Essential role of GluD1 in dendritic spine development and GluN2B to GluN2A NMDAR subunit switch in the cortex and hippocampus reveals ability of GluN2B inhibition in correcting hyperconnectivity

Highlights

• GluD1 loss leads to higher dendritic spine number in the cortex and hippocampus.

• GluD1 loss leads to lower GluN2A/GluN2B expression ratio.

• Inhibition of GluN2B receptors reverse excess spine and behavioral deficits.

Abstract

The glutamate delta-1 (GluD1) receptor is highly expressed in the forebrain. We have previously shown that loss of GluD1 leads to social and cognitive deficits in mice, however, its role in synaptic development and neurotransmission remains poorly understood. Here we report that GluD1 is enriched in the medial prefrontal cortex (mPFC) and GluD1 knockout mice exhibit a higher dendritic spine number, greater excitatory neurotransmission as well as higher number of synapses in mPFC. In addition abnormalities in the LIMK1-cofilin signaling, which regulates spine dynamics, and a lower ratio of GluN2A/GluN2B expression was observed in the mPFC in GluD1 knockout mice. Analysis of the GluD1 knockout CA1 hippocampus similarly indicated the presence of higher spine number and synapses and altered LIMK1-cofilin signaling. We found that systemic administration of an N-methyl-d-aspartate (NMDA) receptor partial agonist d-cycloserine (DCS) at a high-dose, but not at a low-dose, and a GluN2B-selective inhibitor Ro-25-6981 partially normalized the abnormalities in LIMK1-cofilin signaling and reduced excess spine number in mPFC and hippocampus. The molecular effects of high-dose DCS and GluN2B inhibitor correlated with their ability to reduce the higher stereotyped behavior and depression-like behavior in GluD1 knockout mice. Together these findings demonstrate a critical requirement for GluD1 in normal spine development in the cortex and hippocampus. Moreover, these results identify inhibition of GluN2B-containing receptors as a mechanism for reducing excess dendritic spines and stereotyped behavior which may have therapeutic value in certain neurodevelopmental disorders such as autism.

Introduction

The ionotropic glutamate receptor family of proteins is comprised of four subtypes NMDA, AMPA, kainate and delta receptors. Glutamate delta-1 (GluD1) and glutamate delta-2 (GluD2) form the delta family of iGluRs and are distinct from other iGluRs in that they do not exhibit typical agonist-induced ion channel currents (Traynelis et al., 2010, Naur et al., 2007, Yadav et al., 2011, Ady et al., 2014). Instead recent studies have demonstrated a crucial role of the delta receptors in synapse formation by interacting with presynaptic proteins such as Neurexin1 (Uemura et al., 2010, Matsuda et al., 2010, Ryu et al., 2012, Yasumura et al., 2012). Although the synaptic function of GluD2 expressed in Purkinje cells has been extensively studied, the function of GluD1 in native system remains poorly understood. We have previously shown that deletion of GluD1 leads to social deficits and emergence of depression-like and aggressive behavior (Yadav et al., 2012). Furthermore, loss of GluD1 leads to abnormal cognitive functions including, enhanced working memory and deficits in both contextual and cued fear conditioning as well as reversal learning of spatial memory (Yadav et al., 2013). We have also found that social deficits in the GluD1 knockout (KO) mice were amenable to modulation of NMDA receptors by agonist d-cycloserine (Yadav et al., 2012), however, the precise molecular mechanism of this effect is not clear. In agreement with our behavioral studies which suggest that loss of GluD1 leads to emergence of behavioral phenotypes relevant to neuropsychiatric disorders, single nucleotide polymorphism and copy-number variation studies have demonstrated an association of GRID1 gene, that codes for the GluD1 receptor, with autism (Griswold et al., 2012, Glessner et al., 2009, Nord et al., 2011, Smith et al., 2009), as well as schizophrenia and bipolar disorder (Fallin et al., 2005, Edwards et al., 2012, Guo et al., 2007, Greenwood et al., 2011).

GluD1 is highly expressed in the forebrain including the cortex and hippocampus (Lomeli et al., 1993, Gao et al., 2007, Yadav et al., 2012) and recent studies also indicate expression in cerebellar interneurons (Konno et al., 2014). In the cortex and hippocampus high level of GluD1 mRNA and protein appears in pyramidal neurons (Gao et al., 2007; Lein et al., 2007, Konno et al., 2014, Hepp et al., 2014). GluD1 appears to be particularly localized to spine-like structures in the hippocampus that represent excitatory synapses (Hepp et al., 2014). In this study we have found that pyramidal neurons in adult GluD1 KO medial prefrontal cortex (mPFC) and hippocampus have higher dendritic spine number that may occur due to impaired spine pruning or excessive spine generation. We also observed abnormalities in LIMK1-cofilin signaling which is involved in regulating spine dynamics and a lower NMDA receptor GluN2A/GluN2B subunit expression ratio suggesting a potential impairment in the GluN2B to GluN2A developmental switch (Williams et al., 1993). Moreover, inhibition of GluN2B-containing receptors was found to reverse signaling abnormalities and spine density as well as stereotyped behavior and depression-like behavior in GluD1 KO mice. These results for the first time demonstrate a critical role of GluD1 in maintaining spine dynamics and ability of GluN2B inhibition in reversing the higher spine density which may have implications for disorders such as autism where higher number of spines is a commonly observed neuropathology (Penzes et al., 2011).