Podcasts about gcamp

In this episode of Manufacturing an American Century, Matt chats with Ray Zaganto, Partner Relations Manager at IMEC (Illinois Manufacturing Excellence Center), to discuss why the future of our manufacturing sector depends on innovation, collaboration, and a national strategy. Ray didn't hold back in sharing his passion for bringing real, lasting change to the industry. We talked about the crucial role of IMEC and how they've been helping small and medium-sized manufacturers not just survive but thrive by adopting new technologies and building stronger businesses. One of Ray's key points is how we've got to engage the next generation early—starting at the grade-school level, not just with high schoolers. He shared how nonprofits like GCAMP in Chicago are making that connection between students, parents, and the exciting world of modern manufacturing. We also dove into some big challenges, like the decline in casting and forging for defense manufacturing, and why we need to rebuild that capability to keep America's defense industrial base strong and resilient.Ray also stressed the importance of a growth mindset in the manufacturing world. Too many companies hesitate to innovate, but as Ray pointed out, sticking your head in the sand is not a strategy. You've got to invest in your future. We wrapped up by talking about the need for a cultural shift—manufacturing has to become a core part of our communities, and companies need to stay engaged with their ecosystem of support to succeed. This episode is packed with insights on how we can all work together to strengthen American manufacturing for the long haul, thanks Ray!AMCC's podcast is made possible in part by the expertise of Mike McAllen, founder of Podcasting4Associations. Are you part of an association also looking to produce a podcast? Let us get you in touch with Mike.Thank you to the Economic Development Administration for their partnership in producing this podcast. This podcast was prepared in part using Federal funds under award 3070145 from the Economic Development Administration, U.S. Department of Commerce. The statements, findings, conclusions, and recommendations are those of the author(s) and do not necessarily reflect the views of the Economic Development Administration or the U.S. Department of Commerce.Participants:Ray Ziganto, Partner Relations Manager, Illinois Manufacturing Excellence CenterMatt Bogoshian: Executive Director, American Manufacturing Communities Collaborative and host of the podcast.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.06.531342v1?rss=1 Authors: Ding, J., Peng, L., Moon, S., Lee, H. J., Patel, D. S., Lu, H. Abstract: In living organisms, changes in calcium flux are integral to many different cellular functions and are especially critical for the activity of neurons and myocytes. Genetically encoded calcium indicators (GECIs) have been popular tools for reporting changes in calcium levels in vivo. In particular, GCaMP, derived from GFP, are the most widely used GECIs and have become an invaluable toolkit for neurophysiological studies. Recently, new variants of GCaMP, which offer a greater variety of temporal dynamics and improved brightness, have been developed. However, these variants are not readily available to the Caenorhabditis elegans research community. This work reports a set of GCaMP6 and jGCaMP7 reporters optimized for C. elegans studies. Our toolkit provides reporters with improved dynamic range, varied kinetics, and targeted subcellular localizations. Besides optimized routine uses, this set of reporters are also well-suited for studies requiring fast imaging speeds and low magnification or low-cost platforms. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.30.525700v1?rss=1 Authors: Zhou, J., Whitmire, M., Chen, Y., Seidemann, E. Abstract: Neuronal representations throughout the primate visual system display a wide range of nonlinear dynamics in response to static visual stimuli. To understand the neural basis of visual perception, it is important to quantify these nonlinearities and to develop general models that allow one to predict response dynamics to visual stimuli with arbitrary temporal waveform. Voltage-sensitive dye imaging (VSDI) is a powerful method for measuring neural population responses from the cortex of awake, behaving, subjects. Here we used VSDI to measure the dynamics of neural population responses in macaque V1 to visual stimuli with a wide range of time courses. We found that beyond clear nonlinearities for briefly presented visual stimuli, stimulus-evoked VSDI responses are surprisingly near-additive in time. These results are qualitatively different from neural dynamics to similar stimuli previously measured in human visual cortex using fMRI and electrocorticography (ECoG), which show strong sub-additivity in time. To test whether this discrepancy is specific to VSDI, a signal dominated by sub-threshold neural activity, we repeated our measurements using a genetically encoded calcium indicator (GCaMP), a signal dominated by spiking activity. We found that GCaMP signals in macaque V1 are also near additive. Therefore, the discrepancies in the degree of additivity between these different measurements are not attributed to the difference between sub- and supra-threshold neural response. Finally, we show that a simple yet flexible delayed normalization model can capture the dynamics of all of these measurements, suggesting that dynamic gain-control is an important mechanism contributing to neural processing in the brain. