CFMEs with a ~60 μm protruding tip were prepared as described before.29 (link) Preparation is detailed in the Supporting Information. A Waveneuro FSCV potentiostat (5 MΩ headstage, Pine Research Instrument) and a PCIe-6363 multifunctional I/O device (National Instruments) were used to apply the waveform and to collect data. HDCV software (provided by R. M. Wightman, University of North Carolina) was used to collect data and for background subtraction. The dopamine waveform (−0.4 V to 1.3 V and back to −0.4 V versus chloridized Ag wire reference electrode) was applied to the CFME every 100 ms at a scan rate of 400 V/s for conditioning and measurements. Electrodes were pre-calibrated and post-calibrated with 1 μM dopamine in a flow cell injection system. For picospritzing, an empty glass capillary was pulled, trimmed, and filled with acetylcholine (ACh) for stimulation. A Picospritzer III (Parker Hannfin) was used to pressure eject ACh into the brain tissue. Capillaries were calibrated by picospritzing a droplet of ACh in oil and the diameter of the pressure-ejected droplet was measured using DS-Qi2 monochrome CMOS camera and NIS-Elements BR imaging software (Nikon Instruments Inc.). For all experiments, a volume corresponding to 0.2 pmol ACh was ejected using a constant applied pressure of 20 psi.