Calcium Imaging Protocol

(1/14/00, by Yong Lu, modified from Zirpel et al. 1998)

Slice preparation

Chicken embryos (E17-E19) were used to obtain brainstem slices. After decapitating an embryo, a thick coronal section containing the entire brain stem was dissected free into 4ûC ice cold artificial cerebrospinal fluid (ACSF). The components of ACSF were (in mM): 130 NaCl, 3 KCl, 1.25 NaH₂PO₄, 26 NaHCO₃, 1 MgCl₂, 10 glucose, 2 CaCl₂. Three to five coronal slices (300 m m in thickness) containing bilateral nuclei of magnocellularis (NM) were cut by using a vibratome. All manipulations were performed in ACSF. Slices were equilibrated in oxygenated ACSF in a slice container at room temperature for 40 min. After equilibration, slices were loaded by 8 m M fura-2 AM (molecular Probes, Eugene, OR), with 0.24% anhydrous dimethylsulfoxide (DMSO; Aldrich Chemical, Milwaukee, WI), and 0.02% Pluronic F-127 (Molecular Probes) at 37ûC for 25 min. Loading solutions were prepared as followings: 50 m g fura-2 AM were dissolved in 10 m l DMSO to obtain 5 mM stock solution; 4.8 m l fura-2 AM stock solution was mixed with 3 m l Pluronic F-127 20% in DMSO; then 3 ml ACSF were added; solution was vortexed for at least 2 min at each step. Slices then were washed with ACSF at least twice and moved into the slice container. One slice was placed in a Teflon imaging chamber and fixed at position by a single thread nylon-netted plantimium harp. The chamber was superfused with oxygenated ACSF at a rate of about 3 ml/min.

Calcium imaging

Fluorometric imaging techniques were used to monitor the intracellular calcium concentration ([Ca²⁺]_i). One slice containing fura-2-loaded NM cells was placed in the imaging chamber on the stage of a Nikon Diaphot inverted microscope and alternately excited with 340 and 380 nm wavelengths of light attenuated 3 or 10% by an in-line neutral density filter. Fura-2 fluorescence emission was obtained at 510 nm by an intensified charge cooled device (CCD) (Hamamatsu, Japan) using a X40 fluor oil immersion lens (Nikon) and a 480-nm long-pass barrier filter. Paired 340/380 fluorescence ratio images were acquired every 3 s for transient [Ca²⁺] _i changes and 180 s for basal level [Ca²⁺] _i. Eight to 20 NM cells with >50 pixel ratio images were randomly chosen for analysis. Neurons were outlined using Image-1/Fluor software (Universal Imaging, West Chester, PA). Ratios (340/380) were converted into [Ca²⁺] _i by the Image-1/Fluor software with an external high-low calibration technique. On-line ratio values were converted to [Ca²⁺] _i by using the calibration curve and assuming the dissociation coefficient (K_d) of fura-2 to be 224 nM (Grynkiewicz et al. 1985).

Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biochem 260:3440-3450.

Zirpel L, Lippe WR, Rubel EW (1998) Activity-dependent regulation of [Ca²⁺]_i in avian cochlear nucleus neurons: roles of protein kinase A and C and relation to cell death. J Neurophysiol 79:2288-2302.