Imperial College London

Professor Thomas KNÖPFEL

Faculty of MedicineDepartment of Brain Sciences

Chair in Optogenetics and Circuit Neurosciences
 
 
 
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Contact

 

+44 (0)20 7594 6898t.knopfel Website

 
 
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Location

 

507Burlington DanesHammersmith Campus

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Summary

 

Publications

Publication Type
Year
to

316 results found

Monakhov MV, Matlashov ME, Colavita M, Song C, Shcherbakova DM, Antic SD, Verkhusha VV, Knöpfel Tet al., 2020, Screening and Cellular Characterization of Genetically Encoded Voltage Indicators Based on Near-Infrared Fluorescent Proteins., ACS Chem Neurosci

We developed genetically encoded voltage indicators using a transmembrane voltage-sensing domain and bright near-infrared fluorescent proteins derived from bacterial phytochromes. These new voltage indicators are excited by 640 nm light and emission is measured at 670 nm, allowing imaging in the near-infrared tissue transparency window. The spectral properties of our new indicators permit seamless voltage imaging with simultaneous blue-green light optogenetic actuator activation as well as simultaneous voltage-calcium imaging when paired with green calcium indicators. Iterative optimizations led to a fluorescent probe, here termed nirButterfly, which reliably reports neuronal activities including subthreshold membrane potential depolarization and hyperpolarization as well as spontaneous spiking or electrically- and optogenetically evoked action potentials. This enables largely improved all-optical causal interrogations of physiology.

Journal article

Quicke P, Howe CL, Song P, Jadan HV, Song C, Knöpfel T, Neil M, Dragotti PL, Schultz SR, Foust AJet al., 2020, Subcellular resolution three-dimensional light-field imaging with genetically encoded voltage indicators, Neurophotonics, Vol: 7, ISSN: 2329-4248

Significance: Light-field microscopy (LFM) enables high signal-to-noise ratio (SNR) and light efficient volume imaging at fast frame rates. Voltage imaging with genetically encoded voltage indicators (GEVIs) stands to particularly benefit from LFM's volumetric imaging capability due to high required sampling rates and limited probe brightness and functional sensitivity. Aim: We demonstrate subcellular resolution GEVI light-field imaging in acute mouse brain slices resolving dendritic voltage signals in three spatial dimensions. Approach: We imaged action potential-induced fluorescence transients in mouse brain slices sparsely expressing the GEVI VSFP-Butterfly 1.2 in wide-field microscopy (WFM) and LFM modes. We compared functional signal SNR and localization between different LFM reconstruction approaches and between LFM and WFM. Results: LFM enabled three-dimensional (3-D) localization of action potential-induced fluorescence transients in neuronal somata and dendrites. Nonregularized deconvolution decreased SNR with increased iteration number compared to synthetic refocusing but increased axial and lateral signal localization. SNR was unaffected for LFM compared to WFM. Conclusions: LFM enables 3-D localization of fluorescence transients, therefore eliminating the need for structures to lie in a single focal plane. These results demonstrate LFM's potential for studying dendritic integration and action potential propagation in three spatial dimensions.

Journal article

Quicke P, Howe CL, Song P, Jadan HV, Song C, Knöpfel T, Neil M, Dragotti PL, Schultz SR, Foust AJet al., 2020, Subcellular resolution 3D light field imaging with genetically encoded voltage indicators, Neurophotonics, Vol: 7, ISSN: 2329-4248

