Spatial control of in vivo optogenetic light stimulation and recording via an imaging fiber bundle (Conference Presentation)

2017 ◽  
Author(s):  
Javier I. Suárez ◽  
Parijat Sengupta ◽  
Jonathan Guo-Han Mun ◽  
Justin Rhodes ◽  
Stephen A. Boppart
Keyword(s):  
Fiber Bundle ◽  
Light Stimulation ◽  
Spatial Control

scholarly journals Genetic Dissection of Rod and Cone Pathways in the Dark-Adapted Mouse Retina

2009 ◽  
Vol 102 (3) ◽  
pp. 1945-1955 ◽  
Author(s):  
Muhammad M. Abd-El-Barr ◽  
Mark E. Pennesi ◽  
Shannon M. Saszik ◽  
Andrew J. Barrow ◽  
Janis Lem ◽  
...  
Keyword(s):  
Bipolar Cell ◽  
Mouse Retina ◽  
Specific Gene ◽  
Natural Environments ◽  
Genetic Dissection ◽  
Light Stimulation ◽  
Response Dynamic ◽  
Chemical Synapses ◽  
Operational Plan

A monumental task of the mammalian retina is to encode an enormous range (>109-fold) of light intensities experienced by the animal in natural environments. Retinal neurons carry out this task by dividing labor into many parallel rod and cone synaptic pathways. Here we study the operational plan of various rod- and cone-mediated pathways by analyzing electroretinograms (ERGs), primarily b-wave responses, in dark-adapted wildtype, connexin36 knockout, depolarizing rod–bipolar cell (DBCR) knockout, and rod transducin alpha-subunit knockout mice [ WT, Cx36(−/−), Bhlhb4(−/−), and Trα(−/−)]. To provide additional insight into the cellular origins of various components of the ERG, we compared dark-adapted ERG responses with response dynamic ranges of individual retinal cells recorded with patch electrodes from dark-adapted mouse retinas published from other studies. Our results suggest that the connexin36-mediated rod–cone coupling is weak when light stimulation is weak and becomes stronger as light stimulation increases in strength and that rod signals may be transmitted to some DBCCs via direct chemical synapses. Moreover, our analysis indicates that DBCR responses contribute about 80% of the overall DBC response to scotopic light and that rod and cone signals contribute almost equally to the overall DBC responses when stimuli are strong enough to saturate the rod bipolar cell response. Furthermore, our study demonstrates that analysis of ERG b-wave of dark-adapted, pathway-specific mutants can be used as an in vivo tool for dissecting rod and cone synaptic pathways and for studying the functions of pathway-specific gene products in the retina.

scholarly journals Prospects and Challenges of Translational Corneal Bioprinting

2020 ◽  
Vol 7 (3) ◽  
pp. 71 ◽  
Author(s):  
Matthias Fuest ◽  
Gary Hin-Fai Yam ◽  
Jodhbir S. Mehta ◽  
Daniela F. Duarte Campos
Keyword(s):  
Tissue Engineering ◽  
Ex Vivo ◽  
Corneal Transplantation ◽  
Human Cornea ◽  
Corneal Tissue ◽  
Full Thickness ◽  
Future Directions ◽  
Spatial Control

Corneal transplantation remains the ultimate treatment option for advanced stromal and endothelial disorders. Corneal tissue engineering has gained increasing interest in recent years, as it can bypass many complications of conventional corneal transplantation. The human cornea is an ideal organ for tissue engineering, as it is avascular and immune-privileged. Mimicking the complex mechanical properties, the surface curvature, and stromal cytoarchitecure of the in vivo corneal tissue remains a great challenge for tissue engineering approaches. For this reason, automated biofabrication strategies, such as bioprinting, may offer additional spatial control during the manufacturing process to generate full-thickness cell-laden 3D corneal constructs. In this review, we discuss recent advances in bioprinting and biomaterials used for in vitro and ex vivo corneal tissue engineering, corneal cell-biomaterial interactions after bioprinting, and future directions of corneal bioprinting aiming at engineering a full-thickness human cornea in the lab.

scholarly journals The Phosphatidylinositol Transfer Protein Domain of Drosophila Retinal Degeneration B Protein Is Essential for Photoreceptor Cell Survival and Recovery from Light Stimulation