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.11.511633v1?rss=1 Authors: Verinaz-Jadan, H., Howe, C. L., Song, P., Lesept, F., Kittler, J., Foust, A. J., Dragotti, P. L. Abstract: Light Field Microscopy (LFM) is an imaging technique that offers the opportunity to study fast dynamics in biological systems due to its rapid 3D imaging rate. In particular, it is attractive to analyze neuronal activity in the brain. Unlike scanning-based imaging methods, LFM simultaneously encodes the spatial and angular information of light in a single snapshot. However, LFM is limited by a trade-off between spatial and angular resolution and is affected by scattering at deep layers in the brain tissue. In contrast, two-photon (2P) microscopy is a point-scanning 3D imaging technique that achieves higher spatial resolution, deeper tissue penetration, and reduced scattering effects. However, point-scanning acquisition limits the imaging speed in 2P microscopy and cannot be used to simultaneously monitor the activity of a large population of neurons. This work introduces a physics-driven deep neural network to image neuronal activity in scattering volume tissues using LFM. The architecture of the network is obtained by unfolding the ISTA algorithm and is based on the observation that the neurons in the tissue are sparse. The deep-network architecture is also based on a novel imaging system modeling that uses a linear convolutional neural network and fits the physics of the acquisition process. To achieve the high-quality reconstruction of neuronal activity in 3D brain tissues from temporal sequences of light field (LF) images, we train the network in a semi-supervised manner using generative adversarial networks (GANs). We use the TdTomato indicator to obtain static structural information of the tissue with the microscope operating in 2P scanning modality, representing the target reconstruction quality. We also use additional functional data in LF modality with GCaMP indicators to train the network. Our approach is tested under adverse conditions: limited training data, background noise, and scattering samples. We experimentally show that our method performs better than model-based reconstruction strategies and typical artificial neural networks for imaging neuronal activity in mammalian brain tissue, considering reconstruction quality, generalization to functional imaging, and reconstruction speed. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.02.505959v1?rss=1 Authors: Wang, Y., Sepers, M. D., Xiao, D., Raymond, L. A., Murphy, T. H. Abstract: Huntington Disease (HD), caused by dominantly inherited expansions of a CAG repeat results in characteristic motor dysfunction. Although gross motor and balance defects have been extensively characterized in multiple HD mouse models using tasks such as rotarod, beam walking and gait analysis, little is known about forelimb deficits. Here we use a high-throughput alternating reward/non-reward water-reaching task conducted daily over ~2 months to simultaneously monitor forelimb impairment and mesoscale cortical changes in GCaMP activity, comparing female zQ175 (HD) and wildtype (WT) littermate mice, starting at ~5.5 months of age. Behavioral analysis of the water-reaching task reveals that HD mice, despite learning the water-reaching task as proficiently as WT mice, take longer to learn the alternating event sequence. Although WT mice displayed no significant changes in cortical activity and reaching trajectory throughout the testing period, HD mice exhibited an increase in cortical activity - especially in the secondary motor and retrosplenial cortices - over time, as well as longer and more variable reaching trajectories by ~7 months of age. HD mice also experienced a progressive reduction in successful performance rates. Tapered beam and rotarod tests before and/or after water-reaching assessment confirmed these early and manifest stages of HD characterized by the absence and presence of failed water-reaching trials, respectively. Reduced DARPP-32 (marker for striatal medium spiny neurons) expression in HD mice further confirmed disease pathology. The water-reaching task can be used to inform HD and potentially other movement disorder onset, therapeutic intervention windows and test drug efficacy. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.29.360214v1?rss=1 Authors: Gielen, V., Mönkemöller, V., Shen, Y., Hofkens, J., Vanden Berghe, P., Campbell, R. E., Moeyaert, B., Dedecker, P. Abstract: Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled analyte quantification (PEAQ) biosensing, a method that leverages photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity. We develop photochromic variants of the popular GCaMP family of calcium ion biosensors, and show that these can be used to resolve dynamic changes in the absolute Ca2+ concentration in live cells. We also show how our method can be expanded to fast imaging with reduced illumination intensities or to situations where the absolute illumination intensities are unknown. In principle, PEAQ biosensing can be applied to other biosensors with photochromic properties, thereby expanding the possibilities for fully quantitative measurements in complex and dynamic systems. Copy rights belong to original authors. Visit the link for more info

Today on Tech Transfer IP, Lisa has the pleasure of speaking with Michael Perham. Mike is the Director of Innovations and External Relations at the Janelia Research Campus in Ashburn, Virginia. At Janelia, Mike leads the Department of Innovation Management and oversees external relations and library services. He handles technology management, IP protection, licensing, and decimation for Janelia with a unique attention to open science initiatives.  Mike shares what led him to tech transfer, his position at Janelia, how Janelia got started, and what they want to accomplish. Mike talks about their fifteen-year research models, how their support teams are used, and the advantages they provide. Mike also discusses products the support team has designed. Listen, as Mike speaks about how his office is structured, product development and dissemination, and his goal of having anyone doing experiments to be able to use the instruments invented at Janelia. He also shares how many invention disclosures and revenue-generating licenses his office receives each year. Mike discusses his offices' biggest successes, the biggest challenges, and what they do for women entrepreneurs and inventors at Janelia. He shares his involvement with AUTM and LES and the benefit he believes these organizations provide tech transfer offices, and what he would wish for if he had three things wishes for his office. In This Episode: [02:39] Welcome to the show, Mike! [02:55] Mike shares his background, which led him to tech transfer. [06:34] Mike speaks about getting the position at Janelia. [08:16] Listen as Mike discusses Janelia, what it's about, and how it started. [11:07] Janelia was modeled after similar facilities at the University of Cambridge and Bell Labs. [14:11] Neuroscience and advanced imaging are areas that Janelia was focused on. [16:21] A key ingredient of Janelia are the people that are recruited. [18:15] Mike speaks about their fifteen-year research models. [21:12] How are your support teams used, and what advantages do they provide to Janelia? [23:36] Mike talks about the microscopes the support team designed. [26:27] Mike shares how his office is structured. [29:03] He enjoys being at Janelia because his office is involved with so many things that he never gets bored. [31:14] Mike speaks about product development practices and dissemination at Janelia. [33:40] Mike discusses wanting to have the experiments using the instrument that was invented at Janelia. [36:56] How many invention disclosures does your office receive each year? [38:17] Mike shares how many revenue-generating licenses they get each year. [40:25] Mike speaks about a success his office has had called GCaMP. [42:22] He discusses some other successes his office has had. [46:22] What are some of your office's biggest challenges? [50:05] Mike talks about what his office does for women entrepreneurs and innovators. [53:35] Mike shares his involvement with AUTM and LES and how they benefit tech transfer. [56:42] Listen as Mike discusses credentialing and whether he believes it makes a difference. [59:54] If you could have three wishes for your office, what would they be? [1:02:53] Thank you for being on the show! Find Michael Email  

Ask local manufacturers what keeps them up at night and you’ll likely hear: “workforce development.” Kathleen Burley, Executive Director of GCAMP—the Golden Corridor Advanced Manufacturing Partnership—sat down with “Strictly Business” host Josh Grodzin for a thought-provoking discussion about the pressing need to fill manufacturing jobs as workers retire. That includes efforts to “lift up manufacturing and make people understand what it truly is right now.”

