Biology subjects

Wietek, J.

Publications and source records attributed to Wietek, J..

9 recordsLinked to original sources

bioRxiv2024

PinkyCaMP a mScarlet-based calcium sensor with exceptional brightness, photostability, and multiplexing capabilities

Genetically encoded calcium (Ca2+) indicators (GECIs) are widely used for imaging neuronal activity, yet current limitations of existing red fluorescent GECIs have constrained their applicability. The inherently dim fluorescence and low signal-to-noise ratio of red-shifted GECIs have posed significant challenges. More critically, several red-fluorescent GECIs exhibit photoswitching when exposed to blue light, thereby limiting their applicability in all-optical experimental approaches. Here, we present the development of PinkyCaMP, the first mScarlet-based Ca2+ sensor that outperforms current red fluorescent sensors in brightness, photostability, signal-to-noise ratio, and compatibility with optogenetics and neurotransmitter imaging. PinkyCaMP is well-tolerated by neurons, showing no toxicity or aggregation, both in vitro and in vivo. All imaging approaches, including single-photon excitation methods such as fiber photometry, widefield imaging, miniscope imaging, as well as two-photon imaging in awake mice, are fully compatible with PinkyCaMP.

neuroscience↗

bioRxiv2024

Oxytocin signaling regulates maternally-directed behavior during early life

Oxytocin is essential in shaping social behavior across the lifespan. While the role of oxytocin signaling in parental care has been widely investigated, little is known about its function in social behavior during early life. This is partly due to the lack of precise technologies for studying the developing brain. Here, we studied the role of oxytocin in pup social behavior under acute separation from and reunion with the mother. We show that the activity of oxytocin neurons was increased by acute maternal separation and returned to baseline after reunion. Behaviorally, maternally-separated pups emitted more ultrasonic vocalizations upon reunion, which were further modulated by nipple attachment behavior. These effects were attenuated by blocking the oxytocin receptor during maternal separation. To investigate the role of oxytocin neurons with higher precision, we established a method for transcranial optogenetic silencing of neuronal activity in untethered and freely behaving pups. Using this approach, we found that silencing of oxytocin neurons during maternal separation disrupted vocal behavior during separation and reunion in a sex-specific manner. Our findings reveal an important role of oxytocin in context-dependent vocal communication in pups, offering new insights into the mechanisms of social behavior during early life.

neuroscience↗

bioRxiv2023

A bistable inhibitory OptoGPCR for multiplexed optogenetic control of neural circuits

Information is transmitted between brain regions through the release of neurotransmitters from long-range projecting axons. Understanding how the activity of such long-range connections contributes to behavior requires efficient methods for reversibly manipulating their function. Chemogenetic and optogenetic tools, acting through endogenous G-protein coupled receptor (GPCRs) pathways, can be used to modulate synaptic transmission, but existing tools are limited in sensitivity, spatiotemporal precision, or spectral multiplexing capabilities. Here we systematically evaluated multiple bistable opsins for optogenetic applications and found that the Platynereis dumerilii ciliary opsin (PdCO) is an efficient, versatile, light-activated bistable GPCR that can suppress synaptic transmission in mammalian neurons with high temporal precision in-vivo. PdCO has superior biophysical properties that enable spectral multiplexing with other optogenetic actuators and reporters. We demonstrate that PdCO can be used to conduct reversible loss-of-function experiments in long-range projections of behaving animals, thereby enabling detailed synapse-specific functional circuit mapping.

neuroscience↗

bioRxiv2022

Temporally extended and reversible neuronal silencing with Aion

Optogenetic silencing allows to reveal the necessity of selected neuronal populations for various neurophysiological functions ranging from specific behaviors to neuronal network activity. An ideal optogenetic silencing tool should be switchable between active and inactive states with precise timing while preserving its activity in the absence of light until switched to an inactive state. Although bistable anion-conducting channelrhodopsins (ACRs) were previously engineered to reach this goal, their conducting state life time was limited to only a few minutes and some ACRs were not fully switchable. Here we report Aion, a bistable ACR displaying a long-lasting open state with a spontaneous closing time constant close to 15 min. Moreover, Aion can be switched between the open and closed state with millisecond precision using blue and orange light, respectively. The long conducting state enables overnight silencing of neurons with minimal light exposure. We further generated trafficking-optimized versions of Aion, which show enhanced membrane localization and allow precisely timed, long-lasting all-optical control of nociceptive responses in larvae of Drosophila melanogaster.

neuroscience↗

bioRxiv2021

Optogenetic silencing of neurotransmitter release with a naturally occurring invertebrate rhodopsin

Information is carried between brain regions through neurotransmitter release from axonal presynaptic terminals. Understanding the functional roles of defined neuronal projection pathways in cognitive and behavioral processes requires temporally precise manipulation of their activity in vivo. However, existing optogenetic tools have low efficacy and off-target effects when applied to presynaptic terminals, while chemogenetic tools are difficult to control in space and time. Here, we show that a targeting-enhanced mosquito homologue of the vertebrate encephalopsin (eOPN3) can effectively suppress synaptic transmission through the Gi/o signaling pathway. Brief illumination of presynaptic terminals expressing eOPN3 triggers a lasting suppression of synaptic output that recovers spontaneously within minutes in vitro as well as in vivo. In freely moving mice, eOPN3-mediated suppression of dopaminergic nigrostriatal afferents leads to an ipsiversive rotational bias. We conclude that eOPN3 can be used to selectively suppress neurotransmitter release at synaptic terminals with high spatiotemporal precision, opening new avenues for functional interrogation of long-range neuronal circuits in vivo.