Significance: Light-field microscopy (LFM) enables high signal-to-noise ratio (SNR) and light efficient volume imaging at fast frame rates. Voltage imaging with genetically encoded voltage indicators (GEVIs) stands to particularly benefit from LFM’s volumetric imaging capability due to high required sampling rates and limited probe brightness and functional sensitivity.Aim: We demonstrate subcellular resolution GEVI light-field imaging in acute mouse brain slices resolving dendritic voltage signals in three spatial dimensions.Approach: We imaged action potential-induced fluorescence transients in mouse brain slices sparsely expressing the GEVI VSFP-Butterfly 1.2 in wide-field microscopy (WFM) and LFM modes. We compared functional signal SNR and localization between different LFM reconstruction approaches and between LFM and WFM.Results: LFM enabled three-dimensional (3-D) localization of action potential-induced fluorescence transients in neuronal somata and dendrites. Nonregularized deconvolution decreased SNR with increased iteration number compared to synthetic refocusing but increased axial and lateral signal localization. SNR was unaffected for LFM compared to WFM.Conclusions: LFM enables 3-D localization of fluorescence transients, therefore eliminating the need for structures to lie in a single focal plane. These results demonstrate LFM’s potential for studying dendritic integration and action potential propagation in three spatial dimensions.

Journal article

Ying L, Tahirbegi B, Magness A, Piersimoni M, Knopfel T, Willison K, Klug Det al., 2020, A novel Aβ40 assembly at physiological concentration, Scientific Reports, Vol: 10, ISSN: 2045-2322

Aggregates of amyloid-β (Aβ) are characteristic of Alzheimer’s disease, but there is no consensus as to either the nature of the toxic molecular complex or the mechanism by which toxic aggregates are produced. We report on a novel feature of amyloid-lipid interactions where discontinuities in the lipid continuum can serve as catalytic centers for a previously unseen microscale aggregation phenomenon. We show that specific lipid membrane conditions rapidly produce long contours of lipid-bound peptide, even at sub-physiological concentrations of Aβ. Using single molecule fluorescence, time-lapse TIRF microscopy and AFM imaging we characterize this phenomenon and identify some exceptional properties of the aggregation pathway which make it a likely contributor to early oligomer and fibril formation, and thus a potential critical mechanism in the etiology of AD. We infer that these amyloidogenic events occur only at areas of high membrane curvature, which suggests a range of possible mechanisms by which accumulated physiological changes may lead to their inception. The speed of the formation is in hours to days, even at 1 nM peptide concentrations. Lipid features of this type may act like an assembly line for monomeric and small oligomeric subunits of Aβ to increase their aggregation states. We conclude that under lipid environmental conditions, where catalytic centers of the observed type are common, key pathological features of AD may arise on a very short timescale under physiological concentration.

Journal article

Buchborn T, Lyons T, Song C, Feilding A, Knöpfel Tet al., 2020, The serotonin 2A receptor agonist 25CN-NBOH increases murine heart rate and neck-arterial blood flow in a temperature-dependent manner., Journal of Psychopharmacology, Vol: 34, Pages: 786-794, ISSN: 0269-8811

BACKGROUND: Serotonin 2A receptors, the molecular target of psychedelics, are expressed by neuronal and vascular cells, both of which might contribute to brain haemodynamic characteristics for the psychedelic state. AIM: Aiming for a systemic understanding of psychedelic vasoactivity, here we investigated the effect of N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine - a new-generation agonist with superior serotonin 2A receptor selectivity - on brain-supplying neck-arterial blood flow. METHODS: We recorded core body temperature and employed non-invasive, collar-sensor based pulse oximetry in anesthetised mice to extract parameters of local blood perfusion, oxygen saturation, heart and respiration rate. Hypothesising an overlap between serotonergic pulse- and thermoregulation, recordings were done under physiological and elevated pad temperatures. RESULTS: N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine (1.5 mg/kg, subcutaneous) significantly increased the frequency of heart beats accompanied by a slight elevation of neck-arterial blood flow. Increasing the animal-supporting heat-pad temperature from 37°C to 41°C enhanced the drug's effect on blood flow while counteracting tachycardia. Additionally, N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine promoted bradypnea, which, like tachycardia, quickly reversed at the elevated pad temperature. The interrelatedness of N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine's respiro-cardiovascular effects and thermoregulation was further corroborated by the drug selectively increasing the core body temperature at the elevated pad temperature. Arterial oxygen saturation was not affected by N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine at either temperature. CONCLUSIONS: Our findings imply that selective serotonin 2A receptor activation modulates systemic cardiovascular functioning in orchestration with thermoregulation and with immediate relevance to brain-imminent

Journal article

Whiteley I, Song C, Knöpfel T, Rowlands CJet al., 2020, Optical readout of voltage indicators using an improved targeted direct patterning concept.

