Korean J Physiol Pharmacol.  2024 Sep;28(5):413-422. 10.4196/kjpp.2024.28.5.413.

Roles of metabotropic glutamate receptor 5 in low [Mg²⁺ ]_o -induced interictal epileptiform activity in rat hippocampal slices

• Yang JS
• Jang HJ
• Sung KW
• Rhie DJ
• Yoon SH

• KMID: 2559134
• DOI: http://doi.org/10.4196/kjpp.2024.28.5.413

Abstract

Group I metabotropic glutamate receptors (mGluRs) modulate postsynaptic neuronal excitability and epileptogenesis. We investigated roles of group I mGluRs on low extracellular Mg²⁺ concentration ([Mg²⁺ ]_o )-induced epileptiform activity and neuronal cell death in the CA1 regions of isolated rat hippocampal slices without the entorhinal cortex using extracellular recording and propidium iodide staining. Exposure to Mg²⁺ -free artificial cerebrospinal fluid can induce interictal epileptiform activity in the CA1 regions of rat hippocampal slices. MPEP, a mGluR 5 antagonist, significantly inhibited the spike firing of the low [Mg²⁺ ]_o -induced epileptiform activity, whereas LY367385, a mGluR1 antagonist, did not. DHPG, a group 1 mGluR agonist, significantly increased the spike firing of the epileptiform activity. U73122, a PLC inhibitor, inhibited the spike firing. Thapsigargin, an ER Ca²⁺ -ATPase antagonist, significantly inhibited the spike firing and amplitude of the epileptiform activity. Both the IP 3 receptor antagonist 2-APB and the ryanodine receptor antagonist dantrolene significantly inhibited the spike firing. The PKC inhibitors such as chelerythrine and GF109203X, significantly increased the spike firing. Flufenamic acid, a relatively specific TRPC 1, 4, 5 channel antagonist, significantly inhibited the spike firing, whereas SKF96365, a relatively non-specific TRPC channel antagonist, did not. MPEP significantly decreased low [Mg²⁺ ] o DMEM-induced neuronal cell death in the CA1 regions, but LY367385 did not. We suggest that mGluR 5 is involved in low [Mg²⁺ ]_o -induced interictal epileptiform activity in the CA1 regions of rat hippocampal slices through PLC, release of Ca²⁺ from intracellular stores and PKC and TRPC channels, which could be involved in neuronal cell death.

Keyword

CA1 region; Hippocampal epilepsy; Low magnesium; Metabotropic glutamate receptor 5; Transient receptor potential channel

Figure

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  Fig. 1 mGluR5 antagonist inhibits the spike firing of low [Mg2+]o-induced interictal epileptiform activity in the CA1 region of rat hippocampal slice. Exposure to Mg2+-free ACSF induced interictal epileptiform activity in the CA1 (A1) and CA3 (B1) regions of rat hippocampal slices. The mGluR5 antagonist MPEP (50 μM) inhibited the spike firing of the low [Mg2+]o-induced interictal epileptiform activity, but the mGluR1 antagonist LY367385 (100 μM) did not affect the interictal epileptiform activity in the CA1 (A1). Graph summarizes the spike firing and amplitude of the low [Mg2+]o- induced epileptiform activity in non (control, n = 7)-, MPEP (n = 6)- and LY367385 (n = 6)-treated groups in the CA1 (A2) and in non (control, n = 6)-, MPEP (n = 7)- and LY367385 (n = 9)-treated groups CA3 (B2) region. Data are expressed as means ± SEM. mGluR, metabotropic glutamate receptor; [Mg2+]o, extracellular Mg2+ concentration; ACSF, artificial cerebrospinal fluid; MPEP, 6-Methyl-2-(phenylethynyl) pyridine. *p < 0.05 relative to respective control (one way ANOVA with Bonferroni’s test).

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  Fig. 2 DHPG enhances the spike firing of low [Mg2+]o-induced epileptiform activity in the CA1 region of rat hippocampal slice. (A) Exposure to low [Mg2+]o induced epileptiform activity in the CA1 region of rat hippocampal slices. The group 1 mGluR agonist DHPG (10 µM) increased the spike firing of the epileptiform activity. (B) Graph summarizes the spike firing and amplitude of the low [Mg2+]o-induced epileptiform activity in non (control, n = 7)-, DHPG (n = 6)-treated groups in the CA1 region. Data are expressed as means ± SEM. DHPG, 3,5-dihydroxyphenylglycine; [Mg2+]o, extracellular Mg2+ concentration; mGluR, metabotropic glutamate receptor. **p < 0.01 relative to respective control (non-paired Student’s t-test).

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  Fig. 3 The phospholipase C inhibitor U73122 inhibits the spike firing of low [Mg2+]o-induced epileptiform activity. (A) Exposure to Mg2+-free ACSF induced epileptiform activity in the CA1 region. U73122 (10 μM) significantly inhibited the spike firing of low [Mg2+]o-induced epileptiform activity. (B) Graph summarizes the spike firing and amplitude of low [Mg2+]o-induced epileptiform activity in non (control, n = 6)- and U73122 (n = 7)-treated groups. Data are expressed as means ± SEM. [Mg2+]o, extracellular Mg2+ concentration; ACSF, artificial cerebrospinal fluid. **p < 0.01 relative to respective control (non-paired Student’s t-test).

