470 nm led

VTA GABA neuron inhibition of NAc CINs was the pathway focus for most of the electrophysiological recordings. In ChAT-ChR2-eYFP mice, CINs could be visualized with eYFP fluorescence. In VGAT-Cre/GAD67-GFP mice, CINs were visualized in the slice preparation as non-GFP neurons amongst predominantly GABA neurons. In mice without fluorescent identification, CINs were identifiable by their large size, morphology, tonic firing (~0.5–3Hz), high membrane capacitance (due to large size), and presence of an I_h current^{40 (link)}. Fluorescent cells were imaged on Olympus BX5 and Nikon Eclipse FNI microscopes with 40x/0.80 n.a. objective lens. The filter cube for GFP detection and ChR2 stimulation was an Olympus GFP U-M6553 cube (Bandpass: 470–490 nm; Barrier: 510–560 nm; dichroic: 505 nm) or Nikon C-FL ENDOW GFP 96343 cube (Bandpass: 450–490 nm; Barrier: 500–550 nm; dichroic: 495 nm). Excitation was performed with a Thorlabs 470 nm LED. Cells were also imaged using infrared differential interference contrast imaging in order to facilitate physiological recordings.

Channelrhodopsin-2 activation was performed at the level of the DRG with an optical fibre (BFL48-400 or BFL48-1000) coupled to a 470 nm LED (Thorlabs, Munich, Germany), unless stated otherwise. Light-intensities, measured with a hand-held power meter (Lasercheck; Coherent, Dieburg, Germany), were 60–80 mW/mm². In VGluT3^−/− mice, dorsal root stimulation was performed by a square current pulse of 0.1 ms duration and at least twice the response threshold, delivered with a suction electrode coupled to a constant current stimulator (A320, WPI, Sarasota, FL, USA), as described previously [27 (link)]. Paired-pulse stimulation was performed every 30 s at paired-pulse intervals of 300 to 500 ms.

Following 3 – 5 weeks of expression, acute slices were prepared as described in Lutas et al., 2019 (link), and widefield fluorescence imaging was performed on an upright microscope (Axioskop 2 plus; Zeiss) equipped with an sCMOS II camera (Prime, Photometrics). Fluorescence excitation for imaging was achieved using a 470 nm LED (Thorlabs). Image acquisition was performed using ImageJ Micro-manager (Edelstein et al., 2014 (link)). Image acquisition frame rate was 2 Hz for cADDis fluorescence imaging. A 10x (Olympus) or 20x (Zeiss) objective was used for all imaging experiments. During imaging, slices were continuously superfused (flow rate: 2–5 ml/min) with oxygenated (95% O₂ and 5% CO₂) artificial cerebrospinal fluid (ACSF) at room temperature. To prevent oxidation of dopamine, 50 μM Na-metabisulfite was included in all ACSF solutions during dopamine application experiments.

In order to select specific whisker regions for imaging and optogenetic inactivation, functional mapping was performed using optical intrinsic signal imaging (ISI) for S1 and a combination of ISI and two-photon calcium imaging for S2. For ISI, the animal was anesthetized with 1.5% isoflurane. The cortical surface was illuminated with a 625-nm LED (Thor Labs). Individual whiskers were stimulated at 10 Hz with a piezo-electric stimulator. Reflectance images were collected through a f = 25mm lens (Navitar) using a CMOS Camera (Hamamatsu; 6.5 um pixel size, 4 x 4 binning, 512 x 512 binned pixels, 30 Hz frame rate). Changes in reflectance during stimulation compared to pre-stimulation were expressed as ΔR/R (150 frame average). Barrel columns were identified as signal minima after averaging intrinsic reflectance signals over 10 trials. A blood vessel map of the cortical area surface was obtained with a 470 nm LED (Thor Labs) for registration and targeting of regions. In situations where ISI maps in S2 were weak, regions were identified and selected for by two-photon calcium imaging of RCamp1.07 signals following whisker stimulation. Whiskers were trimmed to a single row corresponding to the selected imaging or stimulation region.