C2741 79

SCN slices (300-μm thickness) were prepared as described above using a vibrating blade microtome from male Wistar rats (12–16 days of age) and preincubated in a chamber with oxygenated normal ACSF containing (in mM) 124 NaCl, 3 KCl, 2.5 CaCl₂, 1 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃ and 10 glucose for 1 h at room temperature. Subsequently, the slices were transferred to an upright microscope stage (BX-50WI; Olympus, Tokyo, Japan). The recording chamber was perfused with oxygenated normal ACSF at 1 ml/min at room temperature. SCN neurons were visualised through an infrared charge-coupled device (CCD) camera (C2741-79; Hamamatsu Photonics, Hamamatsu, Japan). The electrodes were filled with internal solution containing (in mM) 150 CsCH₃SO₃, 5 KCl, 5 HEPES, 0.1 EGTA, 3 Mg-ATP and 0.4 Na-GTP (pH 7.3; electrode resistances: 3–6 MΩ). Neurons were recorded in the whole-cell voltage-clamp mode with a holding potential of −60 mV using a patch-clamp amplifier (Axopatch 200B; Axon Instruments, Union City, CA). Series resistance was compensated. The output of the amplifier was digitised using an A/D converter board (Digidata 1200; Axon Instruments) with a sampling rate of 10 kHz, and recorded on a hard disk by data acquisition software (pCLAMP 8; Axon Instruments). Membrane potentials were low-pass-filtered at 2 kHz.

For observation of DUM2-L neuronal projections, we performed intracellular staining with Lucifer Yellow, as previously described [29 (link)]. Cell bodies were visualized with a fixed-stage upright microscope (BX50WI or BX51WI; Olympus) equipped with DI differential interference contrast optics and long-working distance objectives (x40 LUMPlanFL/IR or x60 LUMPlanFL/IR water immersion), and a CCD camera (C2741-79; Hamamatsu Photonics) to enhance the contrast. Cell clusters on the brain surface could be clearly visualized with this system, allowing us to select cells for microelectrode insertion. In this study, we intended to stain DUM2-L neurons. Large cell bodies in the DUM cluster of the SOG were visualized and impaled with a microelectrode filled with Lucifer Yellow CH (4% in 0.1 mol/L LiCl₂; Sigma-Aldrich). After impaling the soma, we iontophoretically injected the Lucifer Yellow using a 1 to 10 nA constant hyperpolarizing current for 3 to 5 minutes. This method efficiently stains the neurons. The obtained images were Z-stacked with maximal intensity using ImageJ software.