© OSA 2020 © 2020 The Author(s) An improved method for the optical readout of voltage indicators is presented, whereby fluorescence from the whole surface of a neuron is integrated onto a single detector, dramatically increasing the recording bandwidth over conventional techniques.

Conference paper

Knopfel T, Sweeney Y, Radulescu CI, Zabouri N, Doostdar N, Clopath C, Barnes Set al., 2019, Audio-visual experience strengthens multisensory assemblies in adult mouse visual cortex, Nature Communications, Vol: 10, ISSN: 2041-1723

We experience the world through multiple senses simultaneously. To better understand mechanisms of multisensory processing we ask whether inputs from two senses (auditory and visual) can interact and drive plasticity in neural-circuits of the primary visual cortex (V1). Using genetically-encoded voltage and calcium indicators, we find coincident audio-visual experience modifies both the supra and subthreshold response properties of neurons in L2/3 of mouse V1. Specifically, we find that after audio-visual pairing, a subset of multimodal neurons develops enhanced auditory responses to the paired auditory stimulus. This cross-modal plasticity persists over days and is reflected in the strengthening of small functional networks of L2/3 neurons. We find V1 processes coincident auditory and visual events by strengthening functional associations between feature specific assemblies of multimodal neurons during bouts of sensory driven co-activity, leaving a trace of multisensory experience in the cortical network.

Journal article

Knöpfel T, Song C, 2019, Optical voltage imaging in neurons: moving from technology development to practical tool, Nature Reviews Neuroscience, Vol: 20, Pages: 719-727, ISSN: 1471-0048

A central goal in neuroscience is to determine how the brain’s neuronal circuits generate perception, cognition and emotions and how these lead to appropriate behavioural actions. A methodological platform based on genetically encoded voltage indicators (GEVIs) that enables the monitoring of large-scale circuit dynamics has brought us closer to this ambitious goal. This Review provides an update on the current state of the art and the prospects of emerging optical GEVI imaging technologies.

Journal article

Mollinedo-Gajate I, Song C, Knöpfel T, 2019, Genetically encoded fluorescent calcium and voltage indicators, Handbook of Experimental Pharmacology, Publisher: Springer Verlag

Fluorescent probes that indicate biologically important quantities are widely used for many different types of biological experiments across life sciences. During recent years, limitations of small molecule-based indicators have been overcome by the development of genetically encoded indicators. Here we focus on fluorescent calcium and voltage indicators and point to their applications mainly in neurosciences.

Book chapter

Liu M, Song C, Liang Y, Knöpfel T, Zhou Cet al., 2019, Assessing spatiotemporal variability of brain spontaneous activity by multiscale entropy and functional connectivity, NeuroImage, Vol: 198, Pages: 198-220, ISSN: 1053-8119

Brain signaling occurs across a wide range of spatial and temporal scales, and analysis of brain signal variability and synchrony has attracted recent attention as markers of intelligence, cognitive states, and brain disorders. However, current technologies to measure brain signals in humans have limited resolutions either in space or in time and cannot fully capture spatiotemporal variability, leaving it untested whether temporal variability and spatiotemporal synchrony are valid and reliable proxy of spatiotemporal variability in vivo. Here we used optical voltage imaging in mice under anesthesia and wakefulness to monitor cortical voltage activity at both high spatial and temporal resolutions to investigate functional connectivity (FC, a measure of spatiotemporal synchronization), Multi-Scale Entropy (MSE, a measure of temporal variability), and their relationships to Regional Entropy (RE, a measure of spatiotemporal variability). We observed that across cortical space, MSE pattern can largely explain RE pattern at small and large temporal scales with high positive and negative correlation respectively, while FC pattern strongly negatively associated with RE pattern. The time course of FC and small scale MSE tightly followed that of RE, while large scale MSE was more loosely coupled to RE. fMRI and EEG data simulated by reducing spatiotemporal resolution of the voltage imaging data or considering hemodynamics yielded MSE and FC measures that still contained information about RE based on the high resolution voltage imaging data. This suggested that MSE and FC could still be effective measures to capture spatiotemporal variability under limitation of imaging modalities applicable to human subjects. Our results support the notion that FC and MSE are effective biomarkers for brain states, and provide a promising viewpoint to unify these two principal domains in human brain data analysis.

