Departments of 1General Physiology of Nervous System and
3Department of Neurology, Neuroscience Heart at Dartmouth, Dartmouth Medical College, Lebanon, New Hampshire; and
4State Key Laboratory for Molecular and Mobile Biology, Kiev, Ukraine
2Cellular Membranology, Bogomoletz Institute of Physiology, Kiev;
2Cellular Membranology, Bogomoletz Institute of Physiology, Kiev;
Departments of 1General Physiology of Nervous System and
3Department of Neurology, Neuroscience Heart at Dartmouth, Dartmouth Medical College, Lebanon, New Hampshire; and
4State Key Laboratory for Molecular and Mobile Biology, Kiev, Ukraine
Departments of 1General Physiology of Nervous System and
2Cellular Membranology, Bogomoletz Institute of Physiology, Kiev;
4State Key Laboratory for Molecular and Mobile Biology, Kiev, Ukraine
3Department of Neurology, Neuroscience Heart at Dartmouth, Dartmouth Medical College, Lebanon, New Hampshire; and
2Cellular Membranology, Bogomoletz Institute of Physiology, Kiev;
4State Key Laboratory for Molecular and Mobile Biology, Kiev, Ukraine
Summary
the low Mg2+ mannequin of epilepsy was developed a number of a long time in the past, and since that point has been broadly used as a mannequin to check antiepileptic medication (Albus et al. 2008; Anderson et al. 1986; Coulter and Lee 1993; Sombati and Delorenzo 1995; Walther et al. 1986). This mannequin of epilepsy has medical relevance as Mg2+ deficits can enhance seizure susceptibility to proconvulsant stimuli (Greenberg and Tufts 1934) and even trigger seizures in people (Arnold et al. 1983; Nuytten et al. 1991). There may be additionally proof that Mg2+ focus in serum and cerebrospinal fluid is decrease in sufferers with generalized tonic-clonic seizures (Afzal et al. 1985; Govil et al. 1981). As well as, intravenously injected Mg2+ has an anticonvulsant impact in animal fashions of epilepsy (Borges and Gucer 1978) and is used to deal with seizures, notably in ladies with eclampsia (Duley et al. 2010).
The mechanism of induction and upkeep of seizure-like exercise (SLA) within the low Mg2+ mannequin of seizures has been extensively studied (Albus et al. 2008; Gloveli et al. 1995; Gutierrez et al. 1999; Mody et al. 1987). Era of SLA on this mannequin relies on facilitation of N-methyl-d-aspartate (NMDA) receptors and on a lower within the floor cost screening close to voltage-activated channels on account of discount of positively charged Mg2+ ions within the extracellular resolution (Heinemann et al. 2006). The seizures within the low Mg2+ mannequin of seizures could be stopped throughout the preliminary section (much less then 2 h after the primary SLA) by software of NMDA receptor blockers or by restoration of extracellular Mg2+ focus. Nonetheless, throughout the late section, neither including NMDA blockers nor restoration of Mg2+ focus to manage ranges may suppress epileptic exercise (Anderson et al. 1986; Derchansky et al. 2004; Dichter and Pollard 2006). Whereas the position of NMDA receptors within the low Mg2+ mannequin of epilepsy is effectively established, the position of floor cost on this mannequin is just not effectively understood.
Floor cost on the mobile membrane produced by sialic acid, phosphates, charged lipids, charged amino acids, and different hydrophilic residues of channel proteins creates native electrical fields close to the channel voltage sensor (Messner et al. 1985; Miller et al. 1983; Roberts and Barchi 1987). The impact of floor cost on the membrane channel could be assessed by altering extracellular concentrations of cations, which produce a screening impact on floor cost. Reducing extracellular Ca2+ or growing the quantity of extracellular negatively charged polysialic acids ends in a major hyperpolarizing shift of activation of voltage-gated Na+ channels (VGSC) (Bennett et al. 1997; Frankenhaeuser and Hodgkin 1957; Inexperienced and Andersen 1991; Hanck and Sheets 1992; Isaeva et al. 2010). Manipulations with extracellular Mg2+ concentrations additionally have an effect on Na+ channel gating properties however to a lesser diploma. Ion selectivity of floor cost is just not totally understood, though the distinction in impression on floor cost between divalent and monovalent cations is effectively described by the Grahame equation derived from Gouy-Chapman idea (Grahame 1947). The adjustments in extracellular Mg2+ or Ca2+ concentrations alter motion potential threshold and have an effect on multi-unit exercise, spontaneous synaptic exercise, spontaneous ectopic discharges, and epileptic exercise in in vitro and in vivo fashions (Feng and Durand 2003; Isaev et al. 2007; Peng et al. 2004).
