Optogenetic manipulation was performed using nVoke 2.0 (Inscopix). For optogenetic activation, 20 Hz, 60-pulse trains (5 ms each) of LED light (620 ± 30 nm, 20 mW/mm2) were initiated every 30 s during the light on epoch 48 (link). For optogenetic inhibition, constant LED light (620 ± 30 nm, 5 mW/mm2) was delivered during the light on epoch 49 (link). The EthoVision XT 15 software and a mini-IO box system (Noldus) were used to record live tracking of mice, and to trigger the laser on during specific behaviors.
Nvoke2
Manufactured by Inscopix
NVoke2 is a compact, lightweight, and portable imaging system designed for in vivo neural activity recording. It provides high-resolution, real-time imaging of neuronal dynamics in freely behaving animals. The system utilizes advanced CMOS sensor technology and LED illumination to capture neural signals with high temporal precision.
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9 protocols using nvoke2
1
Optogenetic Manipulation of Mouse Behaviors
2
Optogenetic Manipulation of Rodent Behavior
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Optogenetic manipulation was performed using nVoke 2.0 (Inscopix). For optogenetic activation, 20 Hz, 60-pulse trains (5 ms each) of LED light (620 ± 30 nm, 20 mW/mm2) were initiated every 30 s during the light on epoch48 (link). For optogenetic inhibition, constant LED light (620 ± 30 nm, 5 mW/mm2) was delivered during the light on epoch49 (link). The EthoVision XT 15 software and a mini-IO box system (Noldus) were used to record live tracking of mice, and to trigger the laser on during specific behaviors.
3
Integrated Calcium Imaging and Optogenetics
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For simultaneous recording of GCaMP6s fluorescence signals and light activation of terminals in the SN, a miniature microscope system for integrated calcium imaging and optogenetics was used (nVoke 2.0, Inscopix). Mice were habituated to the miniscope 3 days before the behavioral experiment. The basal level of calcium activity was recorded in the first 5 min after CNO application. Then, the effect of GPeCRHR1 neuron activity was measured by illuminating ChrimsonR-expressing terminals in the SN (590 nm, 10 mW, 20 Hz) for 5 min. Another 5 min later, CNO reached its full effect on hM3Dq-expressing IPACLCRH neurons. Thus, the recorded activity of cells in the next 5 min reflected both light-induced activation of GPeCRHR1 and CNO-induced activation of IPACLCRH neurons. Cell activities recorded in the last 5 min correspond to CNO-activated IPACLCRH neurons. Image acquisition and behavior were synchronized. For imaging data processing and analysis, we used the Inscopix data processing software (version 1.3.0).
4
In Vivo Calcium Imaging and Optogenetic Manipulation
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Following recovery, DAT::Cre mice were individually housed and food restricted for 2 days prior to recording. Prior to the recording day, food-deprived mice were habituated to handling and the nVoke miniaturized microscope (an integrated imaging and optogenetics system, 450 nm blue GCaMP excitation LED, 620 nm amber optogenetic LED, Inscopix, Palo Alto, CA). 24 hrs prior to recordings, mice were habituated in their homecage to a dimly lit recording room containing constant white noise (Marpac Dohm-DS dual speed sound conditioner, Wilmington, NC, USA). On the recording day, mice were attached to the nVoke miniaturized microscope and habituated in their homecage for 15 min. After the 15 min habituation, a 30 min recording session, composed of 10 min OFF-ON-OFF epochs, was initiated. Gray scale images were collected at 10 frames per second using 0.094-0.266 mW / mm2 (estimated light power based on GRIN lens efficiency) of the miniaturized microscope’s 450 nm LED transmission range (nVoke 2.1.5., Inscopix, Palo Alto, CA). During the ON epoch, 20 Hz, 60p (5 ms pulses) trains of 620 nm LED light were initiated every 30 s for the duration of the 10 min epoch.