Journal article

Quicke P, Song C, McKimm EJ, Milosevic MM, Howe CL, Neil M, Schultz SR, Antic SD, Foust AJ, Knopfel Tet al., 2019, Corrigendum: Single-neuron level one-photon voltage imaging with sparsely targeted genetically encoded voltage indicators, Frontiers in Cellular Neuroscience, Vol: 13, ISSN: 1662-5102

Voltage imaging of many neurons simultaneously at single-cell resolution is hampered bythe difficulty of detecting small voltage signals from overlapping neuronal processes inneural tissue. Recent advances in genetically encoded voltage indicator (GEVI) imaginghave shown single-cell resolution optical voltage recordings in intact tissue throughimaging naturally sparse cell classes, sparse viral expression, soma restricted expression,advanced optical systems, or a combination of these. Widespread sparse and strongtransgenic GEVI expression would enable straightforward optical access to a denselyoccurring cell type, such as cortical pyramidal cells. Here we demonstrate that a recentlydescribed sparse transgenic expression strategy can enable single-cell resolution voltageimaging of cortical pyramidal cells in intact brain tissue without restricting expression tothe soma. We also quantify the functional crosstalk in brain tissue and discuss optimalimaging rates to inform future GEVI experimental design.

Journal article

Agrawal V, Chakraborty S, Knöpfel T, Shew WLet al., 2019, Scale-change symmetry in the rules governing neural systems, iScience, Vol: 12, Pages: 121-131, ISSN: 2589-0042

Similar universal phenomena can emerge in different complex systems when those systems share a common symmetry in their governing laws. In physical systems operating near a critical phase transition, the governing physical laws obey a fractal symmetry; they are the same whether considered at fine or coarse scales. This scale-change symmetry is responsible for universal critical phenomena found across diverse systems. Experiments suggest that the cerebral cortex can also operate near a critical phase transition. Thus we hypothesize that the laws governing cortical dynamics may obey scale-change symmetry. Here we develop a practical approach to test this hypothesis. We confirm, using two different computational models, that neural dynamical laws exhibit scale-change symmetry near a dynamical phase transition. Moreover, we show that as a mouse awakens from anesthesia, scale-change symmetry emerges. Scale-change symmetry of the rules governing cortical dynamics may explain observations of similar critical phenomena across diverse neural systems.

Journal article

Quicke P, Song C, McKimm EJ, Milosevic MM, Howe CL, Neil M, Schultz SR, Antic SD, Foust AJ, Knopfel Tet al., 2019, Single-neuron level one-photon voltage imaging with sparsely targeted genetically encoded voltage indicators, Frontiers in Cellular Neuroscience, Vol: 13, ISSN: 1662-5102

Voltage imaging of many neurons simultaneously at single-cell resolution is hampered by the difficulty of detecting small voltage signals from overlapping neuronal processes in neural tissue. Recent advances in genetically encoded voltage indicator (GEVI) imaging have shown single-cell resolution optical voltage recordings in intact tissue through imaging naturally sparse cell classes, sparse viral expression, soma restricted expression, advanced optical systems, or a combination of these. Widespread sparse and strong transgenic GEVI expression would enable straightforward optical access to a densely occurring cell type, such as cortical pyramidal cells. Here we demonstrate that a recently described sparse transgenic expression strategy can enable single-cell resolution voltage imaging of cortical pyramidal cells in intact brain tissue without restricting expression to the soma. We also quantify the functional crosstalk in brain tissue and discuss optimal imaging rates to inform future GEVI experimental design.