Within the current research, we evaluated the contribution of the impact of decreasing Mg2+ within the extracellular resolution on floor cost within the preliminary stage of the low Mg2+ mannequin of epilepsy. Since variations in [Mg2+]o alter each the exercise of NMDA receptors and floor cost, we diverse floor cost screening within the low Mg2+ mannequin by altering [Ca2+]o. As Ca2+ performs an necessary position in synaptic launch, such substitution couldn’t precisely describe the affect of floor cost by Mg2+. Additionally, Mg2+ facilitates transmitter launch, and, when eliminated, facilitates presynaptic Ca2+ accumulation (Mody et al. 1987). Nonetheless, contemplating that synaptic launch (Zucker 1993) and launch likelihood (Bollmann et al. 2000; Dodge and Rahamimoff 1967) will increase with enhancement of [Ca2+]o in evoked synaptic exercise and might facilitate excitatory synaptic transmission of neuronal community (Katz and Miledi 1969; Llinas and Walton 1980), we doubtless underestimated the impact brought on by Mg2+ ions. Within the current research, we investigated the impact of various extracellular concentrations of Mg2+ and Ca2+ on practical properties of VGSC on hippocampal CA1 pyramidal neurons and seizure manifestation within the low Mg2+ mannequin of epilepsy.
MATERIALS AND METHODS
RESULTS
DISCUSSION – “magnesium model”
The position of NMDA receptors within the low Mg2+ mannequin of epilepsy is effectively described. Elevated NMDA currents seem like a important set off of seizure susceptibility on this mannequin (DeLorenzo et al. 1998; Traub et al. 1994). Nonetheless, Mg2+ ions have a potent capacity to display membrane floor cost, and a lower on this screening may facilitate activation of the voltage-gated channels.
Divalent cations have been reported to trigger a voltage-dependent block of Na+ channels (Armstrong and Cota 1991). Certainly, we have now discovered {that a} tenfold enhance in [Ca2+]o or [Mg2+]o ends in a statistically important, however nearly negligible, block of INa peak by ∼5%. Thus, within the mannequin of pyramidal neurons, we totally assist the info by Armstrong and Cota (1991) {that a} direct block of Na+ channels by divalent cations at physiological voltages has no substantial position in INa alteration, a minimum of when examined on the concentrations used right here.
Software of low Mg2+/2 mM Ca2+ resolution evoked rSLA in 2/3 slices examined. Rising [K+]o from 3.5 to five mM, a focus that’s conventionally used with low [Mg2+]o mannequin of seizures on the mind slices (Dreier and Heinemann 1990; Jones and Heinemann 1987; Khosravani et al. 2005; Pohl et al. 1992), considerably elevated seizure incidence (P = 0.025, actual Fisher check). Such an alteration of [K+]o decreases neuronal resting membrane potential by ∼8 mV (Isaev et al. 2007) and facilitates seizure incidence (Isaev et al. 2005). Two sorts of seizure exercise manifestation have been noticed at elevated [K+]o: cSLA and rSLA, which is in settlement with the info obtained beforehand (Dreier and Heinemann 1991; Yaari et al. 1986). Probably the most outstanding distinction in rSLA recorded in 3.5 mM and 5 mM [K+]o was within the interval between rSLA however not rSLA length. It’s attention-grabbing {that a} comparable remark on the low [Mg2+]o mannequin of seizures was made with decreasing of pH (Velísek et al. 1994). rSLA length on this mannequin might depend upon intrinsic properties of the community somewhat than on different elements.
We discovered solely a slight distinction between outcomes obtained within the experiments with growing [K+]o to five mM and decreasing [Ca2+]o to 1 mM. A standard methodology to induce a low [Ca2+]o mannequin of seizure exercise in a hippocampal slice is to perfuse it with ACSF containing a focus of Ca2+ beneath 0.5 mM. It was proven that non-synaptic mechanisms play an necessary position within the era of SLA on this mannequin (Feng and Durand 2003; Haas and Jefferys 1984; Taylor and Dudek 1984). In our research, blockade of voltage-gated Ca2+ channels by CdCl2 stopped SLA induced by perfusion with 1 mM [Ca2+]o (see supplies and strategies). Nonetheless, saturating focus of CdCl2 had no impact on bursting within the low Ca2+ mannequin of seizures (Bikson et al. 1999). These outcomes present that SLA induced in low Mg2+/1 mM Ca2+ ACSF is induced by decreasing Mg2+ somewhat than decreasing Ca2+.