5
In Vivo Calcium Imaging and Optogenetic Manipulation
6
In Vivo Calcium Imaging of Neural Activity
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On each day of behavioral experimentation, the miniscope was mounted on the implanted GRIN lens and baseplate immediately prior to the mouse being placed in the behavioral chamber. Custom-written MATLAB code triggered simultaneous video acquisition and nVoke2 (Inscopix) calcium recording, both recording at 20 Hz sampling rate. Parameters of the miniscope, such as the LED power, the gain, and the electronic focus, were adjusted on a mouse-to-mouse basis but otherwise kept consistent for the 5 sequential days of behavioral testing.
Acquired calcium transients, concatenated per recording day with each recording day consisting of the acclimation and behavior time, were processed in the Inscopix Data Processing Software (IDPS, Inscopix). First, the traces were spatially down sampled by a factor of 2, then pre-processed, spatially filtered, and motion corrected. Individual cells were identified using a constrained non-negative matrix factorization algorithm (CNMF). Traces with abnormal physiological calcium transients (i.e., transients lasting over one minute) were excluded. For co-registration purposes, the motion-corrected video was temporally averaged into an image depicting anatomical landmarks. ROIs generated by the CNMF, and their corresponding calcium traces were also exported.
Acquired calcium transients, concatenated per recording day with each recording day consisting of the acclimation and behavior time, were processed in the Inscopix Data Processing Software (IDPS, Inscopix). First, the traces were spatially down sampled by a factor of 2, then pre-processed, spatially filtered, and motion corrected. Individual cells were identified using a constrained non-negative matrix factorization algorithm (CNMF). Traces with abnormal physiological calcium transients (i.e., transients lasting over one minute) were excluded. For co-registration purposes, the motion-corrected video was temporally averaged into an image depicting anatomical landmarks. ROIs generated by the CNMF, and their corresponding calcium traces were also exported.
7
One-photon Calcium Imaging and Optogenetics
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Imaging data were collected using a miniaturized one-photon microscope (nVoke2; Inscopix). GCaMP7f signals (calcium activity) were detected using 435–460 nm excitation LED (0.1–0.2 mW), and optogenetic stimulation of eNpHR-expressing axons was performed using a second 590–650 nm excitation LED (1–2 mW light power). nVoke2 software (Inscopix) was used to control the microscope and collect imaging data. Images were acquired at 20 frames per second, spatially downsampled (4×), and were stored for offline data processing. An input TTL from a separate ANY-maze computer (Stoelting Europe) to the nVoke2 acquisition software were used to synchronize calcium imaging and mouse behaviour movies.
8
Elevated Platform Exploration Assay
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Mice were connected to the nVoke2 (Inscopix) system and recorded with the Inscopix data acquisition software with a 20-Hz sampling rate while behaviorally recorded simultaneously from above using a camera (The Imaging Source) that is controlled by ANY-maze (v5.23, Stoelting) and sampled up to 20 Hz. The mice were then placed in the enclosed center of the elevated platform and recorded while freely moving for 5 min. The elevated platform is constructed by completely blocking the entrance of the closed arms of an EPM (arms with 7.5 cm in width, 30 cm in length separated by a center that is 7.5 cm in width and length and enclosed by 60-cm-tall cardboard pieces on the two sides not connected to the arms). This elevated platform maximizes and forces the recorded mice to explore the anxiogenic exposed open edges. The larger dimensions of this platform also provided more room for the recorded mice to maneuver.
9
Miniscope-based Fear Conditioning
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Implanted were checked bi-weekly until the area below the lens have cleared and GCaMP6m labeled cells can be visualized. To do so, mice were held by the head-bar on a custom-built running wheel while the miniscope (Inscopix) is attached. ZI calcium transients were recorded by the nVoke2 and nVista2 (Inscopix) systems and visualized through the Inscopix Data Acquisition Software (IDAS). Once cells could be visualized, mice were individually handled and habituated to the experimental room (5 mins per day for 3 consecutive days). Following the last handling day, mice were subjected to a 7-day fear conditioning protocol (pre-conditioning, 5 conditioning days, and test day). On each day, the miniscope is first attached and mice were then placed into either a plexiglass box (20cm wide, 25cm long, 15cm tall) covered with polka-dots (context A, pre-conditioning/test days, cleaned with 1% acetic acid) or a different (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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