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Antiepileptic effects of cobalt, manganese and magnesium on bicuculline-induced epileptiform activity in hippocampal neurons
Brain Research 1732 (2020) 146684
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Research report
Antiepileptic effects of cobalt, manganese and magnesium on bicucullineinduced epileptiform activity in hippocampal neurons
T
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Saft Carstena, , Speckmann Erwin-Josefb a b
Department of Neurology, St. Josef Hospital, Gudrunstraße 56, 44791 Bochum, Germany Institut für Physiology I, Westfälische Wilhelms Universität, Robert-Koch Str. 27a, 48149 Münster, Germany
H I GH L IG H T S
inorganic calcium channel blockers cobalt, manganese and magnesium were able to suppress bicuculline induced epileptiform field potentials in a dosage • The dependent and reversible manner. were carried out by using hippocampal slices of guinea pigs as a model. Potential mechanisms and clinical relevance of findings are discussed. • Experiments • Potential mechanisms and clinical relevance of findings are discussed.
A R T I C LE I N FO
A B S T R A C T
Keywords: Calcium channel blocker Cobalt Manganese Magnesium Epilepsy
Background: Calcium signaling is described as a relevant factor in synchronization of neurons and increased excitability in epileptogenesis. Aim of the present investigations was to test the antiepileptic effect of the classical inorganic calcium channel blockers cobalt (Co2+), manganese (Mn2+) and magnesium (Mg2+). Methods: Experiments were carried out on hippocampal slices of guinea pigs. Epileptiform field potentials (EFP) were elicited by adding bicuculline (10 µmol/l) to the artificial cerebrospinal fluid (CSF). Kalium was elevated from normal (4 mmol/l) to 8 mmol/l. Co2+ (CoCl2; 2, 1, 0.5 and 0.1 mmol/l), Mn2+ (MnCl2; 2, 1, 0.5 and 0.1 mmol/l) and Mg2+ (MgCl2; 8, 6, 5, 4 and 2 mmol/l) were added to the superfusate. Results: Concentrations of 2, 1 and 0.5 mmol/l Co2+, 2 and 1 mmol/l Mn2+ and 8 respectively 6 mmol/l Mg2+ were able to suppress EFP sufficient in a dose dependent manner. In concentrations of 0.1 mmol/l Co2+, 0.5 mmol/l and 0.1 mmol/l Mn2+ and 5 respectively 4 and 2 mmol/l Mg2+ suppression was incomplete. With washout of the inorganic calcium channel blockers the EFP reappeared. Discussion: All tree inorganic calcium channel blockers were able to suppress EFP in a dosage dependent and reversible manner. Weak reappearance of EFP after washout of Co2+ might be due to additional cytotoxic effects. The following mechanisms may contribute: i) blockade of voltage-activated calcium channels in the postsynaptic membrane, ii) changes in the activation of voltage-dependent sodium channels, iii) blockade of synaptic transmission.
1. Introduction Calcium (Ca) signaling is described as a relevant factor in synchronization of neurons and increased excitability in epileptogenesis (Steinlein, 2014). Several mechanisms seem to be involved in generation of epileptic discharges. Discussed are immediate effects on membrane excitability by calcium influx through ion channels, feedback mechanisms including mitochondrial calcium signaling and delayed mechanisms that act through G-protein coupled pathways (Steinlein, 2014).
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Organic blockers of voltage-activated calcium channels have been demonstrated to suppress epileptiform activity induced by gamma aminobutyric acid (GABA) receptor antagonists in various experimental models (Aicardi and Schwartzkroin, 1990; Bingmann et al., 1988; Straub et al., 1990; Straub et al., 1994). Veratridine-induced epileptiform activity was suppressed by both inorganic (Cobalt) and organic (Verapamil) calcium antagonists (Link et al., 2008). These observations led to the hypothesis, that a massive influx of Ca through voltage-sensitive calcium channels is involved in the convulsant action of the antagonist of the GABA receptor. The antiepileptic effect of organic
Corresponding author. E-mail address: [email protected] (S. Carsten).
https://doi.org/10.1016/j.brainres.2020.146684 Received 8 January 2020; Received in revised form 22 January 2020; Accepted 25 January 2020 Available online 27 January 2020 0006-8993/ © 2020 Published by Elsevier B.V.
Brain Research 1732 (2020) 146684
S. Carsten and S. Erwin-Josef
2.3. Dosage dependent and reversible suppression of epileptiform activity by manganese
calcium antagonists, therefore, was mainly attributed to a reduction of postsynaptic calcium currents (Bingmann and Speckmann, 1989). Magnesium supplementation is discussed to reduce seizures in people with epilepsy (Yuen and Sander, 2012). The aim of the present investigation was to test the antiepileptic effect of the inorganic calcium channel blockers cobalt, manganese and magnesium in a model using hippocampal slices of guinea pigs and bicuculline induced epileptiform field potentials (EFP). In contrast to the organic substances the inorganic ones are known to have a more focused effect on calcium channels. Bicuculline, a GABAA receptor antagonist, is assumed to produce paroxysmal depolarisation shifts (PDS) by blocking GABA-mediated synaptic transmission (Pong and Graham, 1972).
When Mn2+ was added to the superfusate, t0.9 were 20 ± 2 min (2 mmol/l; n = 4), 26.6 ± 5 min (1 mmol/l; n = 6) and 190 min (0.1 mmol/l; n = 1/7). In concentrations of 0.1 mmol/l 90% depression could only be found in 1 out of 7 slices, t0.9 was not reached within 4 h in 6 slices. With concentrations of 0.5 mmol/l t0.9 was not reached within 4 h in both slices. When Mn2+ was added to the superfusate, t0.5 were 16.3 ± 1 min (2 mmol/l; n = 4), 15.8 ± 1 min (1 mmol/l; n = 6), 230 min (0.5 mmol/l; n = 1/2) and 97.5 ± 44 min (0.1 mmol/l; n = 4/7). In concentrations of 0.1 mmol/l 50% depression could only be found in 4 out of 7 slices, t0.5 was not reached within 4 h in 3 slices. In concentrations of 0.5 mmol/l 50% depression could only be found in 1 out of 2 slices, t0.5 was not reached within 4 h in 1 slice (see Fig. 2; Table 1). With washout of the inorganic calcium channel blocker Mn2+ EFP reappeared in all slices. After washout from Mn2+ latencies of 50% reappearance (t0.5) were 35 ± 10 min (2 mmol/l; n = 3/4), 30 ± 6 min (1 mmol/l; n = 4/6), 15 ± 0 min (0,5 mmol/l; n = 2) and 19 ± 9 (0.1 mmol/l; n = 4/7). In concentrations of 2 mmol/l 50% reappearance could only be found in 3 out of 4 slices, in concentrations of 1 mmol/l only in 4 out of 6 slices, and in concentrations of 0.1 mmol/l only in 4 out of 7 slices. After washout from Mn2+ latency of 90% reappearance (t0.9) were 30 min (2 mmol/l; n = 1/4), 55 min (0.5 mmol/l; n = 1/2) and 15 ± 5 min (0.1 mmol/l; n = 2/7). In concentrations of 2 mmol/l 90% reappearance could only be found in 1 out of 4 slices, in concentrations of 0.5 mmol/l only in 1 out of 2 slices and in concentrations of 0.1 mmol/l only in 2 out of 7 slices. In concentrations of 1 mmol/l in none of the slices 90% reappearance was reached within a maximum of 2½ hour (all ½-2½) hour (see Table 2).
2. Results 2.1. Induction of epileptiform activity 89 slices, including 5 controls, were investigated. In the 5 controls epileptiform activity was induced by superfusion with artificial cerebrospinal fluid (CSF) with bicuculline (10 μmol/l) and elevated Kalium (K+) without adding a calcium antagonist. Epileptiform activity occurs after 6 ± 4 min and remains stable until washout for 6 h. Field potentials of stratum pyramidale of hippocampal cornu ammonis 1 (CA1) and hippocampal cornu ammonis 3 (CA3) regions were recorded simultaneously in 77 slices, in 10 slices only CA3 region was recorded due to technical reasons. Superfusion with bicuculline containing CSF (Period 2) induces epileptic field potentials (EFP) if K+ was normal in 5 of 89 cases. Because no statistically analysis is possible, results of occurrence of EFP and suppression by inorganic calcium antagonists for these 5 cases are not given in this report. After elevation of K+ to 8 mmol/l (n = 84) EFP occurs in 69 slices after 6.8 ± 3.3 min. No constant EFP occur in 15 slices. EFP occurred simultaneously in CA1 and CA3 region in 60 from all 69 cases. In 5 cases EFP occurred only in CA3 region, in 4 cases only in CA1 region. Because activation occurred simultaneously in all cases mentioned above, slices with only one active region (CA1 or CA3) were also included in the study without making a difference in the following.
2.4. Dosage dependent and reversible suppression of epileptiform activity by magnesium When Mg2+ was added to the superfusate, t0.9 were 13.3 ± 2 min (8 mmol/l; n = 3), 15 ± 0 min (6 mmol/l; n = 3), 52 ± 37 min (5 mmol/l; n = 5/6) and 70 ± 45 min (4 mmol/l; n = 3/5). In concentrations of 5 mmol/l 90% depression could only be found in five of six slices, t0.9 was not reached within 4 h in 1 slice. In concentrations of 4 mmol/l 90% depression could only be found in 3 out of 5 slices, t0.9 was not reached within 4 h in 2 slices. With concentrations of 2 mmol/l (n = 3) t0.9 was not reached within 4 h in 3 slices. When Mg2+ was added to the superfusate, t0.5 were 10 ± 0 min (8 mmol/l; n = 3), 10 ± 0 min (6 mmol/l; n = 3), 50 ± 38 min (5 mmol/l; n = 5) and 25 ± 11 min (4 mmol/l; n = 5). With concentrations of 2 mmol/l (n = 3) t0.5 was not reached within 4 h in 3 slices (see Fig. 3; Table 1). With washout of the inorganic calcium channel blocker Mg2+ EFP reappeared in all slices. After washout from Mg2+ latencies of 50% reappearance (t0.5) were 15 ± 3 min (8 mmol/l; n = 3), 13 ± 3 min (6 mmol/l; n = 3), 14.2 ± 2 min (5 mmol/l; n = 6), 26.3 ± 10 min (4 mmol/l; n = 4/5) and 10 ± 0 (2 mmol/l; n = 3). In concentrations of 4 mmol/l 50% reappearance could only be found in 4 out of 5 slices. After washout from Mg2+ latency of 90% reappearance (t0.9) were 15 ± 0 min (8 mmol/l; n = 2/3), 20 ± 5 min (6 mmol/l; n = 3), 19 ± 3 min (5 mmol/l; n = 5/6), 22.5 ± 3 min (4 mmol/l; n = 2/5) and 10 ± 0 min (2 mmol/l; n = 3). In concentrations of 8 mmol/l 90% reappearance could only be found in 2 of 3 slices, in concentrations of 5 mmol/l only in 5 out of 6 slices and in concentrations of 4 mmol/l only in 2 out of 5 slices (see Table 2).
2.2. Dosage dependent and reversible suppression of epileptiform activity by cobalt When Co2+ was added to the superfusate, latencies of 90% depression (t0.9) were 15.7 ± 1 min (2 mmol/l; n = 7), 21.2 ± 2 min (1 mmol/l; n = 8), 55 ± 5 min (0.5 mmol/l; n = 2) and 162 ± 39 min (0.1 mmol/l; n = 5/8). In concentrations of 0.1 mmol/l 90% depression could only be found in five of eight slices, t0.9 was not reached within 4 h in three slices. Latencies of 50% depression (t0.5) were 11.4 ± 1 min (2 mmol/l; n = 7), 16.2 ± 2 min (1 mmol/l; n = 8), 20 ± 5 min (0.5 mmol/l; n = 2) and 80 ± 22 min (0.1 mmol/l; n = 8: see Fig. 1; Table 1). With washout of the inorganic calcium channel blocker Co2+ EFP reappeared in all slices. After washout from Co2+ latencies of 50% reappearance (t0.5) were 25 min (2 mmol/l; n = 1/7) and 22.5 ± 8 (1 mmol/l; n = 2/8). In concentrations of 2 mmol/l 50% reappearance could only be found in 1 out of 7 slices, in concentrations of 1 mmol/l only in 2 out of 8 slices, in concentrations of 0.5 mmol/l and 0.1 mmol/l in none of the slices 50% reappearance was reached within a maximum of 2 h (all 1–2) hour. After washout from Co2+ 1 mmol/l latency of 90% reappearance (t0.9) was 15 min in only one of eight cases (1 mmol/l; n = 1/8). In concentrations of 2, 0.5 mmol/l and 0.1 mmol/l in none of the slices 90% reappearance was reached within two hours (see Table 2).
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Brain Research 1732 (2020) 146684
S. Carsten and S. Erwin-Josef
Fig. 1. Example of the effect of Co2+ (2, 1, and 0.1 mmol/l) on bicuculline induced epileptiform field potentials (FP). CA3 neuron, hippocampal slice, guinea pig. FP1 and FP2: tracings of field potential by a storage oscilloscope and by an inkwriter, respectively. Recordings related to each other by characters. Times given refer to commencement of the indicated periods.
other concentrations. Because t0.5 and t0.9 could not be reached in the experiments with concentrations 2 mmol/l Mg2+, no statistical analysis was conducted (no data). Comparing the different inorganic calcium channel blockers with each other, there was a significant stronger effect for Co2+ compared with Mn2+ in concentrations of 2 mmol/l for t0.5 (p = 0.021). There were no statistical significant differences comparing Co2+ and Mn2+ in other concentrations, also not comparing Mg2+ with Mn2+ or Co2+ in concentrations of 2 mmol/l (data not shown). Epileptiform activity vanish in all experiments in period 5.
2.5. Statistical analysis In an explorative analysis there was a statistical significant difference in a dose dependent manner for the different concentrations of Co2+, Mn2+ and Mg2+ in period 3. For Co2+ in all concentrations a dose dependent significant effect could be found either for t0.5 or for t0.9. For Mn2+ in concentrations of 2, 1 and 0.1 mmol/l a dose dependent significant effect could be found in t0.5. Because t0.5 could only be reached in 1 of the experiments with concentrations of 0.5 mmol/l Mn2+, no statistical analysis was conducted (no data). Because t0.9 could only be reached in one of the experiments and in none of the experiments with concentrations of 0.5 and 0.1 mmol/l Mn2+, no statistical analysis was conducted (no data). There was no statistical relevant difference between 2 and 1 mmol/l Mn2+ for t0.9. For Mg2+ in concentrations of 8, 6 and 4 mmol/l a dose dependent significant effect could be found in t0.5, but not in t0.9. The effect for Mg2+ in the concentrations of 5 mmol/l was not statistical relevant compared to the
3. Discussion Aim of the present study was to test the antiepileptic effect of the inorganic calcium channel blockers cobalt, manganese and magnesium in a model using hippocampal slices of guinea pigs. Application of organic calcium antagonists such as verapamil, 3
Brain Research 1732 (2020) 146684
S. Carsten and S. Erwin-Josef
Table 1 Latencies of 90% (t0.9) and 50%, (t0.5) depression of EFP after addition of Co2+, Mn2+ and Mg2+ in minutes. ND: no data; if suppression criteria was not reached in all slices, the number of slices with fulfilled depression criteria is given in brackets; t-test (*) was used if data showed a Gaussian distribution and equal variance and the Mann-Whitney rank sum test (**) if data were not normally distributed. All values are given as mean and standard error of mean (mean ± SEM), with p = 0.05 considered to be significant. Statistical comparisons with groups smaller than n = 3 were not performed. Co2+
2 mmol/ln = 7
1 mmol/ln = 8
0.5 mmol/ln = 2
0.1 mmol/ln = 8
t0.9
15.7 ± 1
21.2 ± 2 ** to 2 mmol/l (p = 0.005)
55 ± 5
t0.5
11.4 ± 1
16.2 ± 2 ** to 2 mmol/l (p = 0.02)
20 ± 5
162 ± 39 (n = 5) ** to 1 mmol/l (p < 0.001) ** to 2 mmol/l (p = 0.001) 80 ± 22 ** to 1 mmol/l (p < 0.001) ** to 2 mmol/l (p < 0.001)
Mn2+
2 mmol/l n=4 20 ± 2 16.3 ± 1
1 mmol/l n=6 26.6 ± 5 15.8 ± 1
0.5 mmol/l n=2 ND (n = 0) 230 (n = 1)
mmol/l n=7 190 (n = 1) 97.5 ± 44 (n = 4) ** to 1 mmol/l (p = 0.001) ** to 2 mmol/l (p = 0.014)
t0.9
8 mmol/l n=3 13.3 ± 2
6 mmol/l n=3 15 ± 0
t0.5
10 ± 0
10 ± 0
5 mmol/l; n=6 52 ± 37 (n = 5) 50 ± 38
4 mmol/l; n=5 70 ± 45 (n = 3) 25 ± 11 ** to 6 mmol/l (p = 0.036) ** to 8 mmol/l (p = 0.036)
t0.9 t0.5
Mg2+
2 mmol/l n=7
1 mmol/l n=8
0.5 mmol/l n=2
0.1 mmol/l n=8
t0.5
25 (n = 1)
ND (n = 0)
ND (n = 0)
t0.9 fmax
ND (n = 0) 2 ± 0 6.6%
22.5 ± 8 (n = 2) 15 (n = 1) 15.2 ± 4 50.5%
ND (n = 0) 4 ± 3 7.5%
ND (n = 0) 12.8 ± 3 15.3%
2 mmol/l n=4 35 ± 10 (n = 3) 30 (n = 1)
1 mmol/l n=6 30 ± 6 (n = 4) ND (n = 0)
0.5 mmol/l n=2 15 ± 0 (n = 2) 55 (n = 1)
34.8 ± 13 73%
36.3 ± 8 61%
33.5 ± 7 113%
mmol/l n=7 19 ± 9 (n = 4) 15 ± 5 (n = 2) 41.1 ± 9 95.7%
8 mmol/l; n=3 15 ± 3
6 mmol/l; n=3 13.3 ± 3
5 mmol/l; n=6 14.2 ± 2
15 ± 0 (n = 2) 35.3 ± 13 109.6%
20 ± 5
19 ± 3 (n = 5) 27.8 ± 12 135.4%
fmax
%
2+
Mn t0.5 t0.9
fmax fmax
%
Mg2+ t0.5 t0.9 fmax fmax
%
25.6 ± 10 111.4%
4 mmol/l; n=5 26.3 ± 10 (n = 4) 22.5 ± 3 (n = 2) 75 ± 37 81.5%
ND (n = 0)
