Effect of spermine and N¹-dansyl-spermine on epileptiform activity in mouse cortical slices

Abstract

N¹-dansyl-spermine is a novel polyamine analogue, which has been demonstrated to have an antagonist action at the stimulatory polyamine site on the N-methyl-d-aspartate (NMDA) receptor macrocomplex. Cortical wedges from genetically epilepsy-prone DBA/2 mice demonstrate spontaneous epileptiform activity when perfused with Mg²⁺-free artificial cerebrospinal fluid (aCSF). This epileptiform activity has been demonstrated to be primarily mediated through the NMDA receptor. N¹-dansyl-spermine reduced the spontaneous epileptiform activity at a high dose (100 μM) but had no effect at a lower dose (50 μM). The polyamine, spermine (300 μM) caused an increase in the rate of the spontaneous epileptiform discharges. This effect of spermine was antagonised by administration of the low dose of N¹-dansyl-spermine (50 μM). This further demonstrates the role of the NMDA receptor in the production of spontaneous epileptiform discharges in the cortical wedge preparation and clearly illustrates both the facilitatory action of spermine and the polyamine antagonist action of N¹-dansyl-spermine at the stimulatory polyamine site on the NMDA receptor.

Introduction

Epilepsy is a chronic neurological disorder consisting of the unpredictable occurrence of seizures and as yet there is no cure, only a series of symptomatic drug treatments. The drug treatments for epilepsy vary greatly in their mechanisms of action affecting numerous transmitter systems within the brain (for review see Meldrum, 1996). Briefly, the neurotransmitter systems affected in the treatment of epilepsy include gamma-amino-butyric acid (GABA), voltage activated sodium channels, calcium channels and the N-methyl-d-aspartate (NMDA) receptor, among others. However, the NMDA receptor and the excitatory amino acid, glutamate, have emerged as one of the primary systems involved in epilepsy.

The NMDA receptor has been widely studied and demonstrated to be involved in many varied cellular mechanisms. It is well established that the NMDA receptor has a role in neuronal degeneration caused by anoxic/ischaemic conditions, seizure mediated brain damage and other neurodegenerative diseases in the brain (Choi, 1985, Rothman and Olney, 1986). The NMDA receptor has also been implicated in the production of seizures in epilepsy. This has been widely illustrated by the production of convulsions in mice by both centrally and peripherally administered NMDA (Czuczwar et al., 1985, Kirby et al., 2004, Moreau et al., 1989, Singh et al., 1990, Turski et al., 1990).

The polyamines, spermine, spermidine and putrescine are a family of di-, tri- and tetra-amines found throughout the body, with high, but locally variable, concentrations in the brain (Al-Deen and Shaw, 1978, Seiler and Schmidt-Glenewinkel, 1975). Previous work has demonstrated a pro-convulsant effect of centrally administered spermine, which is thought to be mediated, at least partially, through the NMDA receptor, with the possible involvement, also, of l-type calcium channels (Anderson et al., 1975, Doyle et al., 2005, Doyle and Shaw, 1996, Doyle and Shaw, 1998, Kirby et al., 2004), though other effects such as those on sodium currents (Ran et al., 2003) and kainite receptors (Mott et al., 2003) cannot be ruled out. The polyamines have been widely demonstrated to interact with the NMDA receptor, producing, at physiological concentrations, a potentiation of NMDA activity (Ransom and Stec, 1988, Singh et al., 1990, Sprosen and Woodruff, 1990, Williams et al., 1991). This is thought to be mediated through a stimulatory polyamine site on the extracellular surface of the NMDA receptor macrocomplex. However, high concentrations of polyamines inhibit the activity of the NMDA receptor, demonstrating a dual activity (Sacaan and Johnson, 1990). This led to the theory that there are two extracellular sites on the NMDA receptor macrocomplex, one stimulatory and one inhibitory.

N¹-dansyl-spermine is a novel analogue of the polyamine, spermine, consisting of a polyamine backbone and a single dansyl group at one of the terminal nitrogens. Chao et al. (1997) examined the effects of N¹-dansyl-spermine on cloned NMDA receptors, looking specifically at the inhibitory polyamine site, and found that N¹-dansyl-spermine was a potent blocker of the NMDA receptor by binding to the inhibitory polyamine site (Chao et al., 1997). However, the stimulatory polyamine site is the most relevant physiologically as substantially lower amounts of spermine are required to stimulate the positive site than the negative site. Indeed, it has recently been shown in this laboratory that N¹-dansyl-spermine is a potent polyamine antagonist at the stimulatory polyamine site (Kirby et al., 2004). N¹-dansyl-spermine antagonised both spermine-induced and spermine-enhanced NMDA-induced CNS excitation (Kirby et al., 2004). In addition, N¹-dansyl-spermine has recently been shown to be neuroprotective in the gerbil bilateral carotid artery occlusion model and also in a permanent focal ischaemia model (Kirby and Shaw, 2004, Li et al., 2004).

DBA/2 mice are genetically epilepsy-prone showing susceptibility to audiogenic seizures and as a result have been used in numerous studies examining compounds for potential anti-epileptic activity (Chapman et al., 1984, De Sarro et al., 1999, De Sarro et al., 1988). The cortical wedge preparation from the DBA/2 mouse has been widely used to examine epileptiform activity in vitro. Harrison and Simmonds initially developed the cortical wedge preparation using a brain slice containing cortical efferents (Harrison and Simmonds, 1985). This has since been modified by different groups but is still used to examine the effects of the NMDA receptor on epileptiform activity (Burton et al., 1987, Hu and Davies, 1995, Hu and Davies, 1997, Naish et al., 2002).

The enhancement of NMDA activity by polyamines has been examined using cortical wedges from a Wistar rat, an experimental set-up similar to that with DBA/2 mice. Robichaud and Boxer demonstrated an enhancement of spontaneous discharge frequency following spermine treatment though higher concentrations inhibited spontaneous discharges (Robichaud and Boxer, 1993). This dual action was similar to that demonstrated by Sacaan and Johnson (1990). Therefore, in this study, it was of interest to examine the effect of N¹-dansyl-spermine on the spontaneous epileptiform discharges in the DBA/2 mouse cortical wedge. Also the effect of N¹-dansyl-spermine was examined on the spermine-induced enhancement of spontaneous epileptiform discharges.