Picospritzer

Mice were mounted in a stereotaxic frame (Stoelting, 51600, Wood Dale, IL) under isoflurane anesthesia (2.5–4%) and injected with 500 nL of AAV9 (3x10¹³ GC/mL, VVCC), AAV2retro (2.5x10¹⁴GC/mL, VVCC), or Fast-DiI (2.5 mg/ml in dimethylformamide; ThermoFisher, Waltham, MA). A pulled borosilicate glass pipette with a tip diameter of ~20 μm was attached to a picospritzer (Parker Hannifin, Hollis, NH) and a microsyringe (Hamilton, Reno, NV) by way of a three-way stopcock and the dead space was filled with mineral oil. The pipette was attached to a stereotaxic manipulator (Stoelting) and the tip was advanced 3 mm ventral from the level of the meninges through a small burr hole in the skull positioned 1.5 mm left of midline at a location 0.5 mm caudal from true lambda. The injectate was delivered under pressure, the pipette was slowly removed from the brain after a 5min rest period, and the skin was closed with VetBond (3M, St,Paul, MN). Animals were allowed to recover on a heated pad, and meloxicam (2 mg/kg, s.c.) was given for post-operative analgesia for 3 consecutive days. Tissues were collected for analysis 6 (AAV) or 4 (DiI) weeks after injections.

Pericytes were focally stimulated under an upright Nikon FN1 microscope by a current pulse (7 μA, 2 ms; Grass Technologies) using an electrode filled with Ames solution. Electrodes were pulled from borosilicate glass (WPI, 1B150F-4) with a P-97 Flaming/Brown puller (Sutter Instruments, Novato, CA, USA) and had a measured resistance of 3–5 MΩ. For consistency across all experiments, the electrode was placed near the cell body of the targeted pericyte. During focal “puff” stimulation, the electrode solution was supplemented with a vasoactive compound and delivered with picospritzer (Parker Hannifin) via a broken patch pipette positioned above the targeted pericyte. For the light stimulation experiments, the microscope’s illuminator was used to deliver a spot of light that was centered on the targeted pericyte cell body and focused on the photoreceptor cell layer. The tissue was adapted at 30 cd/m², and the stimulus was 270 cd/m². Light spot flicker (40 µm diameter, 10 Hz) was controlled by a shutter (Uniblitz, Vincent Associates). Responses to stimuli were captured on video or time-lapse photos with a microscope-mounted Sony A7s full-frame camera. Images of blood vessels were analyzed in ImageJ, using a region of interest (ROI) tracing tool. At each experimental condition, the capillary lumen cross-sections were mapped at 2 μm steps along the capillary.

Dams underwent abdominal surgery (1–2 cm) at E11.5 to expose the uterine horns. Using a pulled glass pipette attached to a picospritzer (Parker Hannifin, Cleveland, OH, USA), a solution including plasmid DNA encoding yellow fluorescent protein (pCAG-eYFP, 1.5 µg) and siRNA (3 ng/µL; Negative control #1 4457289 or HOTAIRM1 [n520253], Thermo Fisher Scientific) was injected through the uterine wall into the third ventricle (3V) of mouse embryos. Electroporation of the ventral mesencephalon was accomplished using a triple electrode configuration (Figure 3) [33 (link)]. Two positive electrodes were placed laterally and ventrally to the mesencephalon. A single negative electrode was placed dorsal to the mesencephalon to create a ventral electrical field vector. Five electrical pulses were administered (amplitude, 35 V; duration, 50 ms; intervals, 950 ms) with a CUY21 SC electroporation system (Nepa Gene Co. Ltd., Chiba, Japan). The uterine horns were placed back into the abdominal cavity, and the dam was sutured and allowed to recover. For survival rates and transfection data see Supplementary Materials Table S1. This protocol transfected approximately 8% of all cells in the intermediate and mantle zones at E13, the majority of which were immature DA cells (see Figure S4).

Whole-cell patch-clamp recordings from the GF were carried out in 2–4-day-old female flies as described above. For P2X₂ receptor activation of LC4 or LPLC2 VPNs, a glass capillary pulled to a 1-μm diameter was positioned on the VPN dendrites, which expressed both GFP and the P2X₂ receptor, approximately 50 μm below the surface of the brain. ATP (Sigma A9187, 5 mM) was microinjected (5 psi, 200-ms pulse) under the control of a Picospritzer (Parker Hannifin). To test dorsoventral gradients of functional connectivity between the VPNs and the GF, either the dorsal or ventral part of the lobula was stimulated in an alternating fashion at 90-s intervals to permit recovery between pulses. Whole-cell recording data were analysed as mentioned above. Before calculating the peak amplitudes of the GF response, the membrane potential traces acquired during ATP applications were low-pass filtered and averaged across individual trials as specified in the figure legends.