Blocking effect of 1-naphthyl acetyl spermine on Ca2+-permeable AMPA receptors in cultured rat hippocampal neurons
Introduction
The blocking effect of Joro spider toxin (JSTX), the toxin of the spider Nephila clavata, on glutamatergic neurotransmission was first reported in the lobster neuromuscular junction (Kawai et al., 1982, Abe et al., 1983). It has recently been shown that JSTX and other closely related spider toxins selectively block recombinant α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors lacking the GluR2 subunit (Blaschke et al., 1993, Herlitze et al., 1993, Washburn and Dingledine, 1994). The properties of the AMPA receptor channels assembled without GluR2 are characterized by a strong inward rectification and a high permeability to Ca2+ (Seeburg, 1993, Hollmann and Heinemann, 1994). Evidence is accumulating that JSTX also selectively suppresses the native Ca2+-permeable AMPA receptors expressed in both neurons and glial cells of the rat CNS (Gu et al., 1996Iino et al., 1996Meucci et al., 1996). For example, Iino et al. (1996)have shown that JSTX-3, a synthetic Joro spider toxin, specifically suppresses responses mediated by the inwardly rectifying and Ca2+-permeable AMPA receptors expressed in type II cultured rat hippocampal neurons without affecting the outwardly rectifying and Ca2+-impermeable AMPA receptors in type I neurons.
1-Naphthyl acetyl spermine (NASPM) is a synthetic analogue of JSTX (Fig. 1; Asami et al., 1989). It has been shown that NASPM has a similar blocking action on the glutamate response at the lobster neuromuscular synapse as JSTX, but that the effect of NASPM is reversible whereas that of JSTX is almost irreversible (Asami et al., 1989). NASPM has been shown to block excitatory synaptic potentials evoked by stimulation of parallel fibers in guinea pig cerebellar Purkinje cells (Ajima et al., 1991). In hippocampus, NASPM has been reported to exert a potent and selective suppression of hippocampal epileptic discharges mediated by non-NMDA receptors (Kanai et al., 1992). Very recently, it has been reported that EPSCs in CA1 neurons of the gerbil hippocampus after ischemia are mediated by Ca2+-permeable non-NMDA receptors, and that these abnormal EPSCs are effectively suppressed by NASPM (Tsubokawa et al., 1995). These results suggest that NASPM may be useful as a pharmacological tool for investigating physiological and pathological roles of AMPA receptors, and, furthermore, that it could be used as a parent compound to develop new agents for the treatment of neuronal cell death and epilepsy. In this study, we examined the effects of NASPM on AMPA receptors in cultured rat hippocampal neurons, and compared them with those of JSTX-3 that had been reported previously (Iino et al., 1996).
Section snippets
Culture of hippocampal neurons
Cultured hippocampal neurons were prepared from 17–19 day-old rat embryos as described previously (Iino et al., 1990). The cultured neurons were used for patch clamp recordings between 8 and 14 days after plating.
Electrophysiological recordings
Whole-cell recordings from cultured neurons were made using an EPC-7 patch clamp amplifier (Heca, Darmstadt, Germany). The pClamp system (Axon Instruments, Foster City, CA) was used for data acquisition and analysis. Whole-cell current traces were filtered at 1 kHz and digitized at 2
Blocking effects of NASPM on AMPA receptors in cultured hippocampal neurons
The effect of NASPM on AMPA receptors was tested in a type II cultured hippocampal neuron (Fig. 2). The AMPA receptors in this neuron were characterized by a strong inward rectification (RI<0.25) and a high permeability to Ca2+. Fig. 2(Aa) represents a current trace elicited by ionophoretic application of kainate, a non-desensitizing agonist of AMPA receptors, in the control solution when the membrane potential was held at −60 mV. The duration of the ionophoretic current was 5 ms and the
Selective blockade of Ca2+-permeable AMPA receptors by NASPM
In this study, we have shown that NASPM selectively blocks AMPA receptors in type II cultured rat hippocampal neurons without affecting those in type I neurons. Strong inward rectification and high permeability to Ca2+ in AMPA receptors in type II neurons have been shown to be due to either a lack of or very little expression of the GluR2 subunit in these neurons (Bochet et al., 1994). It is, therefore, most likely that NASPM recognizes the glutamine–arginine (Q–R) site in the pore-forming
Acknowledgements
We thank Professor Terumi Nakajima for the information about chemical properties of spider toxins and their analogues. We also thank Daicel Chemical Industries for generously providing us with 1-naphthyl acetyl spermine. This study was supported by grants from the Human Frontier Science Program and the Ministry of Education, Science and Culture of Japan.
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