Journal article

Quicke P, Howe CL, Song P, Jadan HV, Dragotti PL, Knöpfel T, Foust AJ, Schultz SR, Neil Met al., 2019, Calculation of high numerical aperture lightfield microscope point spread functions

© OSA 2019 © 2019 The Author(s) 3D deconvolution of lightfield images enables high resolution reconstruction of sample volumes. Previous point spread function calculations assume low to moderate NA objectives. Here we present a simple vectorial calculation valid for high NA objectives.

Conference paper

van Opbergen CJM, Koopman CD, Kok BJM, Knopfel T, Renninger SL, Orger MB, Vos MA, van Veen TAB, Bakkers J, de Boer TPet al., 2018, Optogenetic sensors in the zebrafish heart: a novel in vivo electrophysiological tool to study cardiac arrhythmogenesis, THERANOSTICS, Vol: 8, Pages: 4750-4764, ISSN: 1838-7640

Cardiac arrhythmias are among the most challenging human disorders to diagnose and treat due to their complex underlying pathophysiology. Suitable experimental animal models are needed to study the mechanisms causative for cardiac arrhythmogenesis. To enable in vivo analysis of cardiac cellular electrophysiology with a high spatial and temporal resolution, we generated and carefully validated two zebrafish models, one expressing an optogenetic voltage indicator (chimeric VSFP-butterfly CY) and the other a genetically encoded calcium indicator (GCaMP6f) in the heart.Methods: High-speed epifluorescence microscopy was used to image chimeric VSFP-butterfly CY and GCaMP6f in the embryonic zebrafish heart, providing information about the spatiotemporal patterning of electrical activation, action potential configuration and intracellular Ca2+ dynamics. Plotting VSFP or GCaMP6f signals on a line along the myocardial wall over time facilitated the visualization and analysis of electrical impulse propagation throughout the heart. Administration of drugs targeting the sympathetic nervous system or cardiac ion channels was used to validate sensitivity and kinetics of both zebrafish sensor lines. Using the same microscope setup, we imaged transparent juvenile casper fish expressing GCaMP6f, demonstrating the feasibility of imaging cardiac optogenetic sensors at later stages of development.Results: Isoproterenol slightly increased heart rate, diastolic Ca2+ levels and Ca2+ transient amplitudes, whereas propranolol caused a profound decrease in heart rate and Ca2+ transient parameters in VSFP-Butterfly and GCaMP6f embryonic fish. Ikr blocker E-4031 decreased heart rate and increased action potential duration in VSFP-Butterfly fish. ICa,L blocker nifedipine caused total blockade of Ca2+ transients in GCaMP6f fish and a reduced heart rate, altered ventricular action potential duration and disrupted atrial-ventricular electrical conduction in VSFP-Butterfly fish. Imaging of juvenile

Journal article

Song C, Piscopo DM, Niell CM, Knopfel Tet al., 2018, Cortical signatures of wakeful somatosensory processing, Scientific Reports, Vol: 8, ISSN: 2045-2322

Sensory inputs carry critical information for the survival of an organism. In mice, tactile information conveyed by the whiskers is of high behavioural relevance, and is broadcasted across cortical areas beyond the primary somatosensory cortex. Mesoscopic voltage sensitive dye imaging (VSDI) of cortical population response to whisker stimulations has shown that seemingly ‘simple’ sensory stimuli can have extended impact on cortical circuit dynamics. Here we took advantage of genetically encoded voltage indicators (GEVIs) that allow for cell type-specific monitoring of population voltage dynamics in a chronic dual-hemisphere transcranial windowed mouse preparation to directly compare the cortex-wide broadcasting of sensory information in wakening (lightly anesthetized to sedated) and awake mice. Somatosensory-evoked cortex-wide dynamics is altered across brain states, with anatomically sequential hyperpolarising activity observed in the awake cortex. GEVI imaging revealed cortical activity maps with increased specificity, high spatial coverage, and at the timescale of cortical information processing.

