution of neurons that can be inhomogeneous and vary with stimulation intensity and neuronal morphology across layers. We explore strategies to enhance stimulation precision, finding that optimizing stimulator optics may offer more significant improvements than the preferentially somatic expression of the opsin through genetic targeting. Our results indicate that, with the right optical setup, single-photon optogenetics can precisely activate isolated neurons at the scale of functional cortical domains spanning several hundred micrometers.
- MeSH
- kočky MeSH
- modely neurologické MeSH
- mozková kůra fyziologie cytologie MeSH
- neurony fyziologie MeSH
- optogenetika * metody MeSH
- pyramidové buňky fyziologie MeSH
- světelná stimulace metody MeSH
- zvířata MeSH
- Check Tag
- kočky MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.
Schizophrenia research has increased in recent decades and focused more on its neural basis. Decision-making and cognitive flexibility are the main cognitive functions that are impaired and considered schizophrenia endophenotypes. Cognitive impairment was recently connected with altered functions of N-methyl-d-aspartate (NMDAR) glutamatergic receptors, which increased cortical activity. Selective NMDAR antagonists, such as MK-801, have been used to model cognitive inflexibility in schizophrenia. Decreased GABAergic inhibitory activity has been shown elsewhere with enhanced cortical activity. This imbalance in the excitatory/inhibitory may reduce the entrainment of prefrontal gamma and hippocampal theta rhythms and result in gamma/theta band de-synchronization. The current study established an acute MK-801 administration model of schizophrenia-like cognitive inflexibility in rats and used the attentional set-shifting task in which rats learned to switch/reverse the relevant rule. During the task, we used in vivo optogenetic stimulations of parvalbumin-positive interneurons at specific light pulses in the prefrontal cortex and ventral hippocampus. The first experiments showed that acute dizocilpine in rats produced schizophrenia-like cognitive inflexibility. The second set of experiments demonstrated that specific optogenetic stimulation at specific frequencies of parvalbumin-positive interneurons in the prefrontal cortex and ventral hippocampus rescued the cognitive flexibility rats that received acute MK-801. These findings advance our knowledge of the pivotal role of parvalbumin interneurons in schizophrenia-like cognitive impairment and may guide further research on this severe psychiatric disorder.
- MeSH
- dizocilpinmaleát * farmakologie MeSH
- hipokampus metabolismus MeSH
- interneurony metabolismus MeSH
- kognice MeSH
- krysa rodu rattus MeSH
- optogenetika MeSH
- parvalbuminy metabolismus MeSH
- prefrontální mozková kůra metabolismus MeSH
- receptory N-methyl-D-aspartátu metabolismus MeSH
- schizofrenie * MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- mozek * fyziologie MeSH
- myši MeSH
- neurony fyziologie MeSH
- optogenetika * metody MeSH
- savci MeSH
- Check Tag
- myši MeSH
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
Poruchy kognitivních funkcí jsou považovány za klíčový příznak schizofrenie a předpovídají terapeutický výsledek. Velmi významně narušují denní fungování pacientů se schizofrenií, a přesto dosud neexistuje cílená léčba kognitivního deficitu u schizofrenie. V tomto translačním projektu plánujeme objasnit kauzální roli hipokampálně-prefrontálních projekcí v kognitivní koordinace a flexibilitě. Rovněž ukážeme na příčinnou roli frontotemporální theta koherence a synchronie pomocí optogenetické kontroly aktivity PV+ interneuronů u volně pohyblivých potkanů. V klinické části otestujeme vliv frontotemporální synchronizace na kognitivní koordinaci a flexibilitu ve skupině 35 pacientů v remisi a u 35 zdravých kontrol s využitím hrEEG/fMRI měření a testů virtuální reality. Hlavním cílem je objasnění neurobiologického substrátu kognitivního deficitu u schizofrenie, které umožní inteligentní design nových léčebných postupů. Výsledky projektu jednoznačně ukážou složky frontotemporální dysfunkce u schizofrenie a otevřou cestu pro budoucí specifickou terapii kognitivního deficitu.; Disturbances of cognitive functions have been recognized as hallmarks of schizophrenia and predictors of therapeutic outcome. They significantly limit patient ́s functioning, yet there are no specific treatments for cognitive deficits in this disease. In this translational project, we seek to determine the causal role of hippocampal-prefrontal projections in cognitive coordination and flexibility. Moreover, causative role of frontotemporal theta coherence and synchrony will be revealed by controlling PV+ interneuron activity in freely-moving rats. The human part will test relations of frontotemporal synchrony to coordination and flexibility in 35 remitted schizophrenia patients and 35 matched healthy controls using a hrEEG/fMRI measurements and tests of the virtual reality. The overall aim is to elucidate a neuronal substrate for cognitive deficits in schizophrenia for an intelligent design of new treatments. Results of this project will unequivocally show the constituents of frontotemporal dysfunction in schizophrenia and open way for future treatment of cognitive deficits.
