Reply - To comment letter by Ben-Ari (The GluR2 Hypothesis..)

In their letter, Y. Ben-Ari and M. Khrestchatisky take issue with the "GluR2 hypothesis' of delayed neurodegeneration, the topic of our recent review[1xPellegrini-Giampietro, D.E et al. Trends Neurosci. 1997; 20: 464–470Abstract | Full Text | Full Text PDF | PubMed | Scopus (397)See all References[1]. We call it a hypothesis because it is not yet established, and we welcome the opportunity to respond to their concerns. The GluR2 hypothesis states that various neurological insults cause selective reduction in expression of the AMPA receptor GluR2 subunit, which leads to increased formation of Ca2+ -permeable AMPA receptors and enhanced toxicity of endogenous glutamate. Evidence in support of the hypothesis has been reported for animal models of global ischemia and status epilepticus and is summarized in our review1; a recent report demonstrates its applicability to in vitro hypoxia-ischemia[2xYing, H.S et al. J. Neurosci. 1997; 17: 9536–9544PubMedSee all References[2]. Our point by point response follows.(1) Is GluR2 downregulation specific? Our lab has reported selective reduction of GluR2 in three distinct studies; other labs have reported reduction in GluR2 mRNA without evaluation of other subunits. The uniform loss of GluR1–3 subunits in postischemic hippocampal CA1–3 reported by Frank et al.[3xFrank, L et al. Acta Neurol. Scand. 1995; 92: 337–343Crossref | PubMedSee all References[3]could have resulted from non-specific labeling by short RNA probes under conditions of low stringency. Their suggestion of uniform depression of transcription is contradicted by their own report of increase in mGluR4 mRNA, and other mRNAs have also been observed to increase after ischemia. The abstract of Wieloch referred to by Ben-Ari is in an unpublished program of a meeting and gives no information about receptor levels.(2) Does GluR2 downregulation precede cell death? Gorter et al.[4xGorter, J.A et al. J. Neurosci. 1997; 17: 6179–6188PubMedSee all References[4]selected viable neurons 72 h after ischemia by criteria of ability to fire action potentials and normal morphology determined by fura-2 injection. These neurons exhibited enhanced AMPA-receptor-mediated Ca2+ entry, which is a functional measure of increased formation of receptors that lack GluR2. Death defined histologically is not prominent until several days later, and, indeed, CA1 becomes positive for TUNEL staining, indicative of apoptosis[5xSee all References[5].The finding that NBQX administered at reperfusion or 24 h after ischemia is neuroprotective supports a mechanism of AMPA-receptor-mediated cell death. The failure of NBQX to prevent GluR2 downregulation suggests that the drug acts not at the level of transcription, but at the receptor that eventually becomes Ca2+ permeable. NBQX injected intraperitoneally might precipitate in the peritoneal cavity or kidneys[6xLe Peillet, E et al. Brain Res. 1992; 571: 115–120Crossref | PubMed | Scopus (146)See all References[6], and from this "store' it could be released at an active concentration over days.(3) Glutamatergic EPSPs are altered after ischemia. AMPA receptors in postischemic CA1 pyramidal neurons exhibit enhanced sensitivity to polyamine block[7xTsubokawa, H et al. J. Neurophysiol. 1995; 74: 218–225PubMedSee all References[7]and enhanced AMPA-induced Ca2+ influx[4xGorter, J.A et al. J. Neurosci. 1997; 17: 6179–6188PubMedSee all References[4], both of which are consistent with GluR2 downregulation. EPSC duration is increased[7xTsubokawa, H et al. J. Neurophysiol. 1995; 74: 218–225PubMedSee all References[7], a change that has no obvious relation to subunit composition. Although NMDA components of EPSCs may be increased after some paradigms for producing ischemia, AMPA antagonists are neuroprotective following global ischemia and NMDA antagonists are not[8xSheardown, M.J, Suzdak, P.D, and Nordholm, L. Eur. J. Pharmacol. 1993; 236: 347–353Crossref | PubMed | Scopus (154)See all References[8].Inward rectification of AMPA receptors is due to block by intracellular poly-amines, which increases with decreasing number of GluR2 subunits per receptor complex[9xWashburn, M.S et al. J. Neurosci. 1997; 17: 9393–9406PubMedSee all References[9]. The electrical linearity of AMPA responses after ischemia[7xTsubokawa, H et al. J. Neurophysiol. 1995; 74: 218–225PubMedSee all References[7]might reflect a reduction in intracellular polyamines by dialysis against the solution in the recording patch electrode or following the ischemic insult.(4) Ca2+ permeability of AMPA receptors of postischemic neurons is increased. Although Gorter et al.[4xGorter, J.A et al. J. Neurosci. 1997; 17: 6179–6188PubMedSee all References[4]did not measure Ca2+ permeability directly or current/voltage (I/V) relations of postischemic AMPA receptors, the observed Ca2+ entry could only have been through AMPA receptors, because voltage-sensitive Ca2+ channels and NMDA receptors were blocked. Cyclothiazide was used to block desensitization by bath-applied AMPA. Synaptic release of glutamate would presumably be phasic and be effective without blocking desensitization. Pyramidal neurons were identified by their showing marked spike accommodation (data not published); interneurons show much less accommodation. Furthermore, interneurons with normally high Ca2+ permeability would have been seen in controls and at early times after ischemia.(5) Does the GluR2 hypothesis apply to interneurons? Interneurons that normally express AMPA receptors lacking GluR2 must be able to handle the Ca2+ influx through these receptors, presumably by buffering, sequestration or extrusion. Obviously, neurons lacking GluR2 cannot downregulate it after ischemia. Although vulnerability of interneurons to delayed neurodegeneration after ischemia has not been extensively investigated with respect to different cell types, available data indicate that the situation is complex[10xFreund, T.F et al. Exp. Brain Res. 1990; 83: 55–66Crossref | PubMed | Scopus (172)See all References[10]. The observation that hippocampal pyramidal cells are present in the adult GluR2 (−/−) mouse[11xJia, Z et al. Neuron. 1996; 17: 945–956Abstract | Full Text | Full Text PDF | PubMed | Scopus (335)See all References[11]suggests to us that some developmental mechanism permits these cells to survive to adulthood and that for some neurons at least a sudden loss of GluR2 is required to trigger delayed neurodegeneration. It will be interesting to test the GluR2 (−/−) mouse for delayed hippocampal degeneration following global ischemia and kainic acid induced status epilepticus; the GluR2 hypothesis predicts that the pyramidal neurons that are vulnerable in the wild type will be resistant in the mouse lacking this receptor subunit.