1997 ◽  
Vol 139 (2) ◽  
pp. 351-363 ◽  
Author(s):  
Scott C. Milligan ◽  
James G. Alb ◽  
Raya B. Elagina ◽  
Vytas A. Bankaitis ◽  
David R. Hyde
Keyword(s):  
Retinal Degeneration ◽  
Light Response ◽  
Protein Domain ◽  
Inherited Disease ◽  
Light Stimulation ◽  
Transfer Protein ◽  
Phosphatidylinositol Transfer Protein ◽  
Phosphatidylinositol Transfer

The Drosophila retinal degeneration B (rdgB) gene encodes an integral membrane protein involved in phototransduction and prevention of retinal degeneration. RdgB represents a nonclassical phosphatidylinositol transfer protein (PITP) as all other known PITPs are soluble polypeptides. Our data demonstrate roles for RdgB in proper termination of the phototransduction light response and dark recovery of the photoreceptor cells. Expression of RdgB's PITP domain as a soluble protein (RdgB-PITP) in rdgB2 mutant flies is sufficient to completely restore the wild-type electrophysiological light response and prevent the degeneration. However, introduction of the T59E mutation, which does not affect RdgB-PITP's phosphatidylinositol (PI) and phosphatidycholine (PC) transfer in vitro, into the soluble (RdgB-PITP-T59E) or full-length (RdgB-T59E) proteins eliminated rescue of retinal degeneration in rdgB2 flies, while the light response was partially maintained. Substitution of the rat brain PITPα, a classical PI transfer protein, for RdgB's PITP domain (PITPα or PITPα-RdgB chimeric protein) neither restored the light response nor maintained retinal integrity when expressed in rdgB2 flies. Therefore, the complete repertoire of essential RdgB functions resides in RdgB's PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo. Expression of either RdgB-T59E or PITPα-RdgB in rdgB+ flies produced a dominant retinal degeneration phenotype. Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPα-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism. This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.

scholarly journals Bulk-surface coupling reconciles Min-protein pattern formation in vitro and in vivo

2020 ◽  
Author(s):  
Fridtjof Brauns ◽  
Grzegorz Pawlik ◽  
Jacob Halatek ◽  
Jacob Kerssemakers ◽  
Erwin Frey ◽  
...  
Keyword(s):  
Protein Pattern ◽  
Oscillation Mode ◽  
Coupled Systems ◽  
Protein Patterns ◽  
Spatial Control ◽  
Bulk Surface ◽  
Oscillation Modes ◽  
Min Proteins

AbstractSelf-organisation of Min proteins is responsible for the spatial control of cell division in Escherichia coli, and has been studied both in vivo and in vitro. Intriguingly, the protein patterns observed in these settings differ qualitatively and quantitatively. This puzzling dichotomy has not been resolved to date. Using reconstituted proteins in laterally wide microchambers with a well-controlled height, we show that the Min protein dynamics on the membrane crucially depend on bulk gradients normal to the membrane. A theoretical analysis shows that in vitro patterns at low bulk height are driven by the same lateral oscillation mode as pole-to-pole oscillations in vivo. At larger bulk height, additional vertical oscillation modes set in, marking the transition to a qualitatively different in vitro regime. Our work qualitatively resolves the Min system’s in vivo/in vitro conundrum and provides important insights on the mechanisms underlying protein patterns in bulk-surface coupled systems.

scholarly journals LED Zappelin’: An open source LED controller for arbitrary spectrum visual stimulation and optogenetics during 2-photon imaging

2020 ◽  
Author(s):  
M.J.Y. Zimmermann ◽  
A.M. Chagas ◽  
P. Bartel ◽  
S. Pop ◽  
L.L. Prieto Godino ◽  
...  
Keyword(s):  
Fluorescence Detection ◽  
Colour Vision ◽  
Visual Stimulation ◽  
Low Cost ◽  
User Requirements ◽  
Light Stimulation ◽  
Led Driver ◽  
Two Photon ◽  
Neuroscience Research