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.22.262527v1?rss=1 Authors: Zhao, Z., Tian, D., McBride, C. S. Abstract: The mosquito Aedes aegypti is the primary worldwide vector of arboviruses that infect humans, including dengue, Zika, chikungunya, and yellow fever. Recent advances in transgenic technology have yielded important new insight into the biology of this disease vector. The early development of neurogenetic tools, in particular, is beginning to shed light on the neural basis of behaviors that allow Ae. aegypti to thrive in human environments and find and bite human hosts. Despite these advances, a pan-neuronal expression driver remains elusive. Pan-neuronal drivers give researchers genetic access to all neurons and thus provide a critical entry point for circuit dissection. Here, we describe our efforts to generate pan-neuronal drivers in Ae. aegypti via targeted knock-in of in-frame reporter constructs to the native coding sequence of broadly expressed neural genes with CRISPR/Cas9. Two of five attempts were successful, resulting in a Syt1:GCaMP6s strain that expresses synaptically-localized GCaMP in all neurons and a brp-T2A-QF2w driver strain that can be used to drive and amplify expression of any effector in all neurons via the Q binary system. We show that both manipulations broadly and uniformly label the nervous system and have only mild effects on behavior. We envision that these strains will facilitate neurobiological research in Ae. aegypti mosquitoes and provide documentation of both successful and failed manipulations as a roadmap for similar tool development in other non-model species. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.05.238741v1?rss=1 Authors: Frost, N., Haggart, A., Sohal, V. S. Abstract: How neurons encode behavior is a fundamental question. Neuronal ensembles increase or decrease activity during specific behaviors. However, it is unclear whether ensembles encode information solely via changes in activity levels, or whether changes in correlations between neurons carry additional information. We used microendoscopic GCaMP imaging to measure prefrontal activity while mice were either alone or engaged in social interaction. Using neural network classifiers to measure how well prefrontal neurons transmit information about social behavior to downstream neurons, we find that surrogate datasets which preserve dynamic correlations outperform those which preserve ensemble activity but not correlations. Notably, this ability of correlations to enhance the information transmitted by neuronal ensembles is lost in mice lacking the autism-associated gene Shank3. These results show that dynamically modulated correlations create patterns of coactive neurons which are behaviorally-specific and enhance the information transmitted by neuronal ensembles. Furthermore, this process may be disrupted in pathological states. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.17.156430v1?rss=1 Authors: Giachello, C. N. G., Zarin, A. A., Kohsaka, H., Fan, Y. N., Nose, A., Landgraf, M., Baines, R. A. Abstract: Mapping the wired connectivity of a nervous system is a prerequisite for full understanding of function. In this respect, such endeavours can be likened to genome sequencing projects. These projects similarly produce impressive amounts of data which, whilst a technical tour-de-force, remain under-utilised without validation. Validation of neuron synaptic connectivity requires electrophysiology which has the necessary temporal and spatial resolution to map synaptic connectivity. However, this technique is not common and requires extensive equipment and training to master, particularly when applied to the small CNS of the Drosophila larva. Thus, validation of connectivity in this CNS has been more reliant on behavioural analyses and, in particular, activity imaging using the calcium-sensor GCaMP. Whilst both techniques are powerful, they each have significant limitations for this purpose. Here we use electrophysiology to validate an array of driver lines reported to label specific premotor interneurons that the Drosophila connectome project suggests are monosynaptically connected to an identified motoneuron termed the anterior corner cell (aCC). Our results validate this proposition for four selected lines. Thus, in addition to validating the connectome with respect to these four premotor interneurons, our study highlights the need to functionally validate driver lines prior to use. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.18.103234v1?rss=1 Authors: McGovern, D. J., Polter, A. M., Root, D. H. Abstract: Ventral tegmental area (VTA) glutamate neurons signal and participate in reward and aversion-based behaviors. However, the neurochemical mechanisms that underlie how these neurons participate in diverse motivated behaviors is unknown. We used a combination of optical sensors to identify how distinct neurochemical inputs to VTA glutamate neurons participate in motivated behavior. Glutamate inputs to VTA glutamate neurons increased for both reward- and aversion-predicting cues and aversive outcomes, but subpopulations of glutamate inputs were increased or decreased by reward. For all cues and outcomes, GABA inputs to VTA glutamate neurons decreased and GCaMP-measured neuronal activity increased. GCaMP recordings also showed that VTA glutamate neuronal activity discriminated between the omission and receipt of an expected reward, but glutamate and GABA inputs to these neurons did not. Electrophysiological recordings in coordination with our sensor data suggest that glutamate inputs, but not GABA inputs, principally regulate VTA glutamate neuron participation in diverse motivated behaviors. Copy rights belong to original authors. Visit the link for more info

