Protocol

Measurement of Free Ca²⁺ Concentration in the Lumen of Neuronal Endoplasmic Reticulum

Adapted from Imaging in Neuroscience and Development (ed. Yuste and Konnerth). CSHL Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

This protocol describes a technique for the simultaneous monitoring of free calcium concentrations in the cytosol ([Ca²⁺]_i) and within the lumen of the endoplasmic reticulum (ER) ([Ca²⁺]_L) of cultured/freshly isolated dorsal root ganglia (DRG) neurons. The method uses two synthetic fluorescent Ca²⁺ probes (fluo-3 and mag-fura-2) in combination with fluorescence microscopy and a whole-cell patch-clamp technique. This approach has been used successfully in acutely isolated/cultured DRG neurons, in Purkinje neurons, acutely isolated from cerebellar slices, and in cultured astrocytes. In this protocol, isolated neurons are first loaded with the membrane-permeant, low-affinity Ca²⁺ indicator, mag-fura-2, which preferentially, though not exclusively, accumulates in the ER. Cells are then loaded with the membrane-impermeant, high-affinity calcium indicator fluo-3 using the whole-cell patch-clamp configuration. This second loading removes the majority of cytosolic mag-fura-2, replacing it with fluo-3. Mag-fura-2 and fluo-3 signals can be separated by virtue of their distinct excitation properties (340 nm and 380 nm for mag-fura-2, and 488 nm for fluo-3). An equation is provided to determine [Ca²⁺]_L values using the 340/380 nm ratio.