More recently the inorganic calcium channel blocker cobalt was demonstrated to reduce Veratridine-induced epileptiform activity, leading to the hypothesis that veratridine-induced epileptiform activity depends not only on sodium, but also on calcium currents (Link et al., 2008). Thus, there is evidence that transmembraneous inward calcium currents contribute to the generation of the paroxysmal depolarization shifts (PDS) in most epilepsy models (Bingmann and Speckmann, 1989; Speckmann and Walden, 1986; Speckmann et al., 1992; Straub et al., 1997; Witte et al., 1987). Calcium currents starting with the onset of each single PDS were shown to alter intracellular and extracellular calcium concentration (Lucke et al., 1990; Moraidis et al., 1991; Wiemann et al., 1996). More recently beside the direct mechanisms on membrane excitability several other calcium dependent mechanisms, such as G-protein coupled pathways, calcium-dependent gliotransmission, feedback mechanisms between mitochondrial calcium signaling and reactive oxygen species were discussed (Steinlein, 2014). In our study we demonstrate that all tree inorganic calcium channel blockers cobalt, manganese and magnesium are able to suppress epileptic FP in a dosage dependent and reversible manner. Beside more unspecific effects, discussed above, the following mechanisms may contribute: i) blockade of voltage-activated calcium channels in the postsynaptic membrane, ii) changes in the activation of voltage-dependent sodium channels, iii) blockade of synaptic transmission. Beside others Link et al (2008) discussed increased calcium fluxes secondary to neuronal depolarization, which activates voltage-dependent calcium currents and further depolarizes the cell as a possible mechanism and an impaired Na+/Ca+ exchange due to elevated intracellular Na+, which leads to increased intracellular Ca2+ concentration (Adam-Vizi and Ligeti, 1986; Alkadhi and Tian, 1996; Bikson et al., 2002; Link et al., 2008). Calcium is discussed also to contributes to repolarization, as elevated intern Calcium directly limits further calcium inward currents (Eckert and Tillotson, 1981) and activates Ca2+ -dependent Cl−- and K+ -currents (Frings et al., 2000; Hilaire
Table 2 Latencies of 50% (t0.5) and 90%, (t0.9) reappearance of EFP and maximum frequency (fmax) after washout of Co2+, Mn2+ and Mg2+ in minutes. fmax %: percent of frequency compared to 100% frequency in period 2. ND: no data. If reappearance criteria was not reached in all slices, the number of slices with fulfilled depression criteria is given in brackets. All values are given as mean and standard error of mean (mean ± SEM). No statistical comparisons was performed since most groups were smaller than n = 3. Co2+
2 mmol/l n=3 ND (n = 0)
2 mmol/l; n=3 10 ± 0 10 ± 0 35.3 ± 7 137%
flunarizine or nifedipine and the quaternized calcium entry blocker D890 were described to reduced epileptiform activity in several experiments (Bingmann and Speckmann, 1989; Moraidis et al., 1991; Speckmann and Walden, 1986; Straub et al., 1997; Witte et al., 1987). 4
Brain Research 1732 (2020) 146684
S. Carsten and S. Erwin-Josef
Fig. 2. Example of the effect of Mn2+ (2, 1, and 0.1 mmol/l) on bicuculline induced epileptiform field potentials (FP). CA3 neuron, hippocampal slice, guinea pig. FP1 and FP2: tracings of membrane potential by a storage oscilloscope and by an inkwriter, respectively. Recordings related to each other by characters. Times given refer to commencement of the indicated periods.
Magnesium was demonstrated to enhance the antiepileptic efficacy of a subprotective dose of Valproate in a model using pentylenetetrazol (PTZ)-induced convulsions in male Wister rats. As main mechanisms the authors discussed an improved redox balance and modulation of brain amino acids (Safar et al., 2010). In addition magnesium has been used for many years as prophylaxis and treatment of seizures associated with eclampsia (Abdelmalik et al., 2012; Euser and Cipolla, 2009; Gordon et al., 2014; McDonald et al., 2012) and further it is discussed to reduce seizures in patients with epilepsy (Yuen and Sander, 2012). In this context it should be noted that. In children suffering from seizures a markedly reduced Mg2+-content in the CSF during seizure free intervals was described (Becker, 1965; Breyer and Quadbeck, 1965). Thus, deficiency in magnesium, but also in cobalt and manganese are not unusual in the patient population. Therefore, the seizure threshold, in both patient and normal population, may be affected when deficiencies (or possibly also excesses related to exposition from environmental
et al., 2005; Kang et al., 2003; Thompson, 1994). The functional significance of calcium dependent potassium current was shown using apamin (an inhibitor of calcium-gated potassium channels) which increased the duration of veratridine-induced oscillations of intern Ca+ while decreasing their frequency (Link et al., 2008; Lopez et al., 1995). The strongest effect we found was for cobalt 2 mmol, the effect of magnesium was the weakest. Veratridine-induced complexes were suppressed totally by 2 mmol/l and 1 mmol/l Cobalt(II) in a model using the veratridine induced epileptiform activity, which is in line with our findings (Link et al., 2008). In opposite, magnesium blockade might be mainly due to presynaptic synaptic transmission as a main mechanism, since in all experiments a strong initial effect and reduction of the FP frequency and reaching of a “steady state” in limit concentrations could be noted. This mechanism is also described in literature for magnesium (Coan and Collingridge, 1985), but also for manganese and cobalt (Hackett, 1976).
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S. Carsten and S. Erwin-Josef
Fig. 3. Example of the effect of Mg2+ (6, 4, and 2 mmol/l) on bicuculline induced epileptiform field potentials (FP). CA3 neuron, hippocampal slice, guinea pig. FP1 and FP2: tracings of field potential by a storage oscilloscope and by an inkwriter, respectively. Recordings related to each other by characters. Times given refer to commencement of the indicated periods.
Because of the weak reappearance after washout and irregular reactivity (see Fig. 1) after addition of Cobalt, an additional cytotoxic effect could be assumed (Kwon et al., 2009). Furthermore cobalt was described to induce epileptic activity (Colasanti and Craig, 1992). There are insufficient data about manganese and epileptiform activity so far. In a review Gonzalez-Reyes RE and coworkers state that there is no satisfactory explanation for the relationship between low manganese levels and the presence of convulsions. There is, however, a documented correlation between low blood manganese levels and the presence of convulsions in both humans and animals (Gonzalez-Reyes et al., 2007). Beside other side effects manganese intoxication is discussed to be neurotoxic and induce parkinsonism (Chen et al., 2018). Thus, in terms of clinical use, only magnesium, but not cobalt or manganese, might be a potential additional treatment option (Gordon et al., 2014). As a limitation, however, one has to consider that both
hazards) occur (Davidson and Ward, 1988). In a retrospective analysis of 22 cases with drug resistant epilepsy oral Magnesium supplementation was associated with a significant decrease in the number of seizure days per month and seizure freedom in two cases (Abdelmalik et al., 2012). In a rat model the significant rise in brain magnesium concentration was associated with an elevation of the seizure threshold and a marked resistance of the animal to electrically as well as NMDA stimulated hippocampal seizures. Using autoradiography the effect of magnesium sulfate on the NMDA receptor-channel complex, as well as on the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and Kainate receptors, was demonstrated. It was hypothesized that magnesium central activity is mediated, at least in part, via the NMDA receptor. Magnesium sulfate was able to enter the cerebrospinal fluid and brain after systemic administration (Hallak et al., 1994; Hallak, 1998).
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4. Materials and methods
et al., 1997)). Time sections were subdivided into 5 min epochs. One period was switched to the next, if the experimental data were stable (maximum variation of 10%) for at least two successive epochs at the end of the ongoing section. Time reference points were the beginnings of periods 2 through 5, respectively. All times given refer to points in the time with 50% (t0.5) and 90% (t0.9) suppression or reappearance with respect to frequency of occurrence of EFP. Data were compared by using the students unpaired t-test and Mann-Whitney rank sum test. ttest was used if data showed a Gaussian distribution and equal variance and the Mann-Whitney rank sum test if data were not normally distributed. All values are given as mean and standard error of mean (mean ± SEM), with P = 0.05 considered to be significant. Statistical comparisons with groups smaller than n = 3 were not performed. Statistics were carried out by using with Microsoft Excel and SPSS.
