Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain

Abstract

In the cerebellum, GluD2 is exclusively expressed in Purkinje cells, where it regulates synapse formation and regeneration, synaptic plasticity, and motor learning. Delayed cognitive development in humans with GluD2 gene mutations suggests extracerebellar functions of GluD2. However, extracerebellar expression of GluD2 and its relationship with that of GluD1 are poorly understood. GluD2 mRNA and protein were widely detected, with relatively high levels observed in the olfactory glomerular layer, medial prefrontal cortex, cingulate cortex, retrosplenial granular cortex, olfactory tubercle, subiculum, striatum, lateral septum, anterodorsal thalamic nucleus, and arcuate hypothalamic nucleus. These regions were also enriched for GluD1, and many individual neurons coexpressed the two GluDs. In the retrosplenial granular cortex, GluD1 and GluD2 were selectively expressed at PSD-95-expressing glutamatergic synapses, and their coexpression on the same synapses was shown by SDS-digested freeze-fracture replica labeling. Biochemically, GluD1 and GluD2 formed coimmunoprecipitable complex formation in HEK293T cells and in the cerebral cortex and hippocampus. We further estimated the relative protein amount by quantitative immunoblotting using GluA2/GluD2 and GluA2/GluD1 chimeric proteins as standards for titration of GluD1 and GluD2 antibodies. