Single-Cell Resolution Ontogenetic Spiking
(a) Illustration of highly targeted optical stimulation of a single ChR2-mCherry expressing neuron in the mouse somatosensory cortex. Using a small (25 μm diameter), low-powered (3 mW) spot of illumination centred on the target cell, action potentials could be induced in current clamp (top trace), without any indication in voltage clamp of post-synaptic currents caused by spiking in other neurons (bottom trace).
(b) Illustration of illumination with a large (125 μm diameter), high-powered (15 mW) spot. Multiple spikes were induced in current clamp, and voltage clamp traces showed evidence of post-synaptic currents caused by spiking in other neurons.
Matthew Tran & Dr Blake Richards
University of Toronto, Canada.
ChR2 Assisted Circuit Mapping (CRACM)
(A) Image of an acute brain slice prepared from a Thy1-ChR2-EYFP mouse with ChR2 expression in L5 pyramidal neurons. Whole-cell patch clamp recording from a L2/3 cell.
(B) Light-activated excitatory postsynaptic potentials (EPSPs) triggered by patterned illumination of a 10×10 grid with 473 nm LED.
(C) Colormap of the activation pattern.
(D) Histogram of automatically measured responses from all cells in a grid. Objective lens is 10X.
Qiuyu Wu & Dr Alexander Chubykin,
Purdue University, USA.
Optogenetic Stimulation of Synaptic Inputs
Light-evoked inhibitory postsynaptic current (IPSC) recorded in a transgenic mice expressing ChR2 in GABAergic neurons.
The postsynaptic cell is non-GABAergic (ChR2 negative) and blue light stimulates GABAergic afferents expressing ChR2.
Blue bars indicate the time of light illumination.
Spot 1 illuminated by Polygon400 evoked reliable IPSCs whereas Spot 2 caused no response.
Dr Wataru Inoue,
University of Western Ontario, Canada.
Light-Modulated Protein-Protein Interactions on a Coverslip Surface
This video shows a protein-protein interaction that is inhibited by blue light exposure.
One component is coupled to the coverslip through biotin surface chemistry, the other is labeled with mCherry, and binds to the coverslip in the dark.
Patterned illumination with the Polygon400 results in revesible dissociation of the mCherry-tagged protein, which rebinds within minutes of turning off blue light exposure.
The Wittmann lab is working on developing this into a cell adhesion surface that can be controlled by light.
Dr. Torsten Wittmann,
University California San Francisco, USA.
Mapping Optically Induced Depolarization in ChR2-expressing Hippocampal Neurons using Polygon400
E18 Sprague-Dawley rat neurons were transduced with CamKII-ChR2-GFP lentivirus. Somatic activity was recorded via whole-cell patch-clamp electrophysiology.
Each field was illuminated by the Polygon400 at 100% power and 20ms exposure time. Intensity of magenta pattern represents depolarization with respect to the instantaneous resting potential prior to stimulation with the Polygon’s 470nm LED.
In the image, green represents the magnitude of GFP signal and black represents the fluorescence intensity of AlexaFluor 594 backfilled by the patch pipette.
Dr. Jacob Robinson,
Rice University, USA.
Photopatterning of an Acrylate Film
A 50 um film of liquid tetra (ethylen glycol) diacrylate, containg 1 wt% Irgacure 819 photointiator was irradiated through a glass coverslip, using the Polygon400 and a 400nm LED light source to project a pattern onto the film surface.
(A) Projection of CU logo image using the 400nm LED and a 4X objective.
(B) Brightfield image of the resulting pattern in the film. Photopolymerization causes a large change in refractive index in the resin, allowing immediate visualization of the pattern.
Standard development techniques could subsequently be performed by washing the film in solvent to remove the unexposed areas of liquid resin.
University of Colorado, USA.
Achieving Subcellular Resolution of Input Activation in a 250um-thick Acute Cortical Section Using the Polygon400
NMDA-receptor mediated synaptic currents recorded at a +40mV membrane potential were elicited by light activation of channelrhodopsin-expressing terminals of thalamocortical afferents onto an upper layer cortical GABAergic interneuron.