4.1. Materials and methods
Author contributions
Experiments were carried out on slices of the hippocampus of guinea pigs of both sexes weighting ca. 300–400 g. Brain was removed under ether anesthesia. The hippocampus was dissected and transverse slices (350–500 μm thick) were cut in parallel to the alvear fibres by means of a hand-held razor blade. Slices were pre-incubated in a submersion chamber for at least 1 h in 28 °C artificial cerebrospinal fluid (CSF) containing (in mmol/l): NaCl 124, KCl 4, CaCl2 1, NaH2PO4 1.24, MgSO4 1.3, NaHCO3 26 and glucose 10. CSF was equilibrated with 5% CO2 in O2 giving a pH of 7.4. After pre-incubation, slices were transferred to a recording submersion chamber which was continuously perfused by CSF with a temperature kept constant at 32 °C. The ionic composition was the same as during pre-incubation except for the Ca2+ concentration which was elevated to 2.0 mmol/l. Field potentials of stratum pyramidale of CA1 and CA3 regions were recorded simultaneously using glass micropipettes (ca. 1MΩ) (Straub et al., 1990). All experiments and protocols were approved by the North-RhineWestphalia authorities for animal experimentation (Regierungspräsident Münster, Schreiben vom 29.11.1990, AZ 26.0834, 71/90 and 06.09.1993, AZ 23.0834, 71/90), all methods were carried out in accordance with relevant guidelines and regulations. Experiments were carried out between 1995 and 1997 at the Institute of Physiology I, Speckmann lab, Münster, Germany.
C.S. carried out the experiments, analyzed and interpreted results, performed statistics and drafted the manuscript. E.J.S. design the study, provided general support and critically reviewed the manuscript.
hypomagnesemia and hypermagnesemia were found to be associated with increased risk of hospital mortality and prolonged length of hospital stay (Al Alawi et al., 2018) and the United States Food and Nutrition Board recommend only daily intake rates for magnesium 420 mg for adult males and 320 mg for adult females (de Baaij et al., 2015). Given the fact that normal range for magnesium in blood is about 1 mmol/l, the concentration we used in our experiments is not in a physiological range. In summary our data support the role of inorganic calcium antagonists in the treatment of epileptiform activity. Beside other mechanisms it strongly suggests that calcium influxes contribute to generation of epileptic discharges.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgements We thank Elke Naß, Birgit Herrenpoth and Ingrid Winkelhues for technical support and support in carrying out the experiments. References Abdelmalik, P.A., Politzer, N., Carlen, P.L., 2012. Magnesium as an effective adjunct therapy for drug resistant seizures. Can. J. Neurol. Sci. 39, 323–327. Adam-Vizi, V., Ligeti, E., 1986. Calcium uptake of rat brain synaptosomes as a function of membrane potential under different depolarizing conditions. J. Physiol. 372, 363–377. Aicardi, G., Schwartzkroin, P.A., 1990. Suppression of epileptiform burst discharges in CA3 neurons of rat hippocampal slices by the organic calcium channel blocker, verapamil. Exp. Brain Res. 81, 288–296. Al Alawi, A.M., Majoni, S.W., Falhammar, H., 2018. Magnesium and human health: perspectives and research directions. Int. J. Endocrinol. 2018, 9041694. Alkadhi, K.A., Tian, L.M., 1996. Veratridine-enhanced persistent sodium current induces bursting in CA1 pyramidal neurons. Neuroscience 71, 625–632. Becker, H., 1965. Untersuchungen über das Verhalten von Magnesium und (Calcium) im Blutserum und Liquor bei verschiedenen Kranheiten im Kindesalter. In: Doctoral Thesis. vol. 1965. Medical Fakulty, eds., Universitäts- und Landesbibliothek- Katalog, WWU Münster, Germany, p. 108. Bikson, M., Baraban, S.C., Durand, D.M., 2002. Conditions sufficient for nonsynaptic epileptogenesis in the CA1 region of hippocampal slices. J. Neurophysiol. 87, 62–71. Bingmann, D., et al., 1988. Differential antiepileptic effects of the organic calcium antagonists verapamil and flunarizine in neurons of organotypic neocortical explants from newborn rats. Exp. Brain Res. 72, 439–442. Bingmann, D., Speckmann, E.J., 1989. Specific suppression of pentylenetetrazol-induced epileptiform discharges in CA3 neurons (hippocampal slice, guinea pig) by the organic calcium antagonists flunarizine and verapamil. Exp. Brain Res. 74, 239–248. Breyer, U., Quadbeck, G., 1965. The cerebrospinal fluid content of magnesium and other cations in central nervous system diseases. Dtsch. Z. Nervenheilkd. 187, 595–607. Chen, P., Bornhorst, J., Aschner, M., 2018. Manganese metabolism in humans. Front. Biosci. (Landmark Ed) 23, 1655–1679. Coan, E.J., Collingridge, G.L., 1985. Magnesium ions block an N-methyl-D-aspartate receptor-mediated component of synaptic transmission in rat hippocampus. Neurosci. Lett. 53, 21–26. Colasanti, B.K., Craig, C.R., 1992. Reduction of seizure frequency by clonazepam during cobalt experimental epilepsy. Brain Res. Bull. 28, 329–331. Davidson, D.L., Ward, N.I., 1988. Abnormal aluminium, cobalt, manganese, strontium and zinc concentrations in untreated epilepsy. Epilepsy Res. 2, 323–330. de Baaij, J.H., Hoenderop, J.G., Bindels, R.J., 2015. Magnesium in man: implications for health and disease. Physiol. Rev. 95, 1–46. Eckert, R., Tillotson, D.L., 1981. Calcium-mediated inactivation of the calcium conductance in caesium-loaded giant neurones of Aplysia californica. J. Physiol. 314, 265–280. Euser, A.G., Cipolla, M.J., 2009. Magnesium sulfate for the treatment of eclampsia: a brief review. Stroke 40, 1169–1175.
4.2. Experimental protocol The established experimental protocol (Straub et al., 1997) consisted of five periods as follows: Period 1: initial control; superfusion with CSF. Period 2: induction of epileptiform activity; superfusion with CSF with bicuculline (10 μmol/l). If no epileptiform activity occurs within one hour, K+ was elevated from normal (4 mmol/l) to 8 mmol/l. Period 3: test of the antiepileptic calcium antagonism; addition of the inorganic calcium antagonist Co2+ (CoCl2; 2, 1, 0.5 and 0.1 mmol/l), Mn2+ (MnCl2; 2, 1, 0.5 and 0.1 mmol/l) and Mg2+ (MgCl2; 8, 6, 5, 4 and 2 mmol/l) to the solution of period 2 (maximum duration four hours). Period 4: washout of the inorganic calcium antagonist with CFS containing bicuculline (maximum duration two hours). Period 5: final control; superfusion with CSF. 4.3. Analysis and statistics The experiments were evaluated by determining the frequency of occurrence of epileptiform field potentials (EFP). The temporal design of each experiment was determined by defined periods consisting of sections lasting 30 min each as described before (page 174; (Straub 7
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Gynaecol. Obstet. 118, 90–96. Moraidis, I., et al., 1991. Caffeine-induced epileptic discharges in CA3 neurons of hippocampal slices of the guinea pig. Neurosci. Lett. 129, 51–54. Pong, S.F., Graham, L.T., 1972. N-methyl bicuculline, a convulsant more potent than bicuculline. Brain Res. 42, 486–490. Safar, M.M., et al., 2010. Magnesium supplementation enhances the anticonvulsant potential of valproate in pentylenetetrazol-treated rats. Brain Res. 1334, 58–64. Speckmann, E.J., Walden, J., 1986. Anticonvulsant effects of calcium channel blockers in partial and generalized model epilepsies. Funct. Neurol. 1, 521–527. Speckmann, E.J., et al., 1992. Pathophysiology of the epilepsies. In: M. Hoke, S.N. Erne, Y.C. Okada, G.L. Romani, (Eds.), Biomagnetism: Clinical Aspects. International Congress Series, Vol. 988, Elsevier Science Publ. BV, Amsterdam, pp. 45–52. Steinlein, O.K., 2014. Calcium signaling and epilepsy. Cell Tissue Res. 357, 385–393. Straub, H., et al., 1990. Paroxysmal depolarization shifts induced by bicuculline in CA3 neurons of hippocampal slices: suppression by the organic calcium antagonist verapamil. Neurosci. Lett. 111, 99–101. Straub, H., Kohling, R., Speckmann, E.J., 1994. Picrotoxin-induced epileptic activity in hippocampal and neocortical slices (guinea pig): suppression by organic calcium channel blockers. Brain Res. 658, 119–126. Straub, H., Kohling, R., Speckmann, E.J., 1997. Strychnine-induced epileptiform activity in hippocampal and neocortical slice preparations: suppression by the organic calcium antagonists verapamil and flunarizine. Brain Res. 773, 173–180. Thompson, S.H., 1994. Facilitation of calcium-dependent potassium current. J. Neurosci. 14, 7713–7725. Wiemann, M., et al., 1996. Simultaneous blockade of intracellular calcium increases and of neuronal epileptiform depolarizations by verapamil. Brain Res. 734, 49–54. Witte, O.W., Speckmann, E.J., Walden, J., 1987. Motor cortical epileptic foci in vivo: actions of a calcium channel blocker on paroxysmal neuronal depolarizations. Electroencephalogr. Clin. Neurophysiol. 66, 43–55. Yuen, A.W., Sander, J.W., 2012. Can magnesium supplementation reduce seizures in people with epilepsy? A hypothesis. Epilepsy Res. 100, 152–156.