Journal article

Knoepfel T, 2018, Neurotechnology to address big questions, Science, Vol: 361, Pages: 328-329, ISSN: 0036-8075

Emotions, cognition, and consciousness emerge with the processing of neuronal information provided by memory readouts and senses. How this actually works seemed for a long time to be a question too big to ask. Advances in neuroscience have always been driven by methodological inventions, and current efforts to develop neurotechnologies are motivated by experimental strategies, including analytical dissection (that is, inverse engineering), large-scale interrogation, and synthetic reconstruction of the mammalian cerebral cortex (which is important for higher cognitive functions) and connected brain structures (1, 2). As part of this neurotechnological endeavor, mapping the transcriptome of neurons with single-cell resolution and with known three-dimensional tissue localization has been a long-sought enabling technology (3). On page 380 of this issue, Wang et al. (4) present the technology and workflow to access transcriptional states of more than 100 genes from up to 30,000 cells in a cubic millimeter of cortical tissue. This advanced methodology will facilitate studies that improve our understanding of the neuronal hardware and, when combined with other emerging neurotechnologies, will enable big questions in neuroscience to be addressed.

Journal article

Quicke P, Reynolds S, Neil M, Knopfel T, Schultz S, Foust AJet al., 2018, High speed functional imaging with source localized multifocal two-photon microscopy, Biomedical Optics Express, Vol: 9, Pages: 3678-3693, ISSN: 2156-7085

Multifocal two-photon microscopy (MTPM) increases imaging speed over single-focus scanning by parallelizing fluorescence excitation. The imaged fluorescence’s susceptibility to crosstalk, however, severely degrades contrast in scattering tissue. Here we present a source-localized MTPM scheme optimized for high speed functional fluorescence imaging in scattering mammalian brain tissue. A rastered line array of beamlets excites fluorescence imaged with a complementary metal-oxide-semiconductor (CMOS) camera. We mitigate scattering-induced crosstalk by temporally oversampling the rastered image, generating grouped images with structured illumination, and applying Richardson-Lucy deconvolution to reassign scattered photons. Single images are then retrieved with a maximum intensity projection through the deconvolved image groups. This method increased image contrast at depths up to 112 μm in scattering brain tissue and reduced functional crosstalk between pixels during neuronal calcium imaging. Source-localization did not affect signal-to-noise ratio (SNR) in densely labeled tissue under our experimental conditions. SNR decreased at low frame rates in sparsely labeled tissue, with no effect at frame rates above 50 Hz. Our non-descanned source-localized MTPM system enables high SNR, 100 Hz capture of fluorescence transients in scattering brain, increasing the scope of MTPM to faster and smaller functional signals.

Journal article

Fagerholm ED, Dinov M, Knopfel T, Leech Ret al., 2018, The characteristic patterns of neuronal avalanches in mice under anesthesia and at rest: An investigation using constrained artificial neural networks, PLOS ONE, Vol: 13, ISSN: 1932-6203

Journal article

Van Opbergen CJM, Koopman CD, Vos MA, Knopfel T, Van Veen TAB, Bakkers J, De Boer TPet al., 2018, Optogenetic sensors in zebrafish hearts as novel in vivo electrophysiological readout tools to study cardiac arrhythmogenesis, 5th Congress of the ESC-Council-on-Basic-Cardiovascular-Science on Frontiers in Cardio Vascular Biology, Publisher: OXFORD UNIV PRESS, Pages: S81-S81, ISSN: 0008-6363

Conference paper

Buchborn T, Lyons T, Knopfel T, 2018, Tolerance and Tachyphylaxis to Head Twitches Induced by the 5-HT2A Agonist 25CN-NBOH in Mice, Frontiers in Pharmacology, Vol: 9, ISSN: 1663-9812

