Adhesive cap 200

ChAT-ZsGreen neurons were microdissected individually and pressure-catapulted into 0.5 ml tube caps (Adhesive Cap 200, Carl Zeiss) with a single laser pulse using a 40× objective lens and the PALM Microbeam system and Robo Software (Carl Zeiss; https://www.zeiss.com/microscopy/en/products/light-microscopes/laser-microdissection/palm-microbeam.html). Neurons pooled from the MS or the dorsal CPU were stored in the LCM tube caps at −80 °C until RNA extraction. Functionally heterogeneous cholinergic cell groups occur in several compartments in the CPU (39 ). ChINs included in our transcriptomic analysis belong to the dorsal portion of the anterior striatum. Neurons in the shell and the core of the ventral striatum and in the dorsomedial and dorsolateral areas of the posterior striatum were not studied.

Slides were placed into the slide holder of the microscope, and 300 ChINs were microdissected by LCM using a PALM Microbeam system (Zeiss). The cells were pressure-catapulted from the object plane into 0.5 ml tube caps (Adhesive Cap 200, Zeiss) with a single laser pulse using a 40× objective lens. A second control cell pool was prepared from 600 medium-sized neurons most of which corresponded to SPNs. The mean profile areas of ChINs and SPNs were 674.76 µm² and 161.22 µm², respectively. The LCM caps were stored at −80°C until RNA extraction.

We followed our recently published protocol for LCM-assisted dissection of fluorescent neurons. In brief, treated KP-Cre/ZsGreen mice (n=6) were perfused transcardially with 0.5% formaldehyde, followed by 20% sucrose. Brains were snap-frozen and tissue blocks containing the preoptic area were dissected. Then, coronal sections were cut from the preoptic area, collected onto PEN slides (Membrane Slide 1.0 PEN, Carl Zeiss, Göttingen, Germany) and air-dried in the cryostat chamber. Formaldehyde-fixed sections, containing fluorescent KP^RP3V neurons were treated sequentially with 50% EtOH, n-butanol:EtOH and xylene substitution:n-butanol. Three hundred KP-Cre/ZsGreen neurons were microdissected from 12-µm-thick preoptic sections of each mouse. Microdissected cells were pressure-catapulted into 0.5 ml tube caps (Adhesive Cap 200, Carl Zeiss), pooled and were stored at -80 °C until RNA extraction.