Korean J Physiol Pharmacol.  2022 Nov;26(6):531-540. 10.4196/kjpp.2022.26.6.531.

Group 1 metabotropic glutamate receptor 5 is involved in synaptically-induced Ca²⁺ -spikes and cell death in cultured rat hippocampal neurons

• Yang JS
• Jeon S
• Jang HJ
• Yoon SH

• KMID: 2534528
• DOI: http://doi.org/10.4196/kjpp.2022.26.6.531

Abstract

Group 1 metabotropic glutamate receptors (mGluRs) can positively affect postsynaptic neuronal excitability and epileptogenesis. The objective of the present study was to determine whether group 1 mGluRs might be involved in synapticallyinduced intracellular free Ca²⁺ concentration ([Ca²⁺ ] i ) spikes and neuronal cell death induced by 0.1 mM Mg²⁺ and 10 µM glycine in cultured rat hippocampal neurons from embryonic day 17 fetal Sprague–Dawley rats using imaging methods for Ca²⁺ and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays for cell survival. Reduction of extracellular Mg²⁺ concentration ([Mg²⁺ ] o ) to 0.1 mM induced repetitive [Ca²⁺ ] i spikes within 30 sec at day 11.5. The mGluR5 antagonist 6-Methyl-2-(phenylethynyl) pyridine (MPEP) almost completely inhibited the [Ca²⁺ ] i spikes, but the mGluR1 antagonist LY367385 did not. The group 1 mGluRs agonist, 3,5-dihydroxyphenylglycine (DHPG), significantly increased the [Ca²⁺ ] i spikes. The phospholipase C inhibitor U73122 significantly inhibited the [Ca²⁺ ] i spikes in the absence or presence of DHPG. The IP₃ receptor antagonist 2-aminoethoxydiphenyl borate or the ryanodine receptor antagonist 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate also significantly inhibited the [Ca²⁺ ] i spikes in the absence or presence of DHPG. The TRPC channel inhibitors SKF96365 and flufenamic acid significantly inhibited the [Ca²⁺ ] i spikes in the absence or presence of DHPG. The mGluR5 antagonist MPEP significantly increased the neuronal cell survival, but mGluR1 antagonist LY367385 did not. These results suggest a possibility that mGluR5 is involved in synapticallyinduced [Ca²⁺ ] i spikes and neuronal cell death in cultured rat hippocampal neurons by releasing Ca²⁺ from IP₃ and ryanodine-sensitive intracellular stores and activating TRPC channels.

Keyword

Calcium spike; Hippocampus; IP₃ receptor; Magnesium; Metabotropic glutamate receptor

Figure

• 

  Fig. 1 The mGluR5 antagonist MPEP, but not the mGluR1 antagonist LY367385, inhibits 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. (A) 0.1 mM [Mg2+]o induced synchronized and repetitive [Ca2+]i spikes within 30 sec at day 11.5. (B) LY367385 (100 µM) did not inhibit 0.1 mM [Mg2+]o-induced [Ca2+]i responses. (C) MPEP (25 µM) inhibited 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. (D, E) Graph summarizing the frequency (D) and the area under the curve (E) of 0.1 mM [Mg2+]o-induced [Ca2+]i spikes in non-treated (control, n = 28), MPEP-treated (n = 21), and LY367385-treated (n = 27) cells. Relative spike frequencies or area under curves (2nd /1st: drug or non-treatment; 3rd / 1st: 0.1 mM [Mg2+]o wash) were shown as a ratio of an initial [Ca2+]i spike frequency and area under curves for 1st 0.1 mM [Mg2+]o treatment. The frequency and the area under the curve of [Ca2+]i spikes were calculated from data collected during a 10 min window before application of drug or vehicle (1st), a 10 min window at 5 min after application of drug or vehicle (2nd), and a 10 min window at 5 min after wash (3rd). Data are expressed as means ± SEM. mGluR, metabotropic glutamate receptor; MPEP, 6-Methyl-2-(phenylethynyl) pyridine. **p < 0.01 relative to respective control and LY367385 (ANOVA with Bonferroni test).

