Overstimulation of glutamate signals leads to hippocampal transcriptional plasticity in hamsters

Abstract

It's known that neurons in mammalian hibernators are more tolerant to hypoxia than those in non-hibernating species and as a consequence animals are capable of awakening from the arousal state without exhibiting cerebral damages. In addition, evidences have suggested that euthermic hamster neurons display protective adaptations against hypoxia, while those of rats are not capable, even though molecular mechanisms involved in similar neuroprotective strategies have not been yet fully studied. In the present work, overstimulation of glutamatergic receptors NMDA recognized as one of the major death-promoting element in hypoxia, accounted for altered network complexity consistent with a moderate reduction of hippocampal neuronal survival (p < 0.05) in hamsters. These alterations appeared to be featured concomitantly with altered glutamatergic signaling as indicated by significant down-regulation (p < 0.01) of NMDAergic (NR2A) and AMPAergic (GluR1, R2) receptor subtypes together with the metabotropic mGluR5 subtype. Diminished mRNA levels were also reported for NMDA receptor binding factors and namely PSD95 plus DREAM, which exert positive and negative regulatory properties, respectively, on receptor trafficking events. Conversely, involvement of glutamatergic signaling systems on neuronal excitotoxicity was strengthened by the co-activation of GABAAR-mediated effects as indicated by toxic morphological effects being notably reduced along with up-regulated GluR1, GluR2, mGluR5, DREAM, and Homer1c scaffold proteins when muscimol was added. Overall, these results point to a neuroprotective role of the GABAergic system against excitotoxicity episodes via DREAM-dependent inhibition of NMDA receptor and activation of AMPA receptor plus mGluR5, respectively, thus proposing them as novel therapeutic targets against cerebral ischemic damages in humans.

Fig. 1

Evaluation of drugs effects on neuronal survival by in vitro cytotoxic test. MTT assay was performed to examine cell viability in cultures subjected to 1 h of simulated excitotoxicity followed by 24 h of reperfusion at DIV8 with respect to all control groups. Two-tail ANOVA followed by Newman Keul’s multiple range test when p < 0.05; F_(2.24) = 3.82. Asterisks indicate significant differences between drugs-treated and control neurons (*p < 0.05), whereas hash sign indicates significant differences between NMDA- and (NMDA+MUS)-treated neurons (^# p < 0.05)

Fig. 2

Neuronal morphological analysis following acute treatment with NMDA, MUS, and NMDA+MUS at DIV8. SEM images of hippocampal neurons a–h after 24 h of neuronal growth in a, e absence of drugs, b, f MUS treatment, c, g NMDA treatment, d, h NMDA+MUS treatment. Quantitative evaluation of i neurites length and j neuronal area per well were calculated in 10 cell fields (100 × 100 μm²) of all treatment groups and compared to controls. Two-tail ANOVA followed by Newman Keul’s multiple range test when p < 0.05; F_(5.84) = 2.61. Asterisks indicate significant differences between drugs-treated and control neurons (*p < 0.05), whereas hash sign indicates significant differences between NMDA- and (NMDA+MUS)-treated neurons (^# p < 0.05). Scale bar 50 μm (a–d); 100 μm (e–h)

Fig. 3

Effects of NMDA and NMDA+MUS treatments on mRNA levels for GluR1 and GluR2 subunits of AMPAR, NR2A subunit of NMDAR and mGluR5, as well as for scaffold proteins PSD95, DREAM, and Homer1c. Two-tail ANOVA followed by Newman Keul’s multiple range test when p < 0.05; F_(21.42) = 2.48. Asterisks indicate significant differences between drugs-treated and control neurons (*p < 0.05; **p < 0.01; ***p < 0.001), whereas letters indicate significant differences between NMDA- and NMDA+MUS-treated neurons (^a p < 0.05; ^c p < 0.001)