LY-274614

Fluorescence microplate readers for the measurement of cytosolic free Ca(2+) ([Ca(2+)](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.

In this paper, we describe the development of a microplate reader assay for the assessment of [Ca(2+)](i) in a primary culture of rat hippocampal neurons maintained in Neurobasal medium using the fluorescent calcium indicator, fluo-3.

The assay was appropriate for the screening of glutamate receptor agonists and antagonists. Furthermore, lowering the extracellular Mg(2+) concentration ([Mg(2+)](O)) produced consistent oscillations in neuronal [Ca(2+)](i) detected using the fluorescence microplate reader. These oscillations were inhibited by the GABA(B) agonist, baclofen, and the NMDA receptor antagonist, LY274614.

Our results indicate that assessment of the inhibitory effects of agents on spontaneous [Ca(2+)](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 [Ca(2+)](i) (including calcium oscillations) in cultured neurons.

The steady-state mRNA levels of the NMDA receptor NR1 subunit were determined by a quantitative solution hybridization assay in selected CNS regions associated with antinociception in the rat. Tissues were obtained by microdissection from rats treated chronically with morphine alone or in combination with LY274614, a competitive NMDA receptor antagonist. Morphine treatment for 7 days resulted in the development of tolerance to morphine's analgesic effect and produced a significant decrease in the steady-state NR1 mRNA levels in the spinal cord dorsal horn (by 16%), and an elevation in nucleus raphe magnus and medial thalamus (by 26 and 38%, respectively). The NR1 mRNA levels were unchanged in the lateral paragigantocellular nucleus, locus coeruleus, periaqueductal grey, and sensorimotor cortex. NMDA receptor binding in the spinal cord measured with [3H]MK-801 was reduced approximately 50% by chronic morphine treatment. Co-administration of LY274614 (s.c. at 24 mg/kg/24 h via an osmotic pump) not only attenuated the development of morphine tolerance but also prevented the changes in the NR1 mRNA levels induced by chronic morphine administration. Neither a 7-day infusion of LY274614 nor an acute injection of morphine (10 mg/kg, s.c.) changed the NR1 mRNA levels. These results suggest that changes in the expression of the NR1 mRNA induced by chronic morphine in three CNS regions involved in antinociception are associated with the development of morphine tolerance and in the spinal cord, morphine tolerance is associated with the downregulation of NMDA receptors.