neuroscience↗

bioRxiv2020

Lateral gene transfer of anion-conducting channelrhodopsins between green algae and giant viruses

Channelrhodopsins (ChRs) are algal light-gated ion channels widely used as optogenetic tools for manipulating neuronal activity 1,2. Four ChR families are currently known. Green algal 3-5 and cryptophyte 6 cation-conducting ChRs (CCRs), cryptophyte anion-conducting ChRs (ACRs) 7, and the MerMAID ChRs 8. Here we report the discovery of a new family of phylogenetically distinct ChRs encoded by marine giant viruses and acquired from their unicellular green algal prasinophyte hosts. These previously unknown viral and green algal ChRs act as ACRs when expressed in cultured neuroblastoma-derived cells and are likely involved in behavioral responses to light.

microbiology↗

bioRxiv2019

MerMAIDs: A novel family of metagenomically discovered, marine, anion-conducting and intensely desensitizing channelrhodopsins.

Channelrhodopsins (ChRs) are algal light-gated ion channels widely used as optogenetic tools for manipulating neuronal activity. ChRs desensitize under continuous bright-light illumination, resulting in a significant decline of photocurrents. We describe a novel, metagenomically identified family of phylogenetically distinct anion-conducting ChRs (designated MerMAIDs). MerMAIDs almost completely desensitize during continuous illumination due to accumulation of a late non-conducting photointermediate that disrupts the ion permeation pathway. MerMAID desensitization can be fully explained by a single photocycle in which a long-lived desensitized state follows the short-lived conducting state. A conserved cysteine is the critical factor in desensitization, as its mutation results in recovery of large stationary photocurrents. The rapid desensitization of MerMAIDs enables their use as optogenetic silencers for transient suppression of individual action potentials without affecting subsequent spiking during continuous illumination. Our results could facilitate the development of further novel optogenetic tools from metagenomic databases and enhance general understanding of ChR function.

biophysics↗

bioRxiv2018

A unifying photocycle model for light adaptation and temporal evolution of cation conductance in Channelrhodopsin-2

Although Channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light-adaptation, photocurrent inactivation, and alteration of the ion selectivity during continuous illumination are not well-understood from a molecular perspective. Herein, we address these open questions using single turn-over electrophysiology, time-resolved step-scan FTIR and Raman spectroscopy of fully dark adapted ChR2. This yields a unifying parallel photocycle model explaining all data: in dark-adapted ChR2, the protonated Schiff base retinal chromophore (RSBH+) adopts an all-trans,C=N-anti conformation only. Upon light activation, a branching reaction into either a 13-cis,C=N-anti or a 13-cis,C=N-syn retinal conformation occurs. The anti-cycle features sequential H+ and Na+ conductance in a late M-like state and an N-like open-channel state. In contrast, the 13-cis,C=N-syn isomer represents a second closed-channel state identical to the long lived P480-state, which has been previously assigned to a late intermediate in a single photocycle model. Light excitation of P480 induces a parallel syn-photocycle with an open channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P480 and stays deprotonated in the C=N-syn-cycle and we show that deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light-adaptation. Parallel anti- and syn-photocycles explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.\n\nSignificance statementUnderstanding the mechanisms of photoactivated biological processes facilitates the development of new molecular tools, engineered for specific optogenetic applications, allowing the control of neuronal activity with light. Here, we use a variety of experimental and theoretical techniques to examine the precise nature of the light-activated ion channel in one of the most important molecular species used in optogenetics, channelrhodopsin-2. Existing models for the photochemical and photophysical pathway after light absorption by the molecule fail to explain many aspects of its observed behavior including the inactivation of the photocurrent under continuous illumination. We resolve this by proposing a new branched photocycle explaining electrical and photochemical channel properties and establishing the structure of intermediates during channel turnover.

biophysics↗

bioRxiv2017

Artificial anion-conducting channelrhodopsins with tuned spectra, modified kinetics and enhanced light sensitivity

Genetic engineering of natural light-gated ion channels has proven a powerful way to generate optogenetic tools for a wide variety of applications. In recent years, blue light-activated engineered anion conducting channelrhodopsins (eACRs) have been developed, improved, and were successfully applied in vivo. We asked whether the approaches used to create eACRs can be transferred to other well-characterized cation-conducting channelrhodopsins (CCRs) to obtain eACRs with a broad spectrum of biophysical properties. We generated 22 variants using two conversion strategies applied to 11 CCRs and screened them for membrane expression, photocurrents and anion selectivity. We obtained two novel eACRs, Phobos and Aurora, with blue-and red-shifted action spectra and photocurrents similar to existing eACRs. Furthermore, step-function mutations greatly enhanced the cellular operational light sensitivity due to a slowed-down photocycle. These bistable eACRs can be reversibly toggled between open and closed states with brief light pulses of different wavelengths. All new eACRs reliably inhibited action potential firing in pyramidal CA1 neurons. In Drosophila larvae, eACRs conveyed robust and specific light-dependent inhibition of locomotion and nociception.

neuroscience↗