Zirconia sleeve

Mice were allowed to adapt to the fiber patch cord for at least three days prior to experiments and typically not handled on the day of the experiment. Fiber optic cables (200 m diameter; NA: 0.22, 1 m long; Doric Lenses; or, 0.5m long, ThorLabs) were connected to the implanted fiber optic cannulas with zirconia sleeves (Doric Lenses) and coupled to lasers via a fiber optic rotary joint (Doric Lenses). We adjusted the light power of the laser (473 nm; Laserglow or Opto Engine) such that the light power (measured with a fiber optic power meter; PM20A; ThorLabs) at the end of the fiber optic cable was ~10 mW. Using an online light transmission calculator for brain tissue (http://web.stanford.edu/group/dlab/cgi-bin/graph/chart.php), we estimated the light power at the DMH or RPa between 3 and 6 mW/mm2. This is an upper limit due to possible light loss between the fiber optic cable and the implanted optic fiber. Light pulses were controlled by a waveform generator (Arduino) programmed to deliver light pulses. In most experiments (unless otherwise indicated), stimulation was on for 1 s, followed by 3 s off, pulses were 10 ms delivered at 20 Hz. After the completion of experiments, fiber placement and ChR2 expression were assessed. Animals without ChR2 expression or incorrect placement of optic fibers were excluded from analysis.

Patch cords (1 m long, 200 mm core diameter; Doric Lenses) were connected from a rotary joint (Doric Lenses) to monofibre optic cannulae through zirconia sleeves (Doric Lenses). A custom-made lickometer (Arduino) was used to detect licks and drive photostimulation upon each lick (10 Hz, 10 ms pulse width, 1 s duration). Blue light was generated from a laser (473 nm; Laserglow) at an intensity of 8.2–11.3 mW as measured at the tip of the optic cannula. We estimated the light power at the LPBN to be around 9.2–12.67 mW/mm² and at the CeA to be 14.4–21 mW/mm² as calculated by an online light transmission calculator (http://web.stanford.edu/group/dlab/cgi-bin/graph/chart.php).

Fibre optic cables (1.5 m long, 200 mm diameter; Doric Lenses) were firmly attached to the implanted fibre optic cannulae with zirconia sleeves (Doric Lenses). During occlusion and PVH^MC4R→LPBN photostimulation experiments, light pulse trains (10-ms pulses of 20 Hz; 1 second on, 3 seconds off; 5 seconds on, 2 seconds off, respectively) were programmed using a waveform generator (PCGU100; Valleman Instruments or Arduino electronics platform) that provided input to a blue light laser (473 nm; Laserglow). We adjusted the light power of the laser such that the light power exiting the fibre optic cable was at least 10 mW using an online light transmission calculator for brain tissue http://web.stanford.edu/group/dlab/cgi-bin/graph/chart.php. We estimated the light power at the PVH, BNST, LH and LPBN to be 6.01, 4.99, 4.99 and 11.25 mW/mm², respectively. Note that this is probably a high estimation because some light was probably lost at the interface between the fibre optic cable and the implanted fibre optic cannula. After the completion of photostimulation experiments, mice were perfused and the approximate locations of fibre tips were identified based on the coordinates of Franklin and Paxinos.⁶³