Frings, S., Reuter, D., Kleene, S.J., 2000. Neuronal Ca2+ -activated Cl- channels–homing in on an elusive channel species. Prog. Neurobiol. 60, 247–289. Gonzalez-Reyes, R.E., Gutierrez-Alvarez, A.M., Moreno, C.B., 2007. Manganese and epilepsy: a systematic review of the literature. Brain Res. Rev. 53, 332–336. Gordon, R., et al., 2014. Magnesium sulphate for the management of preeclampsia and eclampsia in low and middle income countries: a systematic review of tested dosing regimens. J. Obstet. Gynaecol. Can. 36, 154–163. Hackett, J.T., 1976. Selective antagonism of frog cerebellar synaptic transmission by manganese and cobalt ions. Brain Res. 114, 47–52. Hallak, M., et al., 1994. Magnesium sulfate treatment decreases N-methyl-D-aspartate receptor binding in the rat brain: an autoradiographic study. J. Soc. Gynecol. Invest. 1, 25–30. Hallak, M., 1998. Effect of parenteral magnesium sulfate administration on excitatory amino acid receptors in the rat brain. Magnes. Res. 11, 117–131. Hilaire, C., et al., 2005. K(+) current regulates calcium-activated chloride current-induced after depolarization in axotomized sensory neurons. Eur. J. Neurosci. 22, 1073–1080. Kang, T.M., Markin, V.S., Hilgemann, D.W., 2003. Ion fluxes in giant excised cardiac membrane patches detected and quantified with ion-selective microelectrodes. J. Gen. Physiol. 121, 325–347. Kwon, Y.M., et al., 2009. Dose-dependent cytotoxicity of clinically relevant cobalt nanoparticles and ions on macrophages in vitro. Biomed. Mater. 4, 025018. Link, M.C., Wiemann, M., Bingmann, D., 2008. Organic and inorganic calcium antagonists inhibit veratridine-induced epileptiform activity in CA3 neurons of the guinea pig. Epilepsy Res. 78, 147–154. Lopez, M.G., et al., 1995. Veratridine-induced oscillations of cytosolic calcium and membrane potential in bovine chromaffin cells. J. Physiol. 482 (Pt 1), 15–27. Lucke, A., et al., 1990. Decrease of free calcium concentration at the outer surface of identified snail neurons during paroxysmal depolarization shifts. Neurosci. Lett. 112, 190–193. McDonald, S.D., et al., 2012. A systematic review of maternal and infant outcomes following magnesium sulfate for pre-eclampsia/eclampsia in real-world use. Int. J.
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Research report
Antiepileptic effects of cobalt, manganese and magnesium on bicucullineinduced epileptiform activity in hippocampal neurons
T
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Saft Carstena, , Speckmann Erwin-Josefb a b
Department of Neurology, St. Josef Hospital, Gudrunstraße 56, 44791 Bochum, Germany Institut für Physiology I, Westfälische Wilhelms Universität, Robert-Koch Str. 27a, 48149 Münster, Germany
H I GH L IG H T S
inorganic calcium channel blockers cobalt, manganese and magnesium were able to suppress bicuculline induced epileptiform field potentials in a dosage • The dependent and reversible manner. were carried out by using hippocampal slices of guinea pigs as a model. Potential mechanisms and clinical relevance of findings are discussed. • Experiments • Potential mechanisms and clinical relevance of findings are discussed.
A R T I C LE I N FO
A B S T R A C T
Keywords: Calcium channel blocker Cobalt Manganese Magnesium Epilepsy
Background: Calcium signaling is described as a relevant factor in synchronization of neurons and increased excitability in epileptogenesis. Aim of the present investigations was to test the antiepileptic effect of the classical inorganic calcium channel blockers cobalt (Co2+), manganese (Mn2+) and magnesium (Mg2+). Methods: Experiments were carried out on hippocampal slices of guinea pigs. Epileptiform field potentials (EFP) were elicited by adding bicuculline (10 µmol/l) to the artificial cerebrospinal fluid (CSF). Kalium was elevated from normal (4 mmol/l) to 8 mmol/l. Co2+ (CoCl2; 2, 1, 0.5 and 0.1 mmol/l), Mn2+ (MnCl2; 2, 1, 0.5 and 0.1 mmol/l) and Mg2+ (MgCl2; 8, 6, 5, 4 and 2 mmol/l) were added to the superfusate. Results: Concentrations of 2, 1 and 0.5 mmol/l Co2+, 2 and 1 mmol/l Mn2+ and 8 respectively 6 mmol/l Mg2+ were able to suppress EFP sufficient in a dose dependent manner. In concentrations of 0.1 mmol/l Co2+, 0.5 mmol/l and 0.1 mmol/l Mn2+ and 5 respectively 4 and 2 mmol/l Mg2+ suppression was incomplete. With washout of the inorganic calcium channel blockers the EFP reappeared. Discussion: All tree inorganic calcium channel blockers were able to suppress EFP in a dosage dependent and reversible manner. Weak reappearance of EFP after washout of Co2+ might be due to additional cytotoxic effects. The following mechanisms may contribute: i) blockade of voltage-activated calcium channels in the postsynaptic membrane, ii) changes in the activation of voltage-dependent sodium channels, iii) blockade of synaptic transmission.
1. Introduction Calcium (Ca) signaling is described as a relevant factor in synchronization of neurons and increased excitability in epileptogenesis (Steinlein, 2014). Several mechanisms seem to be involved in generation of epileptic discharges. Discussed are immediate effects on membrane excitability by calcium influx through ion channels, feedback mechanisms including mitochondrial calcium signaling and delayed mechanisms that act through G-protein coupled pathways (Steinlein, 2014).
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Organic blockers of voltage-activated calcium channels have been demonstrated to suppress epileptiform activity induced by gamma aminobutyric acid (GABA) receptor antagonists in various experimental models (Aicardi and Schwartzkroin, 1990; Bingmann et al., 1988; Straub et al., 1990; Straub et al., 1994). Veratridine-induced epileptiform activity was suppressed by both inorganic (Cobalt) and organic (Verapamil) calcium antagonists (Link et al., 2008). These observations led to the hypothesis, that a massive influx of Ca through voltage-sensitive calcium channels is involved in the convulsant action of the antagonist of the GABA receptor. The antiepileptic effect of organic
Corresponding author. E-mail address: [email protected] (S. Carsten).
https://doi.org/10.1016/j.brainres.2020.146684 Received 8 January 2020; Received in revised form 22 January 2020; Accepted 25 January 2020 Available online 27 January 2020 0006-8993/ © 2020 Published by Elsevier B.V.
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S. Carsten and S. Erwin-Josef
2.3. Dosage dependent and reversible suppression of epileptiform activity by manganese
calcium antagonists, therefore, was mainly attributed to a reduction of postsynaptic calcium currents (Bingmann and Speckmann, 1989). Magnesium supplementation is discussed to reduce seizures in people with epilepsy (Yuen and Sander, 2012). The aim of the present investigation was to test the antiepileptic effect of the inorganic calcium channel blockers cobalt, manganese and magnesium in a model using hippocampal slices of guinea pigs and bicuculline induced epileptiform field potentials (EFP). In contrast to the organic substances the inorganic ones are known to have a more focused effect on calcium channels. Bicuculline, a GABAA receptor antagonist, is assumed to produce paroxysmal depolarisation shifts (PDS) by blocking GABA-mediated synaptic transmission (Pong and Graham, 1972).
When Mn2+ was added to the superfusate, t0.9 were 20 ± 2 min (2 mmol/l; n = 4), 26.6 ± 5 min (1 mmol/l; n = 6) and 190 min (0.1 mmol/l; n = 1/7). In concentrations of 0.1 mmol/l 90% depression could only be found in 1 out of 7 slices, t0.9 was not reached within 4 h in 6 slices. With concentrations of 0.5 mmol/l t0.9 was not reached within 4 h in both slices. When Mn2+ was added to the superfusate, t0.5 were 16.3 ± 1 min (2 mmol/l; n = 4), 15.8 ± 1 min (1 mmol/l; n = 6), 230 min (0.5 mmol/l; n = 1/2) and 97.5 ± 44 min (0.1 mmol/l; n = 4/7). In concentrations of 0.1 mmol/l 50% depression could only be found in 4 out of 7 slices, t0.5 was not reached within 4 h in 3 slices. In concentrations of 0.5 mmol/l 50% depression could only be found in 1 out of 2 slices, t0.5 was not reached within 4 h in 1 slice (see Fig. 2; Table 1). With washout of the inorganic calcium channel blocker Mn2+ EFP reappeared in all slices. After washout from Mn2+ latencies of 50% reappearance (t0.5) were 35 ± 10 min (2 mmol/l; n = 3/4), 30 ± 6 min (1 mmol/l; n = 4/6), 15 ± 0 min (0,5 mmol/l; n = 2) and 19 ± 9 (0.1 mmol/l; n = 4/7). In concentrations of 2 mmol/l 50% reappearance could only be found in 3 out of 4 slices, in concentrations of 1 mmol/l only in 4 out of 6 slices, and in concentrations of 0.1 mmol/l only in 4 out of 7 slices. After washout from Mn2+ latency of 90% reappearance (t0.9) were 30 min (2 mmol/l; n = 1/4), 55 min (0.5 mmol/l; n = 1/2) and 15 ± 5 min (0.1 mmol/l; n = 2/7). In concentrations of 2 mmol/l 90% reappearance could only be found in 1 out of 4 slices, in concentrations of 0.5 mmol/l only in 1 out of 2 slices and in concentrations of 0.1 mmol/l only in 2 out of 7 slices. In concentrations of 1 mmol/l in none of the slices 90% reappearance was reached within a maximum of 2½ hour (all ½-2½) hour (see Table 2).
2. Results 2.1. Induction of epileptiform activity 89 slices, including 5 controls, were investigated. In the 5 controls epileptiform activity was induced by superfusion with artificial cerebrospinal fluid (CSF) with bicuculline (10 μmol/l) and elevated Kalium (K+) without adding a calcium antagonist. Epileptiform activity occurs after 6 ± 4 min and remains stable until washout for 6 h. Field potentials of stratum pyramidale of hippocampal cornu ammonis 1 (CA1) and hippocampal cornu ammonis 3 (CA3) regions were recorded simultaneously in 77 slices, in 10 slices only CA3 region was recorded due to technical reasons. Superfusion with bicuculline containing CSF (Period 2) induces epileptic field potentials (EFP) if K+ was normal in 5 of 89 cases. Because no statistically analysis is possible, results of occurrence of EFP and suppression by inorganic calcium antagonists for these 5 cases are not given in this report. After elevation of K+ to 8 mmol/l (n = 84) EFP occurs in 69 slices after 6.8 ± 3.3 min. No constant EFP occur in 15 slices. EFP occurred simultaneously in CA1 and CA3 region in 60 from all 69 cases. In 5 cases EFP occurred only in CA3 region, in 4 cases only in CA1 region. Because activation occurred simultaneously in all cases mentioned above, slices with only one active region (CA1 or CA3) were also included in the study without making a difference in the following.
2.4. Dosage dependent and reversible suppression of epileptiform activity by magnesium When Mg2+ was added to the superfusate, t0.9 were 13.3 ± 2 min (8 mmol/l; n = 3), 15 ± 0 min (6 mmol/l; n = 3), 52 ± 37 min (5 mmol/l; n = 5/6) and 70 ± 45 min (4 mmol/l; n = 3/5). In concentrations of 5 mmol/l 90% depression could only be found in five of six slices, t0.9 was not reached within 4 h in 1 slice. In concentrations of 4 mmol/l 90% depression could only be found in 3 out of 5 slices, t0.9 was not reached within 4 h in 2 slices. With concentrations of 2 mmol/l (n = 3) t0.9 was not reached within 4 h in 3 slices. When Mg2+ was added to the superfusate, t0.5 were 10 ± 0 min (8 mmol/l; n = 3), 10 ± 0 min (6 mmol/l; n = 3), 50 ± 38 min (5 mmol/l; n = 5) and 25 ± 11 min (4 mmol/l; n = 5). With concentrations of 2 mmol/l (n = 3) t0.5 was not reached within 4 h in 3 slices (see Fig. 3; Table 1). With washout of the inorganic calcium channel blocker Mg2+ EFP reappeared in all slices. After washout from Mg2+ latencies of 50% reappearance (t0.5) were 15 ± 3 min (8 mmol/l; n = 3), 13 ± 3 min (6 mmol/l; n = 3), 14.2 ± 2 min (5 mmol/l; n = 6), 26.3 ± 10 min (4 mmol/l; n = 4/5) and 10 ± 0 (2 mmol/l; n = 3). In concentrations of 4 mmol/l 50% reappearance could only be found in 4 out of 5 slices. After washout from Mg2+ latency of 90% reappearance (t0.9) were 15 ± 0 min (8 mmol/l; n = 2/3), 20 ± 5 min (6 mmol/l; n = 3), 19 ± 3 min (5 mmol/l; n = 5/6), 22.5 ± 3 min (4 mmol/l; n = 2/5) and 10 ± 0 min (2 mmol/l; n = 3). In concentrations of 8 mmol/l 90% reappearance could only be found in 2 of 3 slices, in concentrations of 5 mmol/l only in 5 out of 6 slices and in concentrations of 4 mmol/l only in 2 out of 5 slices (see Table 2).
2.2. Dosage dependent and reversible suppression of epileptiform activity by cobalt When Co2+ was added to the superfusate, latencies of 90% depression (t0.9) were 15.7 ± 1 min (2 mmol/l; n = 7), 21.2 ± 2 min (1 mmol/l; n = 8), 55 ± 5 min (0.5 mmol/l; n = 2) and 162 ± 39 min (0.1 mmol/l; n = 5/8). In concentrations of 0.1 mmol/l 90% depression could only be found in five of eight slices, t0.9 was not reached within 4 h in three slices. Latencies of 50% depression (t0.5) were 11.4 ± 1 min (2 mmol/l; n = 7), 16.2 ± 2 min (1 mmol/l; n = 8), 20 ± 5 min (0.5 mmol/l; n = 2) and 80 ± 22 min (0.1 mmol/l; n = 8: see Fig. 1; Table 1). With washout of the inorganic calcium channel blocker Co2+ EFP reappeared in all slices. After washout from Co2+ latencies of 50% reappearance (t0.5) were 25 min (2 mmol/l; n = 1/7) and 22.5 ± 8 (1 mmol/l; n = 2/8). In concentrations of 2 mmol/l 50% reappearance could only be found in 1 out of 7 slices, in concentrations of 1 mmol/l only in 2 out of 8 slices, in concentrations of 0.5 mmol/l and 0.1 mmol/l in none of the slices 50% reappearance was reached within a maximum of 2 h (all 1–2) hour. After washout from Co2+ 1 mmol/l latency of 90% reappearance (t0.9) was 15 min in only one of eight cases (1 mmol/l; n = 1/8). In concentrations of 2, 0.5 mmol/l and 0.1 mmol/l in none of the slices 90% reappearance was reached within two hours (see Table 2).
2
Brain Research 1732 (2020) 146684
S. Carsten and S. Erwin-Josef
Fig. 1. Example of the effect of Co2+ (2, 1, and 0.1 mmol/l) on bicuculline induced epileptiform field potentials (FP). CA3 neuron, hippocampal slice, guinea pig. FP1 and FP2: tracings of field potential by a storage oscilloscope and by an inkwriter, respectively. Recordings related to each other by characters. Times given refer to commencement of the indicated periods.
other concentrations. Because t0.5 and t0.9 could not be reached in the experiments with concentrations 2 mmol/l Mg2+, no statistical analysis was conducted (no data). Comparing the different inorganic calcium channel blockers with each other, there was a significant stronger effect for Co2+ compared with Mn2+ in concentrations of 2 mmol/l for t0.5 (p = 0.021). There were no statistical significant differences comparing Co2+ and Mn2+ in other concentrations, also not comparing Mg2+ with Mn2+ or Co2+ in concentrations of 2 mmol/l (data not shown). Epileptiform activity vanish in all experiments in period 5.