- MeSH
- elektroencefalografie MeSH
- hipokampus MeSH
- kognitivní dysfunkce etiologie patofyziologie MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- magnetická rezonanční tomografie MeSH
- modely nemocí na zvířatech MeSH
- nervový přenos MeSH
- neurozobrazování MeSH
- optogenetika MeSH
- prefrontální mozková kůra MeSH
- schizofrenie diagnóza MeSH
- translační biomedicínský výzkum MeSH
- virtuální realita MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- neurologie
- psychiatrie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
Schizophrenia research arose in the twentieth century and is currently rapidly developing, focusing on many parallel research pathways and evaluating various concepts of disease etiology. Today, we have relatively good knowledge about the generation of positive and negative symptoms in patients with schizophrenia. However, the neural basis and pathophysiology of schizophrenia, especially cognitive symptoms, are still poorly understood. Finding new methods to uncover the physiological basis of the mental inabilities related to schizophrenia is an urgent task for modern neuroscience because of the lack of specific therapies for cognitive deficits in the disease. Researchers have begun investigating functional crosstalk between NMDARs and GABAergic neurons associated with schizophrenia at different resolutions. In another direction, the gut microbiota is getting increasing interest from neuroscientists. Recent findings have highlighted the role of a gut-brain axis, with the gut microbiota playing a crucial role in several psychopathologies, including schizophrenia and autism.There have also been investigations into potential therapies aimed at normalizing altered microbiota signaling to the enteric nervous system (ENS) and the central nervous system (CNS). Probiotics diets and fecal microbiota transplantation (FMT) are currently the most common therapies. Interestingly, in rodent models of binge feeding, optogenetic applications have been shown to affect gut colony sensitivity, thus increasing colonic transit. Here, we review recent findings on the gut microbiota-schizophrenia relationship using in vivo optogenetics. Moreover, we evaluate if manipulating actors in either the brain or the gut might improve potential treatment research. Such research and techniques will increase our knowledge of how the gut microbiota can manipulate GABA production, and therefore accompany changes in CNS GABAergic activity.
- MeSH
- lidé MeSH
- mozek MeSH
- optogenetika MeSH
- osa mozek-střevo MeSH
- probiotika * MeSH
- schizofrenie * terapie MeSH
- střevní mikroflóra * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of ζ-chain in a light controlled manner. We showed that spatial clustering of the ζ-chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of ζ-chain, Zap70, PLCγ, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate ζ-chain phosphorylation upon ζ-chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed ζ-chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.
