Group I metabotropic glutamate receptors reduce excitotoxic injury and may facilitate neurogenesis
Introduction
It has been well established that in many systems, including organotypic hippocampal cultures, glutamate toxicity is mediated by excessive activation of N-methyl-d-aspartate (NMDA) receptors; consequently, application of NMDA induces neuronal death in a concentration-dependent manner (Adamchik and Baskys, 2000). It is less well understood how glutamate applied at low concentrations for a prolonged period and before the NMDA application reduces the excitotoxic damage. This neuroprotective effect appears to involve group I metabotropic glutamate receptors (mGluRs) that couple to phospholipase C (PLC) and can be mimicked by a selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) (Adamchik and Baskys, 2000, Blaabjerg et al., 2003a). In our previous experiments we examined the contribution of mGluR1 and mGluR5 receptors to the DHPG-induced neuroprotection. A selective mGluR1 antagonist LY367385 abolished DHPG neuroprotection in a concentration-dependent manner (1–10 μM), whereas the mGluR5 selective antagonist MPEP (1 μM) had no significant effect (Blaabjerg et al., 2003a). Although it has been shown that DHPG can stimulate internalization of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and NMDA receptors (Snyder et al., 2001), most likely through GTPase Rab5b facilitated endocytosis (Arnett et al., 2004), key elements of signaling pathways involved in group I mGluR-mediated neuroprotection have not yet been fully identified. Group I mGluRs link to the downstream targets through a PLC-dependent pathway (Rhee, 2001, Llansola et al., 2000, Johnson et al., 1999). At the neuronal level, DHPG reduces both evoked and spontaneous excitatory post-synaptic currents (EPSCs), a phenomenon often referred to as metabotropic glutamate receptor-dependent form of long-term depression or mGluR-LTD (Xiao et al., 2001). It is possible that PLC is one of the main intracellular signaling components both in mGluR-LTD and in DHPG-mediated reduction of susceptibility to NMDA injury. On the other hand, it has been reported that reduction of nerve cell death by a mixed group I and II agonist trans-1-amino-1,3-cyclopentanediocarboxylic acid (trans-ACPD) was not prevented by PLC inhibition (Llansola et al., 2000), and that, in CA1 neurons, group I mGluR agonists increased excitability of pyramidal neurons by a PLC-independent mechanism (Ireland and Abraham, 2002). It is possible then, that reduction of excitotoxicity following group I mGluR activation could be PLC-dependent and associated with a reduction of EPSCs through a PLC-dependent mechanism. Alternatively, the protective effect of DHPG could be PLC-independent and may or may not be associated with the reduction of EPSCs.
Three major PLC families (PLCβ, PLCγ and PLCδ) expressed in the brain tissue (Rebecchi and Pentyala, 2000) have been associated with regulation of cell division and proliferation (Nebigil, 1997, Shimohama et al., 1998). Formation of new nerve cells (neurogenesis) occurs in at least two parts of the brain: the subgranular cell layer of the hippocampal formation, and the subventricular zone in the lateral ventricles, and persists into adulthood in rats, mice, tree shrews and humans (Alvarez-Buylla and Garcia-Verdugo, 2002, Cameron and McKay, 2001, Corotto et al., 1993, Eriksson et al., 1998, Gage et al., 1998, Gould et al., 1997, Palmer et al., 2001). Neurogenesis can be modulated by a wide range of different treatments such as epileptic seizure activity induced by amygdala kindling, pilocarpine administration or electroconvulsive shock (Parent et al., 1998, Parent et al., 1997, Scharfman et al., 2001), and growth factors such as epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2) (Gage et al., 1998, Kuhn et al., 1997) and insulin-like growth factor 1 (IGF-1) (Aberg et al., 2000, O'Kusky et al., 2000) or antidepressants (Malberg et al., 2000). Modulation of the glutamatergic neurotransmitter system by administration of ionotropic glutamate receptor antagonists (e.g. NMDA receptor antagonist MK-801) also affects dentate granule cell neurogenesis (Nacher et al., 2001). It has been shown that both amygdala kindling, electroconvulsive shock and antidepressants, treatments that increase neurogenesis also increase the expression of mGluR1 and 5 in the hippocampus (Akbar et al., 1996, Smialowska et al., 2002) indicating that neurogenesis might play a role in DHPG-induced protection. In this study, we used organotypic hippocampal slice culture preparation to investigate whether mGluR-associated reduction in neuronal susceptibility to injury is PLC-dependent, what are neurophysiological correlates of such reduction, and whether there may be a link between mGluR1 or mGluR5 and neurogenesis. Our data suggest that a prolonged but not a short-term stimulation of group I mGluRs increases nerve cell resistance to excitotoxic injury in a PLC-dependent manner. They further suggest that a prolonged but not a short-term exposure of cultures to DHPG induces a PLC-dependent reduction of excitatory synaptic transmission. Finally, these data suggest that mGluR1 stimulation may be required to facilitate neurogenesis.
A portion of these results was presented as a poster at the American Society for Neuroscience annual meeting (Bayazitov et al., 2004).
Section snippets
Preparation of cultures
Organotypic hippocampal slice cultures were prepared and grown by the interface method (Stoppini et al., 1991). Seven-day-old Wistar rat pups (Charles River, Raleigh, NC, USA) were anesthetized with halothane, decapitated, and the brains placed into an ice-cold stabilization medium [50% minimal essential medium (MEM) with no bicarbonate or glutamate, 50% calcium and magnesium-free Hanks' balanced salt solution, 7.5 mM d-glucose, and 20 mM N-2-(hydroxyethyl)-piperazine-N′-(2-ethanesulfonic acid)
Results
As it has been shown earlier (Blaabjerg et al., 2003a), a 2-h treatment of cultures with DHPG (10 μM) prior to their exposure to NMDA (50 μM, 30 min) produced a marked reduction of PI uptake in DHPG-treated cultures compared to PI uptake produced by NMDA alone (Fig. 1A, B, 56 ± 3%, P < 0.001, n = 7 experiments) measured 24 h following NMDA (50 μM, 30 min) exposure. However, a short-term (10 min) DHPG treatment-induced reduction was not statistically significant at 10 or at 100 μM of DHPG (in % to NMDA alone:
Discussion
These experiments confirmed our previous finding of DHPG-induced neuroprotection (Blaabjerg et al., 2003a, Arnett et al., 2004). They also showed that the neuroprotective effect of DHPG occurs after a prolonged but not short-term exposure to the drug, requires PLC activation and is associated with a reduction of AMPA receptor-mediated responses in a PLC-dependent manner. Further, they showed that neurogenesis in organotypic hippocampal cultures requires mGluR1 activation, raising a possibility
Acknowledgements
Dr. Susan Hocksfield, Yale University, generously provided the TOAD-64 antibody. For technical assistance Dorte Lyholmer is gratefully acknowledged. The study was supported by EU 5th framework program (QLRT-CT-2001-004017), the Danish MRC, the Foundation for Research in Mental Diseases, Eli Lilly's Foundation for Psychiatric Research, The Novo Nordisk Foundation, the Foundation for Neurological Research, the Psychiatric Research Foundation and Overlægerådets legatudvalg, VISN 22 Mental Illness
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