A Specific Role for NR2A-Containing NMDA Receptors in the Maintenance of Parvalbumin and GAD67 Immunoreactivity in Cultured Interneurons

Abstract

Several lines of evidence suggest that a hypoglutamatergic condition may induce a phenotypic loss of cortical parvalbumin (PV)-positiveGABAergic interneurons, such as that observed in brain tissue of schizophrenic subjects. However, it is not known whether the loss of PV interneurons is a consequence of the hypoglutamatergic condition or a secondary aspect of the disease. We characterized the signaling and subunit expression of NMDA receptors in cultured cortical PV interneurons and determined whether a hypoglutamatergic condition, created by direct application of sublethal concentrations of ketamine or subunit-selective NMDA receptor antagonists, can affect the expression of the GABAergic markers as observed in vivo. Real-time PCR performed on mRNA isolated from single neurons showed that PV interneurons present a fivefold higher NR2A/NR2B ratio than pyramidal neurons. Brief, nontoxic, exposure to NMDA led to an increase in ERK1/2 (extracellular signal-regulated kinase 1/2) and cAMP response element-binding protein phosphorylation in PV interneurons, and this increase was blocked by the NR2A-selective antagonist NVP-AAM077. Application of the nonselective NMDA receptor antagonist ketamine, at sublethal concentrations, induced a time and dose-dependent decrease in parvalbumin and GAD67 immunoreactivity specifically in PV interneurons. These effects were reversible and were also observed with the NR2A-selective antagonist, whereas the NR2B-selective antagonist Ro-25-6981 only partially reduced GAD67 immunoreactivity. Coexposure to the calcium channel opener BayK, or the group I metabotropic glutamate receptor agonist DHPG [(RS)-3,5-dihydroxyphenylglycine] attenuated the decrease in GAD67 and parvalbumin induced by the NMDA receptor antagonists. These results suggest that the activity of NR2A-containing NMDA receptors play a pivotal role in the maintenance of the GABAergic function of PV interneurons.

Does schizophrenia arise from oxidative dysregulation of parvalbumin-interneurons in the developing cortex?

“…However, 24 h continuous exposure of three week old cultured neurons to ketamine produces a substantial reduction in GAD67 immunoreactivity (Kinney et al, 2006). These results suggest that PV-interneurons are more resistant to the effects of NMDA-R antagonists during the rodent equivalent to the adolescence period in humans.…”

“…Blockade of NMDA-Rs during the third week of development in culture leads to the loss of the GABAergic phenotype of PV-interneurons, and this loss can be prevented by strategies that increase intracellular calcium levels (Kinney et al, 2006). As noted above, PVinterneurons express GluR2-less AMPA receptors and are permeable to Ca 2+ .…”

“…However, although the effects of repetitive exposures to NMDA-R antagonist resemble more accurately the dysfunction of PV-interneurons observed in schizophrenia patients, these effects are slowly reversible in both the in vitro and in vivo models, requiring 48 h in culture (Kinney et al, 2006) or several days in the absence of drug in vivo . Thus, although causing persistent effects, repetitive adult exposures to NMDA-R antagonists do not produce the irreversible changes in inhibitory circuitry observed in schizophrenia.…”

“…Regarding group 1 metabotropic glutamate receptors, cortical and hippocampal PV-interneurons preferentially express mGluR5 (Cauli et al, 1997;van Hooft et al, 2000). On the other hand, NMDA-Rs in PV-interneurons have a subunit composition that differs from neighboring pyramidal neurons, with NR2A and NR2C subunits being highly expressed (Kinney et al, 2006;Xi et al, 2009). NMDA-Rs exert a tight control of the basal excitability in PV-interneurons, and are highly sensitive to NMDA-R antagonists (Grunze et al, 1996;Middleton et al, 2008).…”

“…Furthermore, the increase in excitatory transmission caused by disinhibition should activate group I metabotropic glutamate receptors present in PV-interneurons and also lead to increases in intracellular Ca2+, now through release from intracellular stores. However, coexposure to a calcium-channel opener or an activator of group I metabotropic receptors was needed to preserve the GABAergic phenotype of PV-interneurons in the presence of NMDA-R antagonists (Kinney et al, 2006). This lack of response of AMPA and mGluR5 receptors to the increased excitatory transmission after disinhibition led to the hypothesis that prolonged blockade of NMDA-Rs in PV-interneurons results in an enduring change in AMPA and mGluR5-mediated responses to glutamate (Behrens et al, 2007).…”