• 

  Fig. 4 Inhibitory effects of the ER Ca2+-ATPase inhibitor thapsigargin, the IP3 receptor antagonist 2-APB, and the ryanodine receptor antagonist dantrolene on low [Mg2+]o-induced epileptiform activity. (A1) Exposure to Mg2+-free ACSF induced epileptiform activity in the CA1 region of acute hippocampal slices. Thapsigargin (10 μM), which induces depletion of ER Ca2+ stores, significantly inhibited the spike firing and amplitude of low [Mg2+]o-induced epileptiform activity. (A2) Graph summarizes the spike firing and amplitude of the epileptiform activity in non (control, n = 6)-, thapsigargin (n = 6)-treated groups. (B1) IP3 receptor antagonist 2-APB (30 μM) and ryanodine receptor antagonist dantrolene (30 μM) significantly inhibited the spike firing of low [Mg2+]o-induced epileptiform activity. (B2) Graph summarizes the spike firing and amplitude of the epileptiform activity in non (control, n = 8)-, 2-APB (n = 6)-, and dantrolene (n = 6)-treated groups. Data are expressed as means ± SEM. ER, endoplasmic reticulum; IP3, inositol-1,4,5-trisphosphate; 2-APB, 2-aminoethoxydiphenyl borate; [Mg2+]o, extracellular Mg2+ concentration; ACSF, artificial cerebrospinal fluid. *p < 0.05 relative to respective control (non-paired Student’s t-test), **p < 0.01 relative to respective control (non-paired Student’s t-test), #p < 0.05 relative to respective control (one way ANOVA with Bonferroni’s test).

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  Fig. 5 The protein kinase C inhibitors GF109203X and chelerythrine increase the spike firing of low [Mg2+]o-induced epileptiform activity. (A) GF109203X (1 μM) and chelerythrine (10 μM) significantly increased the spike firing of low [Mg2+]o-induced interictal epileptiform activity. (B) Graph summarizes the spike firing and amplitude of the interictal epileptiform activity in non (control, n = 8)-, GF109203X (n = 7)- and chelerythrine (n = 5)-treated groups. Data are expressed as means ± SEM. [Mg2+]o, extracellular Mg2+ concentration. *p < 0.05 relative to control, **p < 0.01 relative to control (one way ANOVA with Bonferroni’s test).

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  Fig. 6 The effects of the TRPC channel inhibitors such as flufenamic acid and SKF96365 on low [Mg2+]o-induced epileptiform activity. (A) Flufenamic acid (100 μM) significantly inhibited the spike firing of low [Mg2+]o-induced epileptiform activity, but SKF96365 (10 μM) did not affect inhibited the spike firing of the epileptiform activity. (B) Graph summarizes the spike firing and amplitude of low [Mg2+]o-induced epileptiform activity in non (control, n = 7)-, flufenamic acid (n = 7)- and SKF96365 (n = 7)-treated groups. Data are expressed as means ± SEM. TRPC, transient receptor potential canonical; [Mg2+]o, extracellular Mg2+ concentration. *p < 0.05 relative to respective control (one way ANOVA with Bonferroni’s test).

• 

  Fig. 7 The mGluR5 antagonist MPEP inhibits low [Mg2+]o-induced PI incorporation in the CA1 regions of rat hippocampal slices. Hippocampal slices were exposed to normal [Mg2+]o or low [Mg2+]o DMEM solution (See METHODS) for 24 h in the absence or presence of mGluR antagonists such as MPEP (50 µM) and LY367385 (100 µM). The viability of neuronal cells was assessed by PI incorporation after 30 min exposure to the red fluorescent dye, PI. PI incorporation was shown as a percentage of 70% ethanol-induced maximal PI. (A) Bar graph shows PI incorporation in non (control: normal [Mg2+]o, n = 12; low [Mg2+]o, n = 14)-, MPEP (normal [Mg2+]o, n = 7; low [Mg2+]o, n = 6)- and LY367385 (normal [Mg2+]o, n = 6; low [Mg2+]o, n = 6)-treated groups in the CA1 and CA3 regions. Exposure to normal [Mg2+]o DMEM solution induced a basal PI incorporation. Exposure to low [Mg2+]o DMEM solution significantly increased PI incorporation in the CA1 and CA3 regions. MPEP significantly decreased the low [Mg2+]o-induced PI incorporation in the CA1 region. However, MPEP or LY367385 did not affect the low [Mg2+]o-induced PI incorporation in the CA3 region. (B) Representative phasecontrast photomicrographs showing rat hippocampal slices 24 h after exposure to normal [Mg2+]o or low [Mg2+]o DMEM solution in the absence or presence of mGluR antagonists. Data are expressed as means ± SEM. mGluR, metabotropic glutamate receptor; MPEP, 6-Methyl-2-(phenylethynyl) pyridine; [Mg2+]o, extracellular Mg2+ concentration; PI, propidium iodide; DMEM, Dulbecco’s modified eagle media. **p < 0.01 relative to respective control, ##p < 0.01 relative to normal [Mg2+]o. Scale bars: 100 μm.

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