Dm lfs

We used wide-field epifluorescence microscopy for all live imaging experiments (Fig. 1A). An Optoscan monochromator (Cairn Research, Faversham, UK) uniformly illuminated the preparations with 488 ± 15-nm light using either a Leica DM-LFS (Leica Microsystems) or an Olympus X50WI compound microscope (Olympus, Center Valley, PA). Emitted light passed through standard green fluorescent protein (GFP) emission filters before reaching an Andor DU897 EMCCD camera (Andor Technologies, Belfast, UK). Images were captured at 5 or 10 Hz with Andor IQ software and constant gain settings. Optical and electrical recordings were synchronized with pulses generated during each camera exposure. Images were stabilized against lateral shifts in ImageJ (NIH, Bethesda, MD). Fluorescence values were extracted from regions of interest (ROIs) in thoracic (T2–T3) and abdominal (A1–A8/9) ganglia with ImageJ or custom MATLAB scripts. T1 and the SOG were obscured by the brain and were not analyzed. Extracted optical signals were analyzed in ImageJ, MATLAB, and Spike2. Signals are expressed as the percent change in fluorescence from baseline, ΔF/F. Changes of 50% ΔF/F were typical in all segments.

Recordings were obtained from brain slices collected from 4–9 week-old male Long-Evans rats (Charles River). Brains were quickly extracted and submerged into an ice cold, high-sucrose artificial cerebrospinal fluid cutting solution (ACSF; saturated with 95% O₂ and 5% CO₂, pH ≈7.4) containing (in mM) 250 sucrose, 2 KCl, 1.25 NaH₂PO₄, 7 MgCl₂, 26 NaHCO₃, 0.5 CaCl₂ and 10 dextrose. All drugs were obtained from Sigma-Aldrich unless indicated otherwise. Horizontal slices containing the entorhinal region were obtained using a vibratome (300 μM thick; WPI, Vibroslice, Sarasota, USA). There was a recovery period of at least one hour in normal ACSF containing (in mM) 124 NaCl, 5 KCl, 1.25 NaH₂PO₄, 2 MgSO₄, 2 CaCl₂, 26 NaHCO₃, and 10 dextrose (pH ≈7.4; 300–310 mOsm; ~22 ºC). During recordings, individual slices were submerged in ASCF (2 ml/min) and fixed using a nylon net, and were visualized using an upright microscope (Leica, DM-LFS) equipped with a 40x objective and differential interference contrast optics. Layer II of the lateral entorhinal cortex was distinguished from layers I and III based on the presence of clusters of cells [1 (link)].

Experiments were performed using Ca_V1.3 knockout mice (Ca_V1.3^–/–) on a C57BL/6N background and control mice (littermate heterozygous or C57BL/6N). Mice from both sexes were used and ranging from postnatal day 4 (P4) to P35. The semicircular canals with their ampullae and cochleae (apical turn) were dissected out from the inner ear as reported previously (Jeng et al., 2020a (link),2021 (link); Spaiardi et al., 2020a (link); Carlton et al., 2021 (link)), using an extracellular solution composed of (in mM) 135 NaCl, 5.8 KCl, 1.3 CaCl₂, 0.9 MgCl₂, 0.7 NaH₂PO₄, 5.6 D-glucose, and 10 HEPES-NaOH. Sodium pyruvate (2 mM), amino acids, and vitamins were added from concentrates (Thermo Fisher Scientific, United Kingdom). The pH was adjusted to 7.5 (osmolality ∼308 mmol kg^–1). The dissected organs were fixed at the bottom of the recording chamber by a nylon-meshed silver ring and were continuously perfused with the above extracellular solution (0.5 ml/min) using a peristaltic pump (Masterflex L/S, Cole Palmer, United States). Hair cells were viewed using a upright microscopes (Olympus BX51; Leica DM-LFS) equipped with Nomarski Differential Interface Contrast (DIC) optics with a 60X or 64X water immersion objective and x15 eyepieces.