2.5. Statistical analysis In an explorative analysis there was a statistical significant difference in a dose dependent manner for the different concentrations of Co2+, Mn2+ and Mg2+ in period 3. For Co2+ in all concentrations a dose dependent significant effect could be found either for t0.5 or for t0.9. For Mn2+ in concentrations of 2, 1 and 0.1 mmol/l a dose dependent significant effect could be found in t0.5. Because t0.5 could only be reached in 1 of the experiments with concentrations of 0.5 mmol/l Mn2+, no statistical analysis was conducted (no data). Because t0.9 could only be reached in one of the experiments and in none of the experiments with concentrations of 0.5 and 0.1 mmol/l Mn2+, no statistical analysis was conducted (no data). There was no statistical relevant difference between 2 and 1 mmol/l Mn2+ for t0.9. For Mg2+ in concentrations of 8, 6 and 4 mmol/l a dose dependent significant effect could be found in t0.5, but not in t0.9. The effect for Mg2+ in the concentrations of 5 mmol/l was not statistical relevant compared to the
3. Discussion Aim of the present study was to test the antiepileptic effect of the inorganic calcium channel blockers cobalt, manganese and magnesium in a model using hippocampal slices of guinea pigs. Application of organic calcium antagonists such as verapamil, 3
Brain Research 1732 (2020) 146684
S. Carsten and S. Erwin-Josef
Table 1 Latencies of 90% (t0.9) and 50%, (t0.5) depression of EFP after addition of Co2+, Mn2+ and Mg2+ in minutes. ND: no data; if suppression criteria was not reached in all slices, the number of slices with fulfilled depression criteria is given in brackets; t-test (*) was used if data showed a Gaussian distribution and equal variance and the Mann-Whitney rank sum test (**) if data were not normally distributed. All values are given as mean and standard error of mean (mean ± SEM), with p = 0.05 considered to be significant. Statistical comparisons with groups smaller than n = 3 were not performed. Co2+
2 mmol/ln = 7
1 mmol/ln = 8
0.5 mmol/ln = 2
0.1 mmol/ln = 8
t0.9
15.7 ± 1
21.2 ± 2 ** to 2 mmol/l (p = 0.005)
55 ± 5
t0.5
11.4 ± 1
16.2 ± 2 ** to 2 mmol/l (p = 0.02)
20 ± 5
162 ± 39 (n = 5) ** to 1 mmol/l (p < 0.001) ** to 2 mmol/l (p = 0.001) 80 ± 22 ** to 1 mmol/l (p < 0.001) ** to 2 mmol/l (p < 0.001)
Mn2+
2 mmol/l n=4 20 ± 2 16.3 ± 1
1 mmol/l n=6 26.6 ± 5 15.8 ± 1
0.5 mmol/l n=2 ND (n = 0) 230 (n = 1)
mmol/l n=7 190 (n = 1) 97.5 ± 44 (n = 4) ** to 1 mmol/l (p = 0.001) ** to 2 mmol/l (p = 0.014)
t0.9
8 mmol/l n=3 13.3 ± 2
6 mmol/l n=3 15 ± 0
t0.5
10 ± 0
10 ± 0
5 mmol/l; n=6 52 ± 37 (n = 5) 50 ± 38
4 mmol/l; n=5 70 ± 45 (n = 3) 25 ± 11 ** to 6 mmol/l (p = 0.036) ** to 8 mmol/l (p = 0.036)
t0.9 t0.5
Mg2+
2 mmol/l n=7
1 mmol/l n=8
0.5 mmol/l n=2
0.1 mmol/l n=8
t0.5
25 (n = 1)
ND (n = 0)
ND (n = 0)
t0.9 fmax
ND (n = 0) 2 ± 0 6.6%
22.5 ± 8 (n = 2) 15 (n = 1) 15.2 ± 4 50.5%
ND (n = 0) 4 ± 3 7.5%
ND (n = 0) 12.8 ± 3 15.3%
2 mmol/l n=4 35 ± 10 (n = 3) 30 (n = 1)
1 mmol/l n=6 30 ± 6 (n = 4) ND (n = 0)
0.5 mmol/l n=2 15 ± 0 (n = 2) 55 (n = 1)
34.8 ± 13 73%
36.3 ± 8 61%
33.5 ± 7 113%
mmol/l n=7 19 ± 9 (n = 4) 15 ± 5 (n = 2) 41.1 ± 9 95.7%
8 mmol/l; n=3 15 ± 3
6 mmol/l; n=3 13.3 ± 3
5 mmol/l; n=6 14.2 ± 2
15 ± 0 (n = 2) 35.3 ± 13 109.6%
20 ± 5
19 ± 3 (n = 5) 27.8 ± 12 135.4%
fmax
%
2+
Mn t0.5 t0.9
fmax fmax
%
Mg2+ t0.5 t0.9 fmax fmax
%
25.6 ± 10 111.4%
4 mmol/l; n=5 26.3 ± 10 (n = 4) 22.5 ± 3 (n = 2) 75 ± 37 81.5%
ND (n = 0)
More recently the inorganic calcium channel blocker cobalt was demonstrated to reduce Veratridine-induced epileptiform activity, leading to the hypothesis that veratridine-induced epileptiform activity depends not only on sodium, but also on calcium currents (Link et al., 2008). Thus, there is evidence that transmembraneous inward calcium currents contribute to the generation of the paroxysmal depolarization shifts (PDS) in most epilepsy models (Bingmann and Speckmann, 1989; Speckmann and Walden, 1986; Speckmann et al., 1992; Straub et al., 1997; Witte et al., 1987). Calcium currents starting with the onset of each single PDS were shown to alter intracellular and extracellular calcium concentration (Lucke et al., 1990; Moraidis et al., 1991; Wiemann et al., 1996). More recently beside the direct mechanisms on membrane excitability several other calcium dependent mechanisms, such as G-protein coupled pathways, calcium-dependent gliotransmission, feedback mechanisms between mitochondrial calcium signaling and reactive oxygen species were discussed (Steinlein, 2014). In our study we demonstrate that all tree inorganic calcium channel blockers cobalt, manganese and magnesium are able to suppress epileptic FP in a dosage dependent and reversible manner. Beside more unspecific effects, discussed above, the following mechanisms may contribute: i) blockade of voltage-activated calcium channels in the postsynaptic membrane, ii) changes in the activation of voltage-dependent sodium channels, iii) blockade of synaptic transmission. Beside others Link et al (2008) discussed increased calcium fluxes secondary to neuronal depolarization, which activates voltage-dependent calcium currents and further depolarizes the cell as a possible mechanism and an impaired Na+/Ca+ exchange due to elevated intracellular Na+, which leads to increased intracellular Ca2+ concentration (Adam-Vizi and Ligeti, 1986; Alkadhi and Tian, 1996; Bikson et al., 2002; Link et al., 2008). Calcium is discussed also to contributes to repolarization, as elevated intern Calcium directly limits further calcium inward currents (Eckert and Tillotson, 1981) and activates Ca2+ -dependent Cl−- and K+ -currents (Frings et al., 2000; Hilaire
Table 2 Latencies of 50% (t0.5) and 90%, (t0.9) reappearance of EFP and maximum frequency (fmax) after washout of Co2+, Mn2+ and Mg2+ in minutes. fmax %: percent of frequency compared to 100% frequency in period 2. ND: no data. If reappearance criteria was not reached in all slices, the number of slices with fulfilled depression criteria is given in brackets. All values are given as mean and standard error of mean (mean ± SEM). No statistical comparisons was performed since most groups were smaller than n = 3. Co2+
2 mmol/l n=3 ND (n = 0)
2 mmol/l; n=3 10 ± 0 10 ± 0 35.3 ± 7 137%
flunarizine or nifedipine and the quaternized calcium entry blocker D890 were described to reduced epileptiform activity in several experiments (Bingmann and Speckmann, 1989; Moraidis et al., 1991; Speckmann and Walden, 1986; Straub et al., 1997; Witte et al., 1987). 4
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Fig. 2. Example of the effect of Mn2+ (2, 1, and 0.1 mmol/l) on bicuculline induced epileptiform field potentials (FP). CA3 neuron, hippocampal slice, guinea pig. FP1 and FP2: tracings of membrane potential by a storage oscilloscope and by an inkwriter, respectively. Recordings related to each other by characters. Times given refer to commencement of the indicated periods.
Magnesium was demonstrated to enhance the antiepileptic efficacy of a subprotective dose of Valproate in a model using pentylenetetrazol (PTZ)-induced convulsions in male Wister rats. As main mechanisms the authors discussed an improved redox balance and modulation of brain amino acids (Safar et al., 2010). In addition magnesium has been used for many years as prophylaxis and treatment of seizures associated with eclampsia (Abdelmalik et al., 2012; Euser and Cipolla, 2009; Gordon et al., 2014; McDonald et al., 2012) and further it is discussed to reduce seizures in patients with epilepsy (Yuen and Sander, 2012). In this context it should be noted that. In children suffering from seizures a markedly reduced Mg2+-content in the CSF during seizure free intervals was described (Becker, 1965; Breyer and Quadbeck, 1965). Thus, deficiency in magnesium, but also in cobalt and manganese are not unusual in the patient population. Therefore, the seizure threshold, in both patient and normal population, may be affected when deficiencies (or possibly also excesses related to exposition from environmental
et al., 2005; Kang et al., 2003; Thompson, 1994). The functional significance of calcium dependent potassium current was shown using apamin (an inhibitor of calcium-gated potassium channels) which increased the duration of veratridine-induced oscillations of intern Ca+ while decreasing their frequency (Link et al., 2008; Lopez et al., 1995). The strongest effect we found was for cobalt 2 mmol, the effect of magnesium was the weakest. Veratridine-induced complexes were suppressed totally by 2 mmol/l and 1 mmol/l Cobalt(II) in a model using the veratridine induced epileptiform activity, which is in line with our findings (Link et al., 2008). In opposite, magnesium blockade might be mainly due to presynaptic synaptic transmission as a main mechanism, since in all experiments a strong initial effect and reduction of the FP frequency and reaching of a “steady state” in limit concentrations could be noted. This mechanism is also described in literature for magnesium (Coan and Collingridge, 1985), but also for manganese and cobalt (Hackett, 1976).
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Fig. 3. Example of the effect of Mg2+ (6, 4, and 2 mmol/l) on bicuculline induced epileptiform field potentials (FP). CA3 neuron, hippocampal slice, guinea pig. FP1 and FP2: tracings of field potential by a storage oscilloscope and by an inkwriter, respectively. Recordings related to each other by characters. Times given refer to commencement of the indicated periods.
