Colibri system

The FISH protocol used was described previously by Durand et al. [6 (link)]. Briefly, 3%-formaldehyde fixed dissected tissues were embedded in polyethylene glycol distearate-1-hexadecanol (9: 1) resin (Sigma, St. Louis, MO). Resin blocks were then cut into 6–10 μm sections using an RM 2165 microtome (Reichert-Jung, Germany). Sections were hybridized using several published probes (Table 2). The probe sequences have been compared to our sequences to check their specificity and determine their mismatches. The hybridization temperature was the same for all samples treated (46°C). Observations and imaging were performed using an ApoTome Axio Imager Z with a COLIBRI system (Zeiss, Jena, Germany) using ZEN software (Zeiss, Jena, Germany).

Seb1-FRB-GFP localization was detected using fluorescence microscopy as previously described^{7 (link)}. Briefly, liquid cultures were grown in EMM to early log phase (OD_600nm ∼ 0.3) then rapamycin or an equal volume of DMSO was added to a final concentration of 2.5 μg/ml. After 2 h incubation, nuclei were stained using Hoechst 33342 for 15 min (0.2 mg/ml) and live cells were mounted on 1.2% agarose patches. GFP-tagged proteins and nuclei were detected at 470 nm and 365 nm, respectively, using a Colibri system (Carl Zeiss Canada, Toronto, ON, Canada) on a Zeiss Axio Observer Z1 inverted microscope with a ×100/1.4 oil objective. Data were analyzed using the ZEN black software (Carl Zeiss Canada).

After 14–24 days, in vitro spontaneous neuronal activity was optically recorded by calcium imaging. For the experiments, cells were rinsed three times with preheated extracellular patch solution (E-patch) and incubated with 4 μM Fluo-4 AM (Invitrogen) diluted in E-patch for 45 min in the dark at RT. The E-patch contains CaCl₂ (2 mM), HEPES (10 mM), KCl (3 mM), MgCl₂ (1 mM), and NaCl (120 mM), and the pH value of the solution was adjusted with 1M NaOH to 7.3. In case, the osmolarity of the culture medium exceeded the osmolarity of E-patch by more than 10 mOsmol/kg, the osmolarity of E-patch was adjusted to match the value of the medium directly before the experiment with d-(+)-Glucose (Sigma). After incubation with Fluo-4 AM, the substrate was rinsed twice with E-patch, and the final volume of 2 mL E-patch was added to the 35 mm Petri dish. The samples were imaged after an additional rest time of ~10 min in the dark at RT. A Zeiss Observer.Z1 equipped with a Zeiss Colibri system and a PCO.edge 5.5 sCMOS camera was used for sequence acquisition. Time sequences with a length of 15–30 s were recorded with the Zeiss ZEN blue software at an exposure time of 5 ms and a frame rate of 200 frames/s. To achieve this temporal resolution, a 2 × 2 binning and an image size of 512 × 480 pixels (1.30 μm/pixel) were chosen. For video analysis, the sequences were converted to AVI files.

Pac1-FRB-GFP localization was detected by using fluorescence microscopy as previously described (28 (link)). Briefly, liquid cultures were grown in EMM to early log phase (OD600nm 0.3) then rapamycin or an equal volume of DMSO was added to a final concentration of 2.5 μg/ml. After two hours incubation, nuclei were stained using Hoechst 33342 for 15 min (0.2 mg/ml) and live cells were mounted on 1.2% agarose patches. GFP-tagged proteins and nuclei were detected at 470 nm and 365 nm, respectively, using a Colibri system (Carl Zeiss Canada, Toronto, ON, Canada) on a Zeiss Axio Observer Z1 inverted microscope with a ×60/1.4 oil objective. Data were analyzed using the ZEN black software (Carl Zeiss Canada).