Moving the 470nm rectangular illumination to the right or the left of the cell activates inputs of different strength innervating different somatodendritic domains of the cell.
In black and red are the individual and the average traces respectively.
Dr. Theofanis Karayannis,
University of Zurich, Switzerland.
In Vivo Optogenetic Control of Zebrafish Larva Using Polygon400 Illuminator
A) Light-evoked response of a head-fixed larva expressing channelrhodopsin (right). Photostimulation site was indicated by a blue circle (470nm).
B) A schematic diagram of the Polygon400 patterned illumination in in vivo optogenetic mapping system.
Dr. Sachiko Tsuda,
Saitama University, Japan.
Uncaging RuBi Glutamate with the Polygon400 to Study Neuronal Networks in the Cerebellar Cortex
An example of connectivity mapping that allow to reproduce some results from in Valera et al. (elife, 2016).
100um RuBi glutamate was uncaged at various locations in the granular layer with 20ms pulses (blue bar), exciting notably granule cells.
We can then measure the spatial organization of the granule cells to Purkinje cell (PC) connections by recording PCs in whole cell patch clamp. Granule cells triggers both monosynaptic excitatory current onto PCS (left map, measured a -60mW), but also disynaptic inhibitory currents via molecular layer interneurons (right map, measured at 0 mW).
Average evoked responses at one location (dotted blue square) are shown at the bottom of the figure. Data storage, measurements, and map representations can be made online, using a homemade software in python.
Dr. Antoine Valera & Dr. Angus Silver,
University College London, UK.
Optogenetic Activation of Channelrhodopsin in Transfected Hippocampal Neurons Using Mightex’s Polygon400
A) Patch-clamp recording of the current generated by the channelrhodopsin when activated with the blue LED light from the Polygon400 illuminator as indicated by the blue bar.
B) Activation of an action potential (upper trace) and the corresponding current under voltage-clamp conditions (lower trace).
The action potential could be evoked by a 0.5ms stimulation of the blue LED light from the Polygon400 illuminator at 60% intensity.
Dr. Hans van Hooft,
University of Amsterdam, Netherland.
Using Polygon400 to Stimulate Hippocampal Slices Acutely Prepared from Mice Transgenically Expressing ChR2 in the Dentate Gyrus
The first figure a repetitive stimulation with a larger circular pattern which elicited increasing responses until an action potential is generated.
The second figure is increasing the intensity of stimulation from 30% to 100% and keeping the size of the pattern the same.
Here we’re driving the cell at 10Hz at 100% and one can see that the stimulation is sufficient to drive doublets of action potentials during each bout of depolorization.
Dr. Geoffrey G. Murphy,
University of Michigan, USA.
Local Photostimulation of Channelrhodopsin-2 Using Polygon400 Illuminator
A) Acute brain slice from a YFP-channelrhodopsin-2 (ChR-2) mouse depicting its expression in cortical L5 pyramidal neurons.
B) L5 pyramidal neuron from the somatosensory cortex filled with Alexa 594 to allow visualization of neuronal compartments without stimulating ChR-2.
Blue dots indicate on the illuminated areas (Blue LED – 470 nm) along the apical dendrite. Expansion of the marked areas depicting the delicate dendrites that were stimulated.
C) Electrophysiological (patch clamp) current recordings from the soma, corresponding to local photostimulation of ChR-2 by blue light.
The numbers in the bottom of the trace corresponds to the stimulated dot numbers as indicated in B.
Dr. Yossi Buskila,
University of Western Sydney, Australia.
Purkinje Cell Firing Properties Unveiled by Optogenetic Activation of the Cerebellar Granular Layer Using Variable Light Patterns with Mightex Polygon400
Purkinje cell (PC) firing is monitored, while various light stimulation patterns are delivered to the granular layer (gl) and/or molecular layer (ml) of acute mouse cerebellar slices.
The mice cerebellum was injected with AAVs carrying the ChR2 construct. The figure shows the firing frequency increase caused in a PC unit (scale bar 500ms) by optogenetic activation of a granular layer ROI (blue rectangle).
Dr. Lisa Mapelli & Dr. Simona Tritto,
University of Pavia, Italy.