Because of the weak reappearance after washout and irregular reactivity (see Fig. 1) after addition of Cobalt, an additional cytotoxic effect could be assumed (Kwon et al., 2009). Furthermore cobalt was described to induce epileptic activity (Colasanti and Craig, 1992). There are insufficient data about manganese and epileptiform activity so far. In a review Gonzalez-Reyes RE and coworkers state that there is no satisfactory explanation for the relationship between low manganese levels and the presence of convulsions. There is, however, a documented correlation between low blood manganese levels and the presence of convulsions in both humans and animals (Gonzalez-Reyes et al., 2007). Beside other side effects manganese intoxication is discussed to be neurotoxic and induce parkinsonism (Chen et al., 2018). Thus, in terms of clinical use, only magnesium, but not cobalt or manganese, might be a potential additional treatment option (Gordon et al., 2014). As a limitation, however, one has to consider that both
hazards) occur (Davidson and Ward, 1988). In a retrospective analysis of 22 cases with drug resistant epilepsy oral Magnesium supplementation was associated with a significant decrease in the number of seizure days per month and seizure freedom in two cases (Abdelmalik et al., 2012). In a rat model the significant rise in brain magnesium concentration was associated with an elevation of the seizure threshold and a marked resistance of the animal to electrically as well as NMDA stimulated hippocampal seizures. Using autoradiography the effect of magnesium sulfate on the NMDA receptor-channel complex, as well as on the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and Kainate receptors, was demonstrated. It was hypothesized that magnesium central activity is mediated, at least in part, via the NMDA receptor. Magnesium sulfate was able to enter the cerebrospinal fluid and brain after systemic administration (Hallak et al., 1994; Hallak, 1998).
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4. Materials and methods
et al., 1997)). Time sections were subdivided into 5 min epochs. One period was switched to the next, if the experimental data were stable (maximum variation of 10%) for at least two successive epochs at the end of the ongoing section. Time reference points were the beginnings of periods 2 through 5, respectively. All times given refer to points in the time with 50% (t0.5) and 90% (t0.9) suppression or reappearance with respect to frequency of occurrence of EFP. Data were compared by using the students unpaired t-test and Mann-Whitney rank sum test. ttest was used if data showed a Gaussian distribution and equal variance and the Mann-Whitney rank sum test if data were not normally distributed. All values are given as mean and standard error of mean (mean ± SEM), with P = 0.05 considered to be significant. Statistical comparisons with groups smaller than n = 3 were not performed. Statistics were carried out by using with Microsoft Excel and SPSS.
4.1. Materials and methods
Author contributions
Experiments were carried out on slices of the hippocampus of guinea pigs of both sexes weighting ca. 300–400 g. Brain was removed under ether anesthesia. The hippocampus was dissected and transverse slices (350–500 μm thick) were cut in parallel to the alvear fibres by means of a hand-held razor blade. Slices were pre-incubated in a submersion chamber for at least 1 h in 28 °C artificial cerebrospinal fluid (CSF) containing (in mmol/l): NaCl 124, KCl 4, CaCl2 1, NaH2PO4 1.24, MgSO4 1.3, NaHCO3 26 and glucose 10. CSF was equilibrated with 5% CO2 in O2 giving a pH of 7.4. After pre-incubation, slices were transferred to a recording submersion chamber which was continuously perfused by CSF with a temperature kept constant at 32 °C. The ionic composition was the same as during pre-incubation except for the Ca2+ concentration which was elevated to 2.0 mmol/l. Field potentials of stratum pyramidale of CA1 and CA3 regions were recorded simultaneously using glass micropipettes (ca. 1MΩ) (Straub et al., 1990). All experiments and protocols were approved by the North-RhineWestphalia authorities for animal experimentation (Regierungspräsident Münster, Schreiben vom 29.11.1990, AZ 26.0834, 71/90 and 06.09.1993, AZ 23.0834, 71/90), all methods were carried out in accordance with relevant guidelines and regulations. Experiments were carried out between 1995 and 1997 at the Institute of Physiology I, Speckmann lab, Münster, Germany.
C.S. carried out the experiments, analyzed and interpreted results, performed statistics and drafted the manuscript. E.J.S. design the study, provided general support and critically reviewed the manuscript.
hypomagnesemia and hypermagnesemia were found to be associated with increased risk of hospital mortality and prolonged length of hospital stay (Al Alawi et al., 2018) and the United States Food and Nutrition Board recommend only daily intake rates for magnesium 420 mg for adult males and 320 mg for adult females (de Baaij et al., 2015). Given the fact that normal range for magnesium in blood is about 1 mmol/l, the concentration we used in our experiments is not in a physiological range. In summary our data support the role of inorganic calcium antagonists in the treatment of epileptiform activity. Beside other mechanisms it strongly suggests that calcium influxes contribute to generation of epileptic discharges.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgements We thank Elke Naß, Birgit Herrenpoth and Ingrid Winkelhues for technical support and support in carrying out the experiments. References Abdelmalik, P.A., Politzer, N., Carlen, P.L., 2012. Magnesium as an effective adjunct therapy for drug resistant seizures. Can. J. Neurol. Sci. 39, 323–327. Adam-Vizi, V., Ligeti, E., 1986. Calcium uptake of rat brain synaptosomes as a function of membrane potential under different depolarizing conditions. J. Physiol. 372, 363–377. Aicardi, G., Schwartzkroin, P.A., 1990. Suppression of epileptiform burst discharges in CA3 neurons of rat hippocampal slices by the organic calcium channel blocker, verapamil. Exp. Brain Res. 81, 288–296. Al Alawi, A.M., Majoni, S.W., Falhammar, H., 2018. Magnesium and human health: perspectives and research directions. Int. J. Endocrinol. 2018, 9041694. Alkadhi, K.A., Tian, L.M., 1996. Veratridine-enhanced persistent sodium current induces bursting in CA1 pyramidal neurons. Neuroscience 71, 625–632. Becker, H., 1965. Untersuchungen über das Verhalten von Magnesium und (Calcium) im Blutserum und Liquor bei verschiedenen Kranheiten im Kindesalter. In: Doctoral Thesis. vol. 1965. Medical Fakulty, eds., Universitäts- und Landesbibliothek- Katalog, WWU Münster, Germany, p. 108. Bikson, M., Baraban, S.C., Durand, D.M., 2002. Conditions sufficient for nonsynaptic epileptogenesis in the CA1 region of hippocampal slices. J. Neurophysiol. 87, 62–71. Bingmann, D., et al., 1988. Differential antiepileptic effects of the organic calcium antagonists verapamil and flunarizine in neurons of organotypic neocortical explants from newborn rats. Exp. Brain Res. 72, 439–442. Bingmann, D., Speckmann, E.J., 1989. Specific suppression of pentylenetetrazol-induced epileptiform discharges in CA3 neurons (hippocampal slice, guinea pig) by the organic calcium antagonists flunarizine and verapamil. Exp. Brain Res. 74, 239–248. Breyer, U., Quadbeck, G., 1965. The cerebrospinal fluid content of magnesium and other cations in central nervous system diseases. Dtsch. Z. Nervenheilkd. 187, 595–607. Chen, P., Bornhorst, J., Aschner, M., 2018. Manganese metabolism in humans. Front. Biosci. (Landmark Ed) 23, 1655–1679. Coan, E.J., Collingridge, G.L., 1985. Magnesium ions block an N-methyl-D-aspartate receptor-mediated component of synaptic transmission in rat hippocampus. Neurosci. Lett. 53, 21–26. Colasanti, B.K., Craig, C.R., 1992. Reduction of seizure frequency by clonazepam during cobalt experimental epilepsy. Brain Res. Bull. 28, 329–331. Davidson, D.L., Ward, N.I., 1988. Abnormal aluminium, cobalt, manganese, strontium and zinc concentrations in untreated epilepsy. Epilepsy Res. 2, 323–330. de Baaij, J.H., Hoenderop, J.G., Bindels, R.J., 2015. Magnesium in man: implications for health and disease. Physiol. Rev. 95, 1–46. Eckert, R., Tillotson, D.L., 1981. Calcium-mediated inactivation of the calcium conductance in caesium-loaded giant neurones of Aplysia californica. J. Physiol. 314, 265–280. Euser, A.G., Cipolla, M.J., 2009. Magnesium sulfate for the treatment of eclampsia: a brief review. Stroke 40, 1169–1175.
4.2. Experimental protocol The established experimental protocol (Straub et al., 1997) consisted of five periods as follows: Period 1: initial control; superfusion with CSF. Period 2: induction of epileptiform activity; superfusion with CSF with bicuculline (10 μmol/l). If no epileptiform activity occurs within one hour, K+ was elevated from normal (4 mmol/l) to 8 mmol/l. Period 3: test of the antiepileptic calcium antagonism; addition of the inorganic calcium antagonist Co2+ (CoCl2; 2, 1, 0.5 and 0.1 mmol/l), Mn2+ (MnCl2; 2, 1, 0.5 and 0.1 mmol/l) and Mg2+ (MgCl2; 8, 6, 5, 4 and 2 mmol/l) to the solution of period 2 (maximum duration four hours). Period 4: washout of the inorganic calcium antagonist with CFS containing bicuculline (maximum duration two hours). Period 5: final control; superfusion with CSF. 4.3. Analysis and statistics The experiments were evaluated by determining the frequency of occurrence of epileptiform field potentials (EFP). The temporal design of each experiment was determined by defined periods consisting of sections lasting 30 min each as described before (page 174; (Straub 7
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