In our patch-clamp experiments, the shift in half-activation of INa between 2 mM and 1 mM [Ca2+]o was roughly −6.3 mV. This hyperpolarizing shift results in a lower within the threshold of spike era in addition to depolarization of neuronal membrane by growing [K+]o, produces, when it comes to decreased threshold, comparable impact. The noticed slight distinction between 1 mM [Ca2+]o and 5 mM [K+]o within the SLA onset (P = 0.14) and interburst intervals of rSLA (P = 0.13) tentatively could be defined by an elevated inhibitory affect of GABA(A) currents with mobile depolarization (Isaev et al. 2005, 2007). Alternatively, excessive [K+]o will increase synaptic transmitter launch, and decreasing of [Ca2+]o has an impact on the steadiness between excitation and inhibition (Jones and Heinemann 1987). On the identical time, a rise of [Ca2+]o to three mM resulted in optimistic shift of the Na+ channel activation and required a stronger mobile depolarization for seizure era. On this situation, we weren’t capable of evoke SLA by low Mg2+ ACSF with 3.5 mM or 5mM [K+]o. Moreover, variations between elevated [K+]o and decreased [Ca2+]o may very well be defined by direct influences of Okay+ and Ca2+ on synaptic transmission. Stimulus-induced synaptic potentials lower with elimination of Ca2+ from the extracellular membrane (Rausche et al. 1990). The [Ca2+]o at which synaptic transmission is blocked elevated with growing [Mg2+]o and decreased when [K+]o was modified from 3 to five mM each in CA1 and the dentate gyrus. Nonetheless, with an elevation of [K+]o from 5 to eight mM, synaptic transmission elevated in CA1, however was decreased within the dentate gyrus (Rausche et al. 1990).
Mobile depolarization by [K+]o in resolution containing 1 mM [Ca2+]o didn’t result in adjustments in both the delay of the SLA onset (P = 0.84) or intervals between rSLA (P = 0.4). This result’s in settlement with beforehand obtained knowledge by Pan and Stringer (1997) the place the authors discovered a 100% likelihood of inducing mobile bursting in 1 mM [Ca2+]o and ≥4 mM [K+]o resolution. The one important distinction in rSLA manifestation on this situation was a ratio of tonic exercise to rSLA. In earlier research, we reported that oversialylation of mobile membrane induced by blockade of neuraminidase, a particular enzyme that cleaves extracellular polysialic acids, with the precise blocker NADNA (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid), considerably will increase the length of the tonic section of SLA induced by decreasing Mg2+ in hippocampal slice tradition (Isaeva et al. 2010). The tonic section of SLA is related to depolarization of each the dendritic and somatic areas of the cell, whereas the clonic section is related to synchronized rhythmic burst era mediated by the activation of sluggish spikes within the dendrites (reviewed by McCormick and Contreras 2001). We suggest that the mixed impact of mobile depolarization and shift of Na+ channel activation to extra adverse voltages ends in synchronization of dendritic and somatic discharges.
We discovered that after a 1-h software of low Mg2+ ACSF in ∼30% of slices in room temperature and 40% in 33°C, SLA turned persistent even to three mM [Ca2+]o. We advise that mechanisms concerned in transition from preliminary seizures to the late stage (Dichter and Pollard 2006) participate from the onset of the seizure discharges.
Our knowledge recommend that each elimination of Mg2+ block of NMDA channels and a lower within the floor cost screening are required situations to induce seizures within the low Mg2+ mannequin of epilepsy. Each mobile depolarization and hyperpolarizing shift of the voltage-gated channel activation trigger comparable results on neuronal community exercise as a result of decreased voltage distinction between the resting membrane potential and potentials the place Na+ channels are activated. Sole change in both the resting membrane potential or within the floor cost have an effect on delay time between software of low Mg2+ resolution and SLA, frequency of rSLA and manifestation of seizure exercise, however not length of rSLA.
These observations present proof that Mg2+ can scale back cerebral excitability by screening floor cost and assist the usefulness of Mg2+ as an anti-seizure and anti-epileptic agent. This research additional means that floor cost might symbolize a novel therapeutic goal within the remedy of epilepsy.
GRANTS
This work was supported by the Science and Know-how Heart in Ukraine Grant #5214, Nationwide Institute of Neurological Issues and Stroke Grants NS-041595 and NS-073083, and Christopher Donalty and Kyle Coggins Memorial Grant from Residents United for Analysis in Epilepsy (CURE).
DISCLOSURES
No conflicts of curiosity, monetary or in any other case, are declared by the creator(s).