Examiner z1

Images were acquired using an Olympus FV1000 confocal microscope equipped with a 20 and 40x water and 60X oil immersion objectives using the 405, 473 and 543 nm laser lines or using an upright microscope (Examiner Z1, Zeiss) equipped with a spinning disk head (CSU-X1, Yokogawa) and a modular laser light source (LasterStack, 3i Intelligent Imaging Innovations). To determine the overlap of GFP in the Tg(pkd2l1:GCaMP5G)icm07 transgenic embryos and larvae with pkd2l1, sst1.1 FISH or GABA and 5-HT IHC, fish were mounted laterally in 1, 5% agarose covered of Vectashield Mounting Medium (Vectorlabs, CA, USA). To analyze apical extensions, slices were transferred into Vectashield Mounting Medium as well (Vectorlab, CA, USA).

Brain slices were prepared with a Vibratome (VT1200s, Leica) in ice-cold, oxygenated artificial CSF (aCSF) containing 110 mM NaCl, 0.5 mM CaCl2, 2.5 mM KCl, 7 mM MgCl2, 1.3 mM NaH2PO4, 1.3 mM Na-ascorbate, 0.6 m M Na-pyruvate, 25 mM NaHCO3, and 20 mM Glucose, and then incubated in warm oxygenated aCSF (34 °C) for 1 hour. Brain slices (350 μm) containing the hippocampal region were transferred into the recording chamber and super-fused (2 ml/min) with oxygenated aCSF at room temperature (22–25 °C). Whole-cell patch recordings were performed with a computer-controlled amplifier (MultiClamp 700B, Molecular Devices). The pipettes for cell-attached recording (3–4 MΩ) contained bath solution and the seal resistance was ∽200 MΩ. GFP-positive neurons were targeted in the DG area under the visual guidance of green, fluorescent signals using an upright microscope (Examiner Z1, Zeiss). CNO (10 μM) dissolved in aCSF was released to the recording area with a small pressure using an 8-channel drug-delivery system (MPS-1, Inbio Life Science Instrument).

Stained embryos were mounted in 75% glycerol at 4  ${}^{\circ }$ C and imaged using a Zeiss Examiner Z1 confocal microscope with a $20\times$ plan NA 0.8 objective. All depicted images show a maximum projection of a z-stack unless differently stated in the figure legend.

After transcardial perfusion, brains were post fixed in 4% PFA for at least 24 h. Medullary slices were cut at a thickness of 150 µm using a Leica VT1200s vibratome. After imbedding in Dako Fluorescence Mounting (Agilent), slices were placed under an upright epifluorescence microscope Examiner Z1 (Zeiss, Göttingen, Germany), illuminated with a SOLA SE lamp (Lumencor, Beaverton, OR, USA) and a dual band filter (EGFP/mCherry; AHF Analysentechnik AG, Tübingen, Germany). Images were captured with an iPhone SE (Apple, Cupertino, CA, USA), which was attached to the ocular with a smartphone adapter (Bresser GmbH, Rhede, Germany).

Whole cell patch clamp experiments were made at 32 °C in a submersion recording chamber under an Examiner Z1 (Zeiss, Germany) upright microscope. Slices were bathed in the saline described above (without glutathion and sodium pyruvate), at a rate of 3 mL/min. Whole-cell recording pipettes (4–6 MΩ) contained (in mM) 140 K-gluconate, 11 EGTA, 10 HEPES, 1 CaCl₂, 2 ATP-Mg, and 0.4 Na-GTP for the current clamp experiments in the cortex. They contained an otherwise similar solution to the exception of K-gluconate, replaced by CsCl, and addition of 2 mM QX314, for the voltage clamp experiments performed in the STN. In the subset of experiments that established the I.V. curve and conductance of AMPA EPSCs, a specific intra-pipette solution was used. Its composition was (in mM): 120 cesium methanesulfonate, 10 NaCl, and 5 TEACl, in addition to 11 EGTA, 10 HEPES, 1 CaCl₂, 2 ATP-Mg, 0.4 Na-GTP and 2 QX314. In all cases, the osmolarity of intra-pipette solutions was between 280 and 300 mOsm and pH was adjusted to 7.25. Cells were visualized under IR-DIC and fluorescence microscopy. Experiments were conducted with Axon Instruments hardware and software (Axopatch-1D, Digidata 1322 A, and PClamp V9.2; Molecular Devices, USA), with the amplifier filter set at 5 kHz, and the digitization rate at 20 kHz. Voltages were corrected off line for liquid junction potentials.