The serotonin (5-HT) 2A receptor is the primary molecular target of serotonergic hallucinogens, which trigger large-scale perturbations of the cortex. Our understanding of how 5-HT2A activation may cause the effects of hallucinogens has been hampered by the receptor unselectivity of most of the drugs of this class. Here we used 25CN-NBOH (N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine), a newly developed selective 5-HT2A agonist, and tested it with regard to the head-twitch-response (HTR) model of 5-HT2A activity and effects on locomotion. 25CN-NBOH evoked HTRs with an inverted u-shape-like dose-response curve and highest efficacy at 1.5 mg/kg, i.p. HTR occurrence peaked within 5 min after agonist injection, and exponentially decreased to half-maximal frequency at ~11 min. Thorough habituation to the experimental procedures (including handling, saline injection, and exposure to the observational boxes 1 day before the experiment) facilitated the animals' response to 25CN-NBOH. 25CN-NBOH (1.5 mg/kg, i.p.) induced HTRs were blocked by the 5-HT2A antagonist ketanserin (0.75 mg/kg, 30 min pre), but not by the 5-HT2C antagonist SB-242084 (0.5 mg/kg, i.p., 30 min pre). SB-242084 instead slightly increased the number of HTRs occurring at a 3.0-mg/kg dose of the agonist. Apart from HTR induction, 25CN-NBOH also modestly increased locomotor activity of the mice. Repeated once-per-day injections (1.5 mg/kg, i.p.) led to reduced occurrence of 25CN-NBOH induced HTRs. This intermediate tolerance was augmented when a second (higher) dose of the drug (3.0 mg/kg) was interspersed. Short-interval tolerance (i.e., tachyphylaxis) was observed when the drug was injected twice at intervals of 1.0 and 1.5 h at either dose tested (1.5 mg/kg and 0.75 mg/kg, respectively). Inducing ketanserin-sensitive HTRs, which are dependent on environmental valences and which show signs of tachyphylaxis and tolerance, 25CN-NBOH shares striking features common to serotonergic hallucinogens. Gi

Journal article

Tahirbegi B, Magness AJ, Boillat A, Willison KR, Klug DR, Knopfel T, Ying Let al., 2018, Probing synaptic amyloid-beta aggregation promoted by copper release, 62nd Annual Meeting of the Biophysical-Society, Publisher: Biophysical Society, Pages: 430A-430A, ISSN: 0006-3495

Whether or not the metal ions released during synaptic transmission induce amyloid-beta oligomer formation in the vicinity of synapses is a central question pertinent to the molecular mechanism of Alzheimer's disease. Recently, through a combination of experimental kinetics studies and coupled reaction-diffusion simulations, we predicted that Cu(II) rather than Zn(II) plays an important role in the very early stages (i.e., dimer formation) of Aβ aggregation in the synapse. Single molecule photobleaching analysis is a powerful tool to determine the stoichiometry of amyloid-beta oligomers which enables us to examine the time course of small amyloid-beta oligomer formation in solution, immobilised to a solid-phase substrate or artificial lipid membrane, and in live neurons in the presence of Cu(II). Preliminary results indicate that small amyloid-beta oligomers can be locked in their oligomeric state without dissociation on a poly-lysine coated surface and that Cu(II) increases the diversity and abundance of amyloid-beta oligomers.

Conference paper

Johnston CM, Rog-Zielinska EA, Wulfers EM, Houwaart T, Siedlecka U, Knopfel T, Kohl P, Schneider-Warme Fet al., 2017, Optogenetic targeting of cardiac myocytes and non-myocytes: tools, challenges and utility, Progress in Biophysics and Molecular Biology, Vol: 130, Pages: 140-149, ISSN: 0079-6107

In optogenetics, light-activated proteins are used to monitor and modulate cellular behaviour with light. Combining genetic targeting of distinct cellular populations with defined patterns of optical stimulation enables one to study specific cell classes in complex biological tissues. In the current study we attempted to investigate the functional relevance of heterocellular electrotonic coupling in cardiac tissue in situ. In order to do that, we used a Cre-Lox approach to express the light-gated cation channel Channelrhodopsin-2 (ChR2) specifically in either cardiac myocytes or non-myocytes. Despite high specificity when using the same Cre driver lines in a previous study in combination with a different optogenetic probe, we found patchy off-target ChR2 expression in cryo-sections and extended z-stack imaging through the ventricular wall of hearts cleared using CLARITY. Based on immunohistochemical analysis, single-cell electrophysiological recordings and whole-genome sequencing, we reason that non-specificity is caused on the Cre recombination level. Our study highlights the importance of careful design and validation of the Cre recombination targets for reliable cell class specific expression of optogenetic tools.

