Selective mGluR1 antagonist EMQMCM inhibits the kainate-induced excitotoxicity in primary neuronal cultures and in the rat hippocampus

Abstract

Abundant evidence suggests that indirect inhibitory modulation of glutamatergic transmission, via metabotropic glutamatergic receptors (mGluR), may induce neuroprotection. The present study was designed to determine whether the selective antagonist of mGluR1 (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methanesulfonate (EMQMCM), showed neuroprotection against the kainate (KA)-induced excitotoxicity in vitro and in vivo. In in vitro studies on mouse primary cortical and hippocampal neuronal cultures, incubation with KA (150 μM) induced strong degeneration [measured as lactate dehydrogenase (LDH) efflux] and apoptosis (measured as caspase-3 activity). EMQMCM (0.1-100 μM) added 30 min to 6 h after KA, significantly attenuated the KA-induced LDH release and prevented the increase in caspase-3 activity in the cultures. Those effects were dose- and time-dependent. In in vivo studies KA (2.5 nmol/1 μl) was unilaterally injected into the rat dorsal CA1 hippocampal region. Degeneration was calculated by counting surviving neurons in the CA pyramidal layer using stereological methods. It was found that EMQMCM (5-10 nmol/1 μl) injected into the dorsal hippocampus 30 min, 1 h, or 3 h (the higher dose only) after KA significantly prevented the KA-induced neuronal degeneration. In vivo microdialysis studies in rat hippocampus showed that EMQMCM (100 μM) significantly increased γ-aminobutyric acid (GABA) and decreased glutamate release. When perfused simultaneously with KA, EMQMCM substantially increased GABA release and prevented the KA-induced glutamate release. The obtained results indicate that the mGluR1 antagonist, EMQMCM, may exert neuroprotection against excitotoxicity after delayed treatment (30 min to 6 h). The role of enhanced GABAergic transmission in the neuroprotection is postulated.

Fig. 1

a, c The effect of EMQMCM on kainate (150 μM)-induced LDH release in the primary cultures of mouse cortical (a) and hippocampal (c) neurons. LDH was measured 48 h (cortical) or 24 h (hippocampal cultures) after KA administration. EMQMCM (in concentrations 0.1, 1, 10, and 100 μM) was added to the culture medium 30 min, 1 h, 3 h, or 6 h after KA. b, d The effect of EMQMCM on KA-induced increase in caspase-3 activity in mouse primary cortical (b) and hippocampal cultures (d). Caspase-3 was measured 6 h after starting KA intoxication. EMQMCM was added to cultures 30 min after KA. Each bar represents the mean of n ≥ 6 platings ± SEM from 3 to 4 independent experiments. Significant differences marked in the following way: ***P < 0.001 (vs. control cultures) and ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 (vs. KA-treated cultures)

Fig. 2

Selected microphotographs illustrating the fluorescence staining with Hoechst 33342 (a marker of apoptosis) in hippocampal cultures. Cell with bright fragmented nuclei (apoptotic bodies, arrows) showing condensation of chromatin were identified as dying in apoptotic mode. a A control culture with few apoptotic bodies. b A culture after KA exposure (150 μM; 24 h); fewer healthy cells and more apoptotic bodies can be seen. c A culture exposed to KA and EMQMCM (100 μM; 30 min after KA); some neuroprotection can be seen as a decrease in the number of apoptotic bodies in comparison with KA (b)

Fig. 3

Microphotographs of frontal sections of rat brain hippocampi stained with cresyl violet. Pyramidal layer of CA regions is pointed by arrows. Calibrations bars 200 μm. a Loss of neurons and extensive gliosis is seen in CA after KA microinjection (2.5 μm/1 μl). b Contralateral hippocampus is not degenerated. A small glial scar only is seen in the site of the buffer injection. c Neuroprotective effect of EMQMCM (10 nmol) injected into the hippocampus 3 h after KA. The lesion is much smaller than after KA alone

Fig. 4

The effect of intrahippocampal injections of KA (2.5 nmol/1 μl) and KA followed by EMQMCM on the number of neurons in the pyramidal layer of CA regions. The results of stereological counting showed neurodegeneration after KA (50% loss) and neuroprotection induced by EMQMCM given 30 min, 1 h, or 3 h after KA. No protection was seen when EMQMCM was given 6 h after KA. Each bar represents the mean ± SEM of n = 6 per group. ***P < 0.001 KA (ipsilateral) versus contralateral side; ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 KA + EMQMCM (ipsilateral) versus KA-lesioned (ipsilateral) hippocampi

Fig. 5

Effect of EMQMCM (100 μM) on extracellular GLU (a) and GABA (b) level in the rat hippocampus. EMQMCM administration is indicated with an arrow, while horizontal bar shows duration of treatment. Data are means ± SEM (n = 4–10). Repeated measures ANOVA and Tukey’s post hoc test. *P < 0.05, **P < 0.01 in comparison with control

Fig. 6

Effect of EMQMCM (100 μM) and kainic acid (KA 50 μM) on extracellular GLU (a) and GABA (b) level in the rat hippocampus. EMQMCM and KA administration is indicated with an arrow, while horizontal bar shows duration of treatment. Data are means ± SEM (n = 4–10). Repeated measures ANOVA and Tukey’s post hoc test. *P < 0.05, **P < 0.01 in comparison with control; ^P < 0.05, ^^P < 0.01 in comparison to group treated with KA