An optic fiber (0.2 mm core diameter) driven by a blue LED (470 nm, Doric Lenses) was placed ~0.5 mm above the exposed SC to photostimulate ChR2-expressing cells. The intensity of LED light was ~160 mW/mm2 at the tip of the optic fiber in all recordings, which was confirmed to be reliably effective in activating ChR2-expressing neurons (Shi et al., 2017 (link)).
Blue led
Manufactured by Doric
Sourced in Canada
The Blue LED is a light-emitting diode that emits blue light. It is a semiconductor device that converts electrical energy into blue-colored light.
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8 protocols using blue led
1
Optogenetic Activation of ChR2 Neurons
2
Optogenetic Activation of MCH Neurons
3
Photostimulation of ChR2-expressing Cells
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To photostimulate ChR2-expressing cells, we used an optic fiber (0.2 mm core diameter) driven by a blue LED (470 nm, Doric Lenses) placed ~0.5 mm above the exposed SC. The tip of the LED fiber was placed at a similar position in all mice. During recordings, it was buried in the agarose that was applied to reduce the pulsation of the brain and protect the tissue. To prevent direct photostimulation of the eyes by the LED light, the Metabond used for mounting the head plate was prepared with black ink. The agarose surface was painted with black ink, and a piece of thick black paper was carefully placed around the fiber to ensure that light could not be seen from the front and sides, as described before48 (link). The LED was driven by a square wave starting from 500 ms before the onset of each visual stimulus and ending at 100 ms after the offset of each visual stimulus (3600 ms for sweeping bars, as described below). The intensity of LED light was ~160 mW/mm2 at the tip of the optic fiber in all recordings, which was confirmed to be reliably effective in silencing SGS excitatory neurons (Supplementary Fig. 2 ).
4
Photostimulation of ChR2-expressing Cells
5
Optogenetic Manipulation of PBN Neurons
6
Optogenetic Silencing of V1 in Mice
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An optical fiber (1 mm) coupled to a blue LED (470 nm; Doric Lenses) was placed over V1 above the intact skull covered with a thin layer of Krazy glue. The fiber was placed at approximately the retinotopic location corresponding to the stimulus during the initial 350 ms in the task: ~2.3 mm from midline and ~1.3 mm from lambdoid suture. To find these coordinates, we recorded multiunit activity in V1 with the monitor in the same position as during optogenetic silencing (monitor was moved <15 degrees to center the spatial receptive field of the multiunit activity). We used these same approximate coordinates for all of our recordings.
For each animal, the total power was increased until the performance was at chance level when the LED illumination started before the stimulus appeared (3.3–20 mW across animals; p>0.05; Wilcoxon ranksum test on stimulus centering times in the reward zone). To turn the LED on at specific delays after stimulus onset, the photodiode signal detecting the onset of the stimulus was sent to an amplifier (Newark; TWLUX - TW-MF2CAB) and then to an external microprocessor (Mega 1280; Arduino). The microprocessor waited for the amplified photodiode signal to cross a threshold before sending out a digital trigger to the LED driver (Thorlabs). The jitter (s.d.) of the LED onset was 4 ms.
For each animal, the total power was increased until the performance was at chance level when the LED illumination started before the stimulus appeared (3.3–20 mW across animals; p>0.05; Wilcoxon ranksum test on stimulus centering times in the reward zone). To turn the LED on at specific delays after stimulus onset, the photodiode signal detecting the onset of the stimulus was sent to an amplifier (Newark; TWLUX - TW-MF2CAB) and then to an external microprocessor (Mega 1280; Arduino). The microprocessor waited for the amplified photodiode signal to cross a threshold before sending out a digital trigger to the LED driver (Thorlabs). The jitter (s.d.) of the LED onset was 4 ms.
7
Optogenetic Manipulation of PBN Neurons
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The same criteria were used for mouse handling as described above. Prior
to experiments, fiberoptic cannulas implanted into the PBNL were
attached through zirconia sleeves (Doric Lenses) to branching fiberoptic
patchcords (Doric Lenses) connected to a blue LED (Doric Lenses) and a
programmable LED driver (Doric Lenses). Optogenetic stimulation was controlled
by the combination of custom programs written in Bonsai software and Doric
Neuroscience Studio (4.1.5.2) through an Arduino circuit board (Uno, Arduino)
and custom sketches written in Arduino software (1.8.7). Approximately the
initial half of the experiments were performed and analyzed by investigators who
were not blinded to genotype, and the remainder of the experiments were repeated
by investigators who were blinded to genotype.
to experiments, fiberoptic cannulas implanted into the PBNL were
attached through zirconia sleeves (Doric Lenses) to branching fiberoptic
patchcords (Doric Lenses) connected to a blue LED (Doric Lenses) and a
programmable LED driver (Doric Lenses). Optogenetic stimulation was controlled
by the combination of custom programs written in Bonsai software and Doric
Neuroscience Studio (4.1.5.2) through an Arduino circuit board (Uno, Arduino)
and custom sketches written in Arduino software (1.8.7). Approximately the
initial half of the experiments were performed and analyzed by investigators who
were not blinded to genotype, and the remainder of the experiments were repeated
by investigators who were blinded to genotype.
8
Optogenetic Silencing of Visual Cortex
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Visual cortex was silenced by illuminating the V1 craniotomy with a 1mm fiber optic coupled to a blue LED (470 nm; 20 mW total output, Doric) positioned several mm above the craniotomy or through the objective (20x) of a fluorescence microscope with a blue LED (470 nm, 2.3 mW total output, Thorlabs) coupled to the excitation port. The LED turned on 650 ms prior to the onset of a visual stimulus trial and lasted throughout the duration of the visual stimulus. Trials with cortical silencing were interleaved with trials without illumination in which cortical activity was intact. To validate the effectiveness of cortical silencing, spiking responses of V1 neurons in response to drifting gratings were recorded using loose-
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