Journal article

Koopman CD, Zimmermann WH, Knopfel T, de Boer TPet al., 2017, Cardiac optogenetics: using light to monitor cardiac physiology, Basic Research in Cardiology, Vol: 112, ISSN: 0300-8428

Our current understanding of cardiac excitation and its coupling to contraction is largely based on ex vivo studies utilising fluorescent organic dyes to assess cardiac action potentials and signal transduction. Recent advances in optogenetic sensors open exciting new possibilities for cardiac research and allow us to answer research questions that cannot be addressed using the classic organic dyes. Especially thrilling is the possibility to use optogenetic sensors to record parameters of cardiac excitation and contraction in vivo. In addition, optogenetics provide a high spatial resolution, as sensors can be coupled to motifs and targeted to specific cell types and subcellular domains of the heart. In this review, we will give a comprehensive overview of relevant optogenetic sensors, how they can be utilised in cardiac research and how they have been applied in cardiac research up to now.

Journal article

Quicke P, Neil M, Knopfel T, Schultz SR, Foust AJet al., 2017, Source-Localized Multifocal Two-Photon Microscopy for High-Speed Functional Imaging, 71st Annual Meeting of the Society-of-General-Physiologists (SGP) on Optical Revolution in Physiology - From Membrane to Brain, Publisher: ROCKEFELLER UNIV PRESS, Pages: 13A-14A, ISSN: 0022-1295

Conference paper

Song C, Do QB, Antic SD, Knopfel Tet al., 2017, Transgenic Strategies for Sparse but Strong Expression of Genetically Encoded Voltage and Calcium Indicators, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, Vol: 18

Journal article

Song C, Barnes S, Knopfel T, 2017, Mammalian cortical voltage imaging using genetically encoded voltage indicators: a review honoring professor Amiram Grinvald, NEUROPHOTONICS, Vol: 4, ISSN: 2329-423X

Journal article

Shimaoka D, Song C, Knopfel T, 2017, State-dependent modulation of slow wave motifs towards awakening, Frontiers in Cellular Neuroscience, Vol: 11, ISSN: 1662-5102

Slow cortical waves that propagate across the cerebral cortex forming large-scale spatiotemporal propagation patterns are a hallmark of non-REM sleep and anesthesia, but also occur during resting wakefulness. To investigate how the spatial temporal properties of slow waves change with the depth of anesthetic, we optically imaged population voltage transients generated by mouse layer 2/3 pyramidal neurons across one or two cortical hemispheres dorsally with a genetically encoded voltage indicator (GEVI). From deep barbiturate anesthesia to light barbiturate sedation, depolarizing wave events recruiting at least 50% of the imaged cortical area consistently appeared as a conserved repertoire of distinct wave motifs. Toward awakening, the incidence of individual motifs changed systematically (the motif propagating from visual to motor areas increased while that from somatosensory to visual areas decreased) and both local and global cortical dynamics accelerated. These findings highlight that functional endogenous interactions between distant cortical areas are not only constrained by anatomical connectivity, but can also be modulated by the brain state.

Journal article

Quicke P, Barnes SJ, Knöpfel T, 2017, Imaging of Brain Slices with a Genetically Encoded Voltage Indicator., Methods Mol Biol, Vol: 1563, Pages: 73-84

Functional fluorescence microscopy of brain slices using voltage sensitive fluorescent proteins (VSFPs) allows large scale electrophysiological monitoring of neuronal excitation and inhibition. We describe the equipment and techniques needed to successfully record functional responses optical voltage signals from cells expressing a voltage indicator such as VSFP Butterfly 1.2. We also discuss the advantages of voltage imaging and the challenges it presents.

Journal article

Barnes S, Song C, Knoepfel T, 2017, Functional Cortical Connectomics through Co-Expression of Genetically Expressed Voltage and Calcium Indicators, 58th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 340A-341A, ISSN: 0006-3495

Conference paper

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