Effect of spermine and N1-dansyl-spermine on epileptiform activity in mouse cortical slices
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.
Section snippets
Subjects
Male and female DBA/2, genetically epilepsy-prone, mice were obtained from Harlan, UK and subsequently bred in the Bioresources unit, Trinity College. Stock mice were housed five to a cage with standard laboratory food and water available ad libitum. The mice were maintained at an ambient temperature of 21 ± 1 °C under a standard 12 h light/dark cycle (light: 7am–7pm). When used the mice were aged between 21 and 42 days.
Cortical wedge preparation
The method of cortical wedge preparation was similar to that described in Hu
Results
Fig. 1A illustrates a characteristic trace from a cortical wedge and shows the changes that occur when the medium is changed from normal aCSF to Mg2+-free aCSF. After approximately 20 min the first small depolarisations (spike) can be seen and the number increases from there, with time. After 2 h of perfusion with Mg2+-free aCSF, the number of depolarisations becomes stable as can be seen in Fig. 1A inset. Hu and Davies (1997) demonstrated that after 2 h the number of spikes in a 5-min period
Discussion
Removal of Mg2+ from the perfusion solution caused the production of spontaneous epileptiform discharges in wedges prepared from DBA/2 mice. N1-dansyl-spermine was demonstrated to have no effect on these discharges at a low dose but to reduce the discharge frequency at a higher concentration. We have also shown that spermine increases the frequency of depolarisations in the cortical wedge and that N1-dansyl-spermine is effective in reducing the frequency of depolarisations following
Acknowledgements
We would like to thank Ms. Rhona Prendergast for synthesis of N1-dansyl-spermine, Dr. J. Davies and Dr. W. Marsh for their assistance with the cortical wedge preparation. This work was supported by the Department of Pharmacology, Trinity College Dublin postgraduate award and an Enterprise Ireland award.
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