Feelings on Columbus,Ga inspiration and Much More --- This episode is sponsored by · Anchor: The easiest way to make a podcast. https://anchor.fm/app --- Send in a voice message: https://anchor.fm/jamariantekyler/message Support this podcast: https://anchor.fm/jamariantekyler/support

Tracklist: 1. Richard Durand Vs. Huge Euge - Signs (Richard Durand Vs. The World mix) 2. Ferry Corsten - Live Forever (Michael Woods Remix) 3. Tempo Giusto & Ima'Gin - Gemini (Evol Waves Remix) 4. David Forbes & Full Tilt - Incline (Original Mix) 5. Cosmic Gate & Jsomething - Over the Rainbow (W&W Remix) 6. Nathan C - Bad Timing (Original Mix) 7. Stephen Kirkwood - Forever After (Original Mix) 8. W&W - White Label (Original Mix) 9. Marcus Schossow - Never Say Never (Original Mix) 10. Orjan Nilsen - Endymion (Original Mix) 11. Wezz Devall - Stadium (Original Mix) 12. Lee Haslam - Vengeance (Original Mix) Show some love: Like my Facebook page! http://www.facebook.com/pages/G-Camp/151306401601854

Track List: 1. Faruk Sabanci & Liquid Vision - The Last Ottoman (Evol Waves Remix) 2. Marco V - Solid Sounds (Original Mix) 3. Arnej & 8 Wonders - Together We Will Rise (Original Mix) 4. Arnej - True Lies (Original Mix) 5. Gofman & Tsukerman - We Control U (Original Mix) 6. Spartaque - Interception (Original Mix) 7. Tiesto - Flight 643 (Yves V Private Remix) 8. Norin & Rad - Pistol Whip (Original Mix) 9. Evol Waves - Nobody Knows (Original Mix) 10. Sander Van Doorn - Apple (Marcus Schossow Remix) 11. Snatt & Vix - So Far Away (Original Mix) 12. Marcel Woods - Lemon Tree (Marcel Woods Treatment) 13. MaRlo - Showgrounds (Original Mix) 14. Richard Durand Feat. Leah - Stand Again (Original Mix) Check out and LIKE my fanpage if you dig the show!! :) http://www.facebook.com/pages/G-Camp/151306401601854

1. Carlo Calabro - Fight on Sissy Lane 2. George Acosta - To the Sky 3. Marcel Woods - The Bottle (Marcus Schossow Remix) 4. Harvey Anderson - Soviet (Mark Sherry's 128 Edit) 5. Henry John Morgan - Jux 6. W&W - Shotgun 7. Mr. Pit - Injected 8. JOOP - Focus (Down Low Mix) 9. Wezz Devall - Kill of the Year 10. Snatt & Vix - So Far Away (Leon Bolier Remix) 11. Lisa Lashes - Snap Shot 12. Leon Bolier - Me (Extended Mix) 13. Silvio Ecomo - In No Dip (Koen Groeneveld Remix) 14. Dave Blaster - ID Don't forget to check out and "like" my page if you dig the show! :) http://www.facebook.com/pages/G-Camp/151306401601854

Motion vision is of fundamental importance for moving animals from arthropods to mammals. In this thesis I lay ground for the functional analysis of the neural circuit underlying visual motion detection in fruit flies by means of genetic tools. In Drosophila melanogaster transgenic tools allow for both experimental observation and manipulation of neural activity: genetically encoded calcium indicators (GECIs) can be used for the optophysiological characterization of neural activity and transgenes for the inhibition of neural activity can be used to determine these neurons' function. Combined, yet independent use of both tools is a powerful approach for the functional analysis of a neural network. However, GECI signals in vivo generally suffer from poor signal-to-noise ratios and GECI characteristics change dramatically and unpredictably when transfered from the cuvette into neurons of living animals, probably due to interactions with native cellular proteins. Here, I quantified and compared the in vivo response properties of five new (Yellow Cameleon 3.60 & 2.60, D3cpV, TN-XL and TN-XXL) and two more established ratiometric GECIs (Yellow Cameleon 3.3, TN-L15). In addition, I included the single-chromophore probe GCaMP 1.6 in this comparison. The analysis was performed under 2-photon microscopy at presynaptic boutons of neuromuscular junctions in transgenic fly larvae. I quantified action potential induced changes of calcium concentrations by calibrating responses of a synthetic calcium indicator that was microinjected under 2-photon guidance. The observed cytosolic calcium concentration was 31 nM at rest and increased linearly with stimulus frequency by 0.1 to 1.8 uM at sustained activity of 10 and 160 Hz, respectively. This allowed for a quantitative comparison of the responses of GECIs in terms of their steady state response amplitudes, signal-to-noise ratio, response kinetics, calcium affinities and hill coefficients in vivo. The results were then compared to in vitro properties of GECIs measured in cuvettes. The data reveal that a new