AbstractTwo-photon (2P) microscopy is a cornerstone technique in neuroscience research. However, combining 2P imaging with spectrally arbitrary light stimulation can be challenging due to crosstalk between stimulation light and fluorescence detection. To overcome this limitation, we present a simple and low-cost electronic solution based on an ESP32 microcontroller and a TLC5947 LED driver to rapidly time-interleave stimulation and detection epochs during scans. Implemented for less than $100, our design can independently drive up to 24 arbitrary spectrum LEDs to meet user requirements. We demonstrate the utility of our stimulator for colour vision experiments on the in vivo tetrachromatic zebrafish retina and for optogenetic circuit mapping in Drosophila.https://github.com/BadenLab/LED-Zappelin

scholarly journals Spatial control over near-critical-point operation ensures fidelity of ParABS-mediated bacterial genome segregation

2020 ◽  
Author(s):  
Longhua Hu ◽  
Jérôme Rech ◽  
Jean-Yves Bouet ◽  
Jian Liu
Keyword(s):  
Critical Point ◽  
Cell Biology ◽  
Bacterial Genome ◽  
Large Cell ◽  
Spatial Control ◽  
Copy Number Plasmid ◽  
Low Copy Number ◽  
Gradient Based ◽  
System Partitioning

ABSTRACTIn bacteria, most low-copy-number plasmid and chromosomally encoded partition systems belong to the tripartite ParABS partition machinery. Despite the importance in genetic inheritance, the mechanisms of ParABS-mediated genome partition are not well understood. Combining theory and experiment, we provided evidences that the ParABS system – partitioning via the ParA gradient-based Brownian ratcheting – operates near a critical point in vivo. This near-critical-point operation adapts the segregation distance of replicated plasmids to the half-length of the elongating nucleoid, ensuring both cell halves to inherit one copy of the plasmids. Further, we demonstrated that the plasmid localizes the cytoplasmic ParA to buffer the partition fidelity against the large cell-to-cell fluctuations in ParA level. Thus, the spatial control over the near-critical-point operation not only ensures both sensitive adaption and robust execution of partitioning, but sheds light on the fundamental question in cell biology: How do cells faithfully measure cellular-scale distance by only using molecular-scale interactions?

scholarly journals Mechanism of in vivo activation of the MutLγ-Exo1 complex for meiotic crossover formation

2019 ◽  
Author(s):  
Aurore Sanchez ◽  
Céline Adam ◽  
Felix Rauh ◽  
Yann Duroc ◽  
Lepakshi Ranjha ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Double Strand Breaks ◽  
Repair Pathway ◽  
Spatial Control ◽  
Strand Breaks ◽  
Catalytic Function ◽  
Polo Kinase ◽  
Meiotic Crossover ◽  
Temporal And Spatial

AbstractCrossovers generated during the repair of programmed double-strand breaks (DSBs) during homologous recombination are essential for fertility to allow accurate homolog segregation during the first meiotic division. Most crossovers arise through the cleavage of recombination intermediates by the Mlh1-Mlh3 (MutLγ) endonuclease and an elusive non-catalytic function of Exo1, and require the Polo kinase Cdc5. Here we show in budding yeast that MutLγ forms a constitutive complex with Exo1, and in meiotic cells transiently contacts the Msh4-Msh5 (MutSγ) heterodimer, also required for crossover formation. We further show that MutLγ-Exo1 associates with recombination intermediates once they are committed to the crossover repair pathway, and then Exo1 recruits Cdc5 through a direct interaction that is required for activating MutLγ and crossover formation. Exo1 therefore serves as a non-catalytic matchmaker between Cdc5 and MutLγ. We finally show that in vivo, MutLγ associates with the vast majority of DSB hotspots, but at a lower frequency near centromeres, consistent with a strategy to reduce at-risk crossover events in these regions. Our data highlight the tight temporal and spatial control of the activity of a constitutive, potentially harmful, nuclease.

Multicolor in vivo brain imaging with a microscope-coupled fiber-bundle microprobe

2012 ◽  
Vol 101 (23) ◽  
pp. 233702 ◽  
Author(s):  
Lyubov V. Doronina-Amitonova ◽  
Il'ya V. Fedotov ◽  
Olga Efimova ◽  
Maria Chernysheva ◽  
Andrei B. Fedotov ◽  
...  
Keyword(s):  
Brain Imaging ◽  
Fiber Bundle
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