• 

  Fig. 2 The phospholipase C inhibitor U73122 (1 mM) inhibits 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. (A) 0.1 mM [Mg2+]o induced synchronized and repetitive [Ca2+]i spikes within 30 sec at day 11.5. (B) 1 µM U73122 inhibited 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. (C, D) Graph summarizing the frequency (C) and the area under the curve (D) of 0.1 mM [Mg2+]o-induced [Ca2+]i spikes in non-treated (control, n = 25) and U73122-treated (n = 24) cells. Relative spike frequencies or area under curves (2nd / 1st: drug or non-treatment; 3rd / 1st: 0.1 mM [Mg2+]o wash) were shown as a ratio of an initial [Ca2+]i spike frequency and area under curves for 1st 0.1 mM [Mg2+]o treatment. Data are expressed as means ± SEM. **p < 0.01 relative to respective control (non-paired Student’s t-test), *p < 0.05 relative to respective control (non-paired Student’s t-test).

• 

  Fig. 3 The IP3 receptor antagonist 2-APB and the ryanodine receptor antagonist TMB-8 inhibits 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. (A) 0.1 mM [Mg2+]o-induced synchronized and repetitive [Ca2+]i spikes within 30 sec at day 11.5. (B) 2-APB (30 µM) inhibits 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. (C) TMB-8 (10 µM) inhibited 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. (D, E) Graph summarizing the frequency (D) and the area under the curve (E) of 0.1 mM [Mg2+]o-induced [Ca2+]i spikes in non-treated (control, n = 21), 2-APB-treated (n = 21), and TMB-8-treated (n = 21) cells. Relative spike frequencies or area under curves (2nd /1st: drug or non-treatment; 3rd / 1st: 0.1 mM [Mg2+]o wash) were shown as a ratio of an initial [Ca2+]i spike frequency and area under curves for 1st 0.1 mM [Mg2+]o treatment. Data are expressed as means ± SEM. IP3, inositol-1,4,5-trisphosphate; 2-APB, 2-aminoethoxydiphenyl borate; TMB-8, 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate. **p < 0.01 relative to respective control (ANOVA with Bonferroni test), *p < 0.05 relative to respective control (ANOVA with Bonferroni test), ++p < 0.01 relative to respective control and TMB-8 (ANOVA with Bonferroni test).

• 

  Fig. 4 Effects of TRPC channel inhibitors such as SKF96365 and flufenamic acid on 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. (A) 0.1 mM [Mg2+]o induced synchronized and repetitive [Ca2+]i spikes within 30 sec at day 11.5. (B) SKF96365 (10 µM) almost completely inhibited 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. (C) flufenamic acid (100 µM) inhibited 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. (D, E) Graph summarizing the frequency (D) and the area under the curve (E) of 0.1 mM [Mg2+]o-induced [Ca2+]i spikes in non-treated (control, n = 19), SKF96365-treated (n = 22), and flufenamic acid-treated (n = 20) cells. Relative spike frequencies or area under curves (2nd /1st: drug or non-treatment; 3rd / 1st: 0.1 mM [Mg2+]o wash) were shown as a ratio of an initial [Ca2+]i spike frequency and area under curves for 1st 0.1 mM [Mg2+]o treatment. Data are expressed as means ± SEM. **p < 0.01 relative to respective control (ANOVA with Bonferroni test), ##p < 0.01 relative to respective control and flufenamic acid (ANOVA with Bonferroni test).

• 

  Fig. 5 Effects of the mGluR5 antagonist MPEP and the mGluR1 antagonist LY367385 on 0.1 mM [Mg2+]o-induced neurotoxicity in cultured pure rat hippocampal neurons. Cells were exposed to HEPES-buffered HBSS containing 0.1 mM Mg2+ and 10 µM glycine for 24 h in the presence or absence of MPEP (25 µM) or LY367385 (100 µM). Neuronal cell survival was measured by MTT reduction assay at 12 days after culture. Cell survival was shown as a percentage of control value. The absorbance of formazan that had formed in non-treated cells grown in culture medium (control) represented 100% viability. (A) Bar graph showing MTT reduction in HEPES-buffered HBSS (control) (vehicle, n = 6; 0.1 mM Mg2+, n = 6), MPEP (vehicle, n = 6; 0.1 mM Mg2+, n = 6), LY367385 (vehicle, n = 6; 0.1 mM Mg2+, n = 6)-treated cells. (B) Representative phasecontrast photomicrographs showing cultured pure rat hippocampal neurons at 24 h following co-treatment of mGluR antagonists with 0.1 mM [Mg2+]o solution for 11 days in culture. Data are expressed as means ± SEM. mGluR, metabotropic glutamate receptor; MPEP, 6-Methyl-2-(phenylethynyl) pyridine; HBSS, Hank’s balanced salt solution; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. ++p < 0.01 relative to respective vehicle (non-paired Student’s t-test), *p < 0.05 relative to respective control (ANOVA with Bonferroni test).

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