DiD (Thermo Fisher Scientific) was dissolved in 50 μL ethanol to make a saturated solution. This was heated to 55 °C for 5 minutes prior to injection into the fish that had been fixed in 4% paraformaldehyde. Fish were mounted in 2% low melting temperature agarose dissolved in PBS. The dye was pressure injected into the habenula under a compound microscope (Leica DM LFS), using a 20× water immersion objective. For labeling the retina, a saturated solution of DiI (Thermo Fisher Scientific) in chloroform was used. Injections were carried out under a stereomicroscope (Zeiss Stemi 2000). After injections, fish were stored at 4 °C overnight to allow tracing, and then imaged with a 40× water immersion objective on a Zeiss LSM 710 confocal microscope.
CM-DiI (Thermo Fisher Scientific) was dissolved in ethanol (1 μg/μL). Fish were mounted in 2% agarose in E3, injected on a compound microscope, then allowed to recover in E3 at 28 °C for 4 hours.

Fluo-4 AM (Thermo Fisher Scientific, Waltham, MA, USA) and SSip-1 DA (synthesized as described in ref. 32 ) were diluted in a HEPES-buffered saline (HBS; in mM: 137 NaCl, 5.4 KCl, 0.8 MgCl₂, 1.8 CaCl₂, 10 glucose, 10 HEPES (pH 7.4)). DRG neurons were loaded with 5 μM Fluo-4 AM in 0.02% cremophor EL for 45 min at room temperature or with 20 μM SSip-1 DA in 0.02% cremophor EL for 45 min at 37 °C. A coverslip was mounted on an upright microscope (DM LFS, Leica, Heidelberg, Germany) and was perfused with HBS at a rate of 1 ml/min. The recording was started after 15-min perfusion of HBS to let the intracellular probes completely esterized. Fluorescence was recorded every 5 sec with a bandpass filter (excitation at 480/40 nm, emission at 527/30 nm) and a CCD camera (Hamamatsu Photonics, Shizuoka, Japan). Images were acquired using Aquacosmos 2.6 software (Hamamatsu Photonics). Experiments were performed at room temperature.
At the end of the experiments, 50 mM KCl or 30 μM Na₂S₂ was applied to neurons to induce maximal Ca²⁺- or SSip-1-responses, respectively. Unless otherwise described, the amplitudes of Ca²⁺- and SSip-1-responses evoked by tested stimulus were normalized by those to KCl and Na₂S₂. In Ca²⁺ imaging, neurons responded to AITC with the amplitudes over 20% of those to KCl were considered as TRPA1-expressing neurons.

Adult-born GCs expressing RFP or ChR2 were binned in the following age groups: 13–14 dpi (2 wpi), 20–22 dpi (3 wpi), 27–30 dpi (4 wpi), 40–44 dpi (6 wpi), 54–60 dpi (8 wpi) and 75–77 dpi (11 wpi). In previous work we have compared mature neurons born in 15-day-old embryos (which populate the outer granule cell layer), 7-day-old pups and adult mice, finding no functional differences among neuronal groups (Laplagneet al., 2006). Therefore, unlabeled neurons localized in the outer third of the granule cell layer were selected here as unlabeled mature controls. Recorded neurons were visually identified in the granule cell layer by fluorescence (FITC fluorescence optics; DMLFS, Leica) and/or infrared DIC videomicroscopy. Criteria to include cells in the analysis were visual confirmation of fluorescent protein (RFP, Tom, GFP or EYFP) in the pipette tip, attachment of the labeled soma to the pipette when suction is performed, and absolute leak current <100 pA and <250 pAat Vh for GCs and INs, respectively. Since INs are differentially distributed over distinct DG areas, we tried to maintain this proportion on the number of recorded INsin each region (Figure 5; Figures S1 and S2). Recordings shown in Figures 5F–5J and Figure S2 were obtained from SST-Tom localized in the hilus or GCL.