generation of GECIs retain improved signalling characteristics in vivo. Maximum fluorescence changes were 2-3 fold increased in new compared to former ratiometric GECI variants. Small calcium changes in response to 10 Hz stimulation induced fluorescence responses with signal-to-noise ratio above 2 in Yellow Cameleon 2.60 & 3.60, D3cpv and TN-XXL. Kinetics were slowest in Yellow Cameleon 2.60 and fastest in TN-XL. The observed changes between in vitro and in vivo performance revealed systematic differences between GECIs of different types. GECIs in this study employ different calcium sensing molecules: calmodulin-M13 in Yellow Cameleons and GCaMP, a redesigned calmodulin-M13 in D3cpv, and troponin C in TN-indicators. Those indicators comprising calmodulin-M13 as calcium sensors displayed reduced maximum fluorescence changes and reduced hill coefficients in vivo, while troponin-based GECIs and D3cpv showed increased hill coefficients and increased maximum fluorescence changes in vivo. Calcium affinity of all GECIs was increased in vivo. The results demonstrate that there are now suitable GECIs at hand for experimental questions at differing expected calcium regimes. However, in contrast to a synthetic calcium sensor, none of the tested GECIs reported calcium concentration changes related to single action potentials at presynaptic boutons of the neuromuscular junction. In the visual system of Drosophila, optical recordings from motion sensitive neurons while selectively blocking certain classes of columnar neurons will allow for a network analysis of the motion detection circuit. The Gal4-UAS system can be used to express proteins that block neural activity. A similar two-part expression system, based on bacterial protein- DNA interaction (LexA and LexA-operator), can be used in parallel to drive the expression of GECIs. I generated flies expressing TN-XXL or Yellow Cameleon 3.60 under the control of the LexA-operator and demonstrated GECI expression in olfactory receptor neurons. In parallel, I cloned putative genomic enhancers that can be used to drive LexA expression in motion sensitive cells of the flies visual system. Finally, adult fixed flies expressing TN-XXL in motion sensitive neurons were visually stimulated by large field moving gratings. Parallel fluorescence measurements from these neurons showed for the first time directional selective calcium responses in Drosophila. The next step will now be the combination of calcium imaging in these neurons and functional blocking of their presynaptic partners.

Genetically encoded fluorescent indicators of neural activity represent promising tools for systems neuroscience. In the first part of my thesis, a comparative in vivo analysis of ten different genetically encoded calcium indicators as well as the pH-sensitive SynaptopHluorin is presented. The calcium indicators are either based on a single chromophore (GCaMP variants, Camgaroo variants, Pericam variants) or on two chromophores (Yellow Cameleon variants, Troponeon variants). I expressed these indicators in the cytosol of presynaptic boutons of the Drosophila larval neuromuscular junction and analyzed their fluorescence changes upon stimulation. GCaMP 1.3, GCaMP 1.6, Yellow Cameleon 2.0, 2.3, and 3.3, Inverse-Pericam, the troponin C-based calcium sensor TNL 15 and SynaptopHluorin allowed reliable detection of presynaptic fluorescence changes at the level of individual boutons. However, the response characteristics of all of these indicators differed considerably from each other. TNL 15 exhibited the most stable and fastest rising signals at lower activity rates, whereas GCaMP 1.6 produced the fastest signals at high rates of nerve activity with largest fluorescence changes. GCaMP 1.6 and GCaMP 1.3 signals, however, were complicated by bleaching, as was the case for Inverse Pericam. The fluorescence signals of the double-chromophore indicators were in general smaller but more photostable and reproducible. Camgaroo-1 and Camgaroo-2 showed little or no response, and Flash Pericam did not result in any detectable fluorescence. GCaMP 1.3 and YC 3.3 revealed fairly linear fluorescence changes and a corresponding linear increase in the signal-to-noise ratio (SNR) over an expanded range of neural activity. As expected, the expression level of the indicator had an influence on the signal kinetics and the SNR, whereas the signal amplitude was independent. In the second part of my thesis work I fused several genetically encoded calcium indicators to different signal sequences. The targeting of the indicators to distinct parts of the cell such as the membrane, vesicles or ion channels allows detection of calcium ions before they disperse in the cytosol. Specific signals can be extracted more efficiently and in a more relevant physiological context. Tagging of YC 2.3, GCaMP 1.6 and TNL 15 to transmembrane domains or proteins involved in the synaptic vesicle cycle did not result in functional targeting. TN XL fused to the transmembrane domain mCD8 at the N-terminus and eight amino acids from a calcium channel subunit at the C-terminus resulted in membrane association at the NMJ. Fractional fluorescence changes up to 6.5 % were recorded upon stimulation. In cells of the fly visual system scattered fluorescent puncta were observed. This fusion protein has the potential for monitoring calcium dynamics in close proximity of ion influx. The presented data will be useful for in vivo experiments with respect to the selection of an appropriate indicator, as well as for the correct interpretation of optical signals.