- MeSH
- aminokyselinové motivy MeSH
- buněčná membrána metabolismus MeSH
- Cercopithecus aethiops MeSH
- COS buňky MeSH
- cytosol metabolismus MeSH
- difuze MeSH
- fluorescenční spektrometrie MeSH
- fosforylace MeSH
- Jurkat buňky MeSH
- lidé MeSH
- optogenetika MeSH
- receptory antigenů T-buněk chemie metabolismus MeSH
- shluková analýza MeSH
- signální transdukce * MeSH
- světlo MeSH
- tyrosinkinasa p56(lck), specifická pro lymfocyty metabolismus MeSH
- vápník metabolismus MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Cíl: Práce seznamuje čtenáře s inovativními postupy v léčbě sítnicových one-mocnění, které by se mohly v následujících letech dostat do klinické praxe. Po-psány jsou retinální protézy, transplantace retinálního pigmentového epitelu (RPE), genová terapie a optogenetika.Metodika: Literární rešerše zaměřená na charakteristiky a mechanismy jednotlivých druhů terapií. Výsledky: Retinální protézy, transplantace RPE, genová terapie a optogenetika nabízejí dosud plně neprobádané možnosti a jsou považovány za budoucnost léčby sítnicových onemocnění tam, kde klasická farmakoterapie či chirurgické možnosti léčby nestačí. Nicméně všechny tyto metody jsou výzvou nejen pro samotné inovativní technické provedení, ale také pro etickou, administrativní a ekonomickou náročnost. Závěr: V léčbě sítnicových onemocnění nás čeká zajímavá budoucnost a nelze nyní odhadnout, která modalita léčby bude dominantní.
Objective: The aim of this comprehensive paper is to acquaint the readers with innovative approaches in the treatment of retinal diseases, which could in the coming years to get into clinical practice. Retinal prostheses, retinal pigment epithelial (RPE) transplantation, gene therapy and optogenetics will be described in this paper. Methodology: Describing the basic characteristics and mechanisms of different types of therapy and subsequently literary minireview clarifying the current state of knowledge in the area. Results: Retinal prostheses, RPE transplantation, gene therapy and optogenetics offer yet unexplored possibilities and are considered as the future of treatment of retinal diseases where classical pharmacotherapy or surgical treatment are no longer sufficient. However, all these methods challenge not only in the innovative technical implementation itself, but also for the ethical, administrative and economic demands. Conclusion: There will be certainly interesting development in the treatment of retinal diseases, but it is not possible to fully estimate which modality of treatment will be dominant in the future.
- Klíčová slova
- retinální protézy,
- MeSH
- buněčná a tkáňová terapie metody trendy MeSH
- experimentální implantáty MeSH
- genetická terapie metody trendy MeSH
- genetické vektory MeSH
- indukované pluripotentní kmenové buňky MeSH
- klinická studie jako téma MeSH
- lidé MeSH
- nemoci retiny * terapie MeSH
- oftalmologické chirurgické výkony metody MeSH
- optogenetika metody trendy MeSH
- retinální pigmentový epitel MeSH
- transplantace kmenových buněk metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
- MeSH
- lidé MeSH
- management bolesti MeSH
- mapování mozku * MeSH
- mozek * MeSH
- neurony MeSH
- optické zobrazování MeSH
- optogenetika MeSH
- výzkum MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- novinové články MeSH
Generation of an electrochemical proton gradient is the first step of cell bioenergetics. In prokaryotes, the gradient is created by outward membrane protein proton pumps. Inward plasma membrane native proton pumps are yet unknown. We describe comprehensive functional studies of the representatives of the yet noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins. They are inward proton pumps as we demonstrate in model membrane systems, Escherichia coli cells, human embryonic kidney cells, neuroblastoma cells, and rat hippocampal neuronal cells. We also solved the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina (NsXeR) and suggest a mechanism of inward proton pumping. We demonstrate that the NsXeR is a powerful pump, which is able to elicit action potentials in rat hippocampal neuronal cells up to their maximal intrinsic firing frequency. Hence, inwardly directed proton pumps are suitable for light-induced remote control of neurons, and they are an alternative to the well-known cation-selective channelrhodopsins.
- MeSH
- Archaea metabolismus MeSH
- buněčné linie MeSH
- Escherichia coli metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- konformace proteinů MeSH
- lidé MeSH
- liposomy MeSH
- molekulární modely MeSH
- optogenetika * metody MeSH
- protonové pumpy metabolismus MeSH
- protony MeSH
- retina metabolismus MeSH
- rodopsin chemie metabolismus MeSH
- spektrální analýza MeSH
- světlo MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
