Sf 28

The methods for fura-2-based Ca²⁺ imaging using acute SCN slices were described previously12 (link). The methods for YC imaging using SCN slice cultures were also described previously33 (link)47 (link). For SCN2.2YC cell imaging, the cells were seeded onto laminin-coated glass-bottom dishes (35-mm) and cultured as described above in a CO₂ incubator (cell density: 1–3 × 10⁵ cells/dish). The culture medium was gently rinsed from the dishes using standard BSS consisting of (in mM) 128 NaCl, 5 KCl, 2.7 CaCl₂, 1.2 MgCl₂, 1 Na₂HPO₄, 10 glucose and 10 HEPES/NaOH (pH 7.3). SCN2.2YC cells were placed on a microscope stage and continuously perfused with BSS at a flow rate of 2 ml/min through an in-line heater (SF-28; Warner Instruments, Hamden, CT) set at 36°C. 5-HT receptor agonists and antagonists were delivered to the cells by switching the perfusate.

Rats were anesthetized, ventilated, and an infusion cannula (Brain infusion kit 2, Alzet) was stereotactically placed in the right lateral ventricle. A 0.5 mm (diameter) burr hole was drilled (1.3 mm posterior, 1.8 mm lateral to bregma), and a 4 mm (length) brain infusion cannula (Brain infusion kit2, Alzet) was glued in place on the cranium with the cannula placed into the lateral ventricle, through which a pre-heated (37 °C, SF-28, Warner Instruments) HCO₃⁻-aCSF containing 1 mg/ml TRITC-dextran (tetramethylrhodamine isothiocyanate-dextran, MW = 150000; T1287, Sigma) was infused at 9 µl/min. CSF was sampled from cisterna magna at 5 min intervals with a glass capillary (30–0067, Harvard Apparatus pulled by a Brown Micropipette puller, Model P-97, Sutter Instruments) placed at a 5° angle (7.5 mm distal to the occipital bone and 1.5 mm lateral to the muscle-midline). The cisterna magna puncture and continuous fluid sampling prevents elevation of ICP during the procedure. The fluorescent content of CSF outflow was measured in triplicate on a microplate photometer (545 nm, SyneryTM Neo2 Multi-mode Microplate Reader; BioTek Instruments), and the CSF secretion rate was calculated from the equation [56 (link)]: $$Vp=ri\times \frac{Ci-Co}{\mathit{Co}}$$ where V_p = CSF secretion rate (µl/min), r_i = infusion rate (µl/min), C_i = fluorescence of inflow solution, C_o = fluorescence of outflow solution.