Measurement of intracellular Ca²⁺ in cultured rat embryonic hippocampal neurons using a fluorescence microplate reader: potential application to biomolecular screening

Abstract

Introduction: Fluorescence microplate readers for the measurement of cytosolic free Ca²⁺ ([Ca²⁺]_i) are used as a drug screening tool, particularly for immortal cell lines. However, wider application of this methodological approach to more differentiated cells such as neurons would also be useful for the screening of compounds that modulate synaptic transmission. Such an approach has the potential to identify lead compounds for the development of novel drugs for the treatment of epilepsy, pathological pain states, Parkinson's disease, or other neurological disorders. Methods: In this paper, we describe the development of a microplate reader assay for the assessment of [Ca²⁺]_i in a primary culture of rat hippocampal neurons maintained in Neurobasal medium using the fluorescent calcium indicator, fluo-3. Results: The assay was appropriate for the screening of glutamate receptor agonists and antagonists. Furthermore, lowering the extracellular Mg²⁺ concentration ([Mg²⁺]_O) produced consistent oscillations in neuronal [Ca²⁺]_i detected using the fluorescence microplate reader. These oscillations were inhibited by the GABA_B agonist, baclofen, and the NMDA receptor antagonist, LY274614. Discussion: Our results indicate that assessment of the inhibitory effects of agents on spontaneous [Ca²⁺]_i oscillations in neurons may be useful for the identification of agents that act on targets for which specific screening methods are not currently available, or those which act via a previously unknown pathway to inhibit synaptic transmission. This technique also has the potential to increase the productivity of experiments designed to characterize changes in [Ca²⁺]_i (including calcium oscillations) in cultured neurons.

Introduction

Fluorescence microplate readers, particularly those designed for moderate to high throughput screening such as the Fluorescence Imaging Microplate Reader (FLIPR, Molecular Devices, Sunnyvale, CA, USA) and the NOVOstar (BMG LabTechnologies, Offenburg, Germany), are increasingly used in a range of biomolecular screening assays Kassack et al., 2002, Rogers et al., 2002. Fluorescence microplate readers can measure a variety of cellular signals and intracellular ions including Na⁺, H⁺, and Ca²⁺. Ca²⁺ is a particularly useful ion for biomolecular screening assays, because of its important role in processes as diverse as muscle contraction, synaptic transmission, hormone release, and gene transcription Luebke et al., 1993, Monteith & Roufogalis, 1995.

One of the most common uses of microplate readers, which involves the assessment of cytosolic free Ca²⁺ ([Ca²⁺]_i), is the screening of agents for the modulation of G-protein-coupled receptors (GPCRs) (Kassack et al., 2002). Often, a particular GPCR is overexpressed in a convenient cell line that already has the necessary functional downstream signaling cascades (e.g., IP₃-dependent Ca²⁺ store release) (Patel et al., 2001). Agents can then be screened for their ability to activate the GPCR of interest. Such studies are easily adapted for the screening of potential therapeutic antagonists, whereby agents are tested for their ability to block Ca²⁺ transients initiated by a ligand acting at the overexpressed GPCR. These techniques are extremely powerful when the target is known and can be overexpressed.

Studies whereby Ca²⁺ responses are assessed in cells that more closely mimic those found in vivo or in disease states would also be useful. Indeed, a lung carcinoma cell line (NCI-H146) has been reported to be similar to neurons in that these cells express N- and P-type Ca²⁺ channels. Indeed, this cell line has been used to screen the effects of Australian plant extracts on K⁺-depolarization-induced Ca²⁺ influx, to identify novel lead compounds for the treatment of neurological disorders (Rogers et al., 2002). However, such cell lines still fail to mimic the complexity of neuronal networks in vivo. Primary cultured neurons, however, retain many features similar to those found in vivo, such as morphology, development of synapses, together with spontaneous neurotransmitter release and [Ca²⁺]_i oscillations Bacci et al., 1999, Shen et al., 1996. Inhibition of spontaneous Ca²⁺ oscillations in primary cultured neurons has been used to characterize the effects of a wide variety of agents including cannabinoids Shen et al., 1996, Shen & Thayer, 1998 and these oscillations are inhibited by L-type, P/Q-type, and N-type Ca²⁺ channel blockers as well as TTX Hemstapat et al., 2003, Tanaka et al., 1996. Such studies have used fluorescence microscopy to assess [Ca²⁺]_i in individual or very small groups of neurons, a technique unsuited to screening.

In the studies described herein, we sought to determine whether [Ca²⁺]_i could be assessed in neurons cultured in media which promotes high purity (>95%) neurons (Neurobasal medium), using a microplate reader suitable for moderate throughput screening (NOVOstar). We also wanted to determine whether neuronal [Ca²⁺]_i oscillations could be assessed in a fluorescence microplate format, as a tool for the screening of novel inhibitors of synaptic transmission. Our technique, described here in detail, has the potential to be a useful initial screening method to determine if a compound is likely to modulate synaptic transmission. Such an evaluation could be done before a wide array of tests on specific targets is performed. Furthermore, this protocol should identify compounds which modulate synaptic transmission through a mechanism for which screening assays are not yet available, or which act on a target that has not been previously identified.