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.20.049726v1?rss=1 Authors: Fu, T., Arnoux, I., Doering, J., Watari, H., Stasevicius, I., Stroh, A. Abstract: Two-photon (2-P) all-optical approaches combine in vivo 2-P calcium imaging and 2-P optogenetic modulations and have the potential to build a framework for network-based therapies, e.g. for rebalancing maladaptive activity patterns in preclinical models of neurological disorders. Here, our goal was to tailor these approaches for this purpose: Firstly, we combined in vivo juxtacellular recordings and GCaMP6f-based 2-P calcium imaging in layer II/III of mouse visual cortex to tune our detection algorithm towards a 100 % specific identification of AP-related calcium transients. False-positive-free detection was achieved at a sensitivity of approximately 73 %. To further increase specificity, secondly, we minimized photostimulation artifacts as a potential source for false-positives by using extended-wavelength-spectrum laser sources for optogenetic stimulation of the excitatory opsin C1V1. We achieved artifact-free all-optical experiments performing photostimulations at 1100 nm or higher and simultaneous calcium imaging at 920 nm in mouse visual cortex in vivo. Thirdly, we determined the spectral range for maximizing efficacy of optogenetic control by performing 2-P photostimulations of individual neurons with wavelengths up to 1300 nm. The rate of evoked transients in GCaMP6f/C1V1-co-expressing cortical neurons peaked already at 1100 nm. By refining spike detection and defining 1100 nm as the optimal wavelength for artifact-free and effective stimulations of C1V1 in GCaMP-based all-optical interrogations, we increased the translational value of these approaches, e.g. for the use in preclinical applications of network-based therapies. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.10.996116v1?rss=1 Authors: Bollimunta, A., Santacruz, S. R., Eaton, R. W., Xu, P. S., Morrison, J. H., Moxon, K. A., Carmena, J. M., Nassi, J. J. Abstract: A major effort is now underway across the brain sciences to identify, characterize and manipulate mesoscale neural circuits in order to elucidate the mechanisms underlying sensory perception, cognition and behavior. Optical imaging technologies, in conjunction with genetically encoded sensors and actuators, serve as important tools toward these goals, allowing access to large-scale genetically defined neuronal populations. In particular, one-photon miniature microscopes, coupled with genetically encoded calcium indicators and microendoscopic gradient-refractive index (GRIN) lenses, enable unprecedented readout of neural circuit dynamics in cortical and deep subcortical brain regions during active behavior in rodents. This has already led to breakthrough discoveries across a wide array of rodent brain regions and behaviors. However, in order to study the neural circuit mechanisms underlying more complex and clinically relevant human behaviors and cognitive functions, it is crucial to translate this technology to non-human primates. Here, we describe the first successful application of this technology in the rhesus macaque. We identified a viral strategy for robust expression of GCaMP, optimized a surgical protocol for microendoscope GRIN lens insertion, and created a chronic cranial chamber and lens mounting system for imaging in gyral cortex. Using these methods, we demonstrate the ability to perform plug-and-play, head-mounted recordings of cellular-resolution calcium dynamics from over 100 genetically-targeted neurons simultaneously in dorsal premotor cortex while the macaque performs a naturalistic motor reach task with the head unrestrained and freely moving. The recorded population of neurons exhibited calcium dynamics selective to the direction of reach, which we show can be used to decode the animal's trial-by-trial motor behavior. Recordings were stable over several months, allowing us to longitudinally track large populations of individual neurons and monitor their relationship to motor behavior over time. Finally, we demonstrate the ability to conduct simultaneous, multi-site imaging in bilateral dorsal premotor cortices, offering an opportunity to study distributed networks underlying complex behavior and cognition. Together, this work establishes head-mounted microendoscopic calcium imaging in macaque as a powerful new approach for studying the neural circuit mechanisms underlying complex and clinically relevant behaviors, and promises to greatly advance our understanding of human brain function, as well as its dysfunction in neurological disease. Copy rights belong to original authors. Visit the link for more info