Properties of the new fluorescent Na⁺ indicator CoroNa Green: Comparison with SBFI and confocal Na⁺ imaging

Abstract

Neuronal activity causes substantial Na⁺ transients in fine cellular processes such as dendrites and spines. The physiological consequences of such Na⁺ transients are still largely unknown. High-resolution Na⁺ imaging is pivotal to study these questions, and, up to now, two-photon imaging with the fluorescent Na⁺ indicator sodium-binding benzofuran isophthalate (SBFI) has been the primary method of choice. Recently, a new Na⁺ indicator dye, CoroNa Green (CoroNa), that has its absorbance maximum at 492 nm, has become available. In the present study, we have compared the properties of SBFI with those of CoroNa by performing Na⁺ measurements in neurons of hippocampal slices. We show that CoroNa is suitable for measurement of Na⁺ transients using non-confocal wide-field imaging with a CCD camera. However, substantial transmembrane dye leakage and lower Na⁺ sensitivity are clearly disadvantages when compared to SBFI. We also tested CoroNa for its suitability for high-resolution imaging of Na⁺ transients using a confocal laser scanning system. We demonstrate that CoroNa, in contrast to SBFI, can be employed for confocal imaging using a conventional argon laser and report the first Na⁺ measurements in dendrites using this dye. In conclusion, CoroNa may prove to be a valuable tool for confocal Na⁺ imaging in fine cellular processes.

Introduction

The inwardly directed Na⁺ gradient energizes the vast majority of transport systems across the plasma membrane and is critical for homeostasis of intracellular ions such as Ca²⁺ or protons and for reuptake of transmitters in the brain (Maragakis and Rothstein, 2004, Rose, 1997). Consequently, Na⁺ entry is a significant factor in cellular brain damage observed following diverse pathological conditions (Pinelis et al., 1994, Pisani et al., 1998, Chen et al., 1999, Chatton et al., 2000, Sheldon et al., 2004b, Magistretti and Chatton, 2005). Moreover, Na⁺ ions are the major charge carriers during action potentials and excitatory postsynaptic currents in most neurons. Besides their purely homeostatic function, several studies indicate that Na⁺ ions have a signaling function and play a role in activity-dependent synaptic plasticity (Bouron and Reuter, 1996, Chatton et al., 2000, Chinopoulos et al., 2000, Linden et al., 1993, Rishal et al., 2003, Yu and Salter, 1998).

In contrast to large-volume fibers, in which electrical signaling requires only small ionic fluxes and does not change the intracellular Na⁺ concentration ([Na⁺]_i) significantly (e.g. Hodgkin and Huxley, 1952) activity-induced Na⁺ accumulations have been reported from fine cellular processes such as dendrites (Callaway and Ross, 1997, Jaffe et al., 1992, Knöpfel et al., 2000, Lasser-Ross and Ross, 1992). In hippocampal neurons, synaptic stimulation causes [Na⁺]_i transients of about 10 mM in dendrites and of up to 35–40 mM in dendritic spines (Rose et al., 1999, Rose and Konnerth, 2001).

Many questions concerning the physiological consequences of [Na⁺]_i transients and the role of Na⁺ ions in intracellular signaling are still open. Clearly, high-resolution [Na⁺]_i imaging close to synapses and in axons is necessary to answer these questions. In contrast to imaging intracellular Ca²⁺ transients, high-resolution [Na⁺]_i imaging has been, up to date, a rather tedious and difficult task. This is partly due to the scarcity of suitable fluorescent indicator dyes. Imaging with the sodium indicator Sodium Green, which has its absorption maximum around 488 nm, has been proven useful in a variety of studies (Friedman and Haddad, 1994, Senatorov et al., 2000, Winslow et al., 2002). However, interactions of this dye with cellular proteins can hinder reliable measurements (Despa et al., 2000). The best established Na⁺-sensitive fluorescent dye, sodium-binding benzofuran isophthalate (SBFI) (Minta and Tsien, 1989), must be excited below 400 nm, and can only be employed in confocal imaging when special UV-lasers are used. Although conventional fluorescence imaging allows detection of [Na⁺]_i transients in dendrites (Callaway and Ross, 1997, Jaffe et al., 1992, Knöpfel et al., 2000, Lasser-Ross and Ross, 1992, Miyakawa et al., 1992, Ross et al., 1993, Tsubokawa et al., 1999), the analysis of the spatial distribution of Na⁺ signals or measurements in fine dendrites and spines in the intact tissue with SBFI require two-photon imaging (Rose et al., 1999). This technique, however, is not applicable for many laboratories because of its high costs for purchase and maintenance.

Recently, a new, green-fluorescent Na⁺ indicator dye, CoroNa Green (CoroNa), has become available (Invitrogen/Molecular Probes). The absorbance maximum of CoroNa is near 492 nm, which makes it suitable for excitation by argon lasers commonly used in confocal microscopy. According to the manufacturer (Invitrogen), CoroNa is brighter and exhibits larger changes in fluorescence after binding of sodium as compared to Sodium Green. In the present study, we compared the properties of CoroNa with those of SBFI to assess the suitability of the former for imaging of [Na⁺]_i transients in neurons in situ with both wide-field and high-resolution confocal imaging. We demonstrate that CoroNa is a suitable tool for measurement of [Na⁺]_i transients using conventional wide-field imaging and report the first confocal [Na⁺]_i measurements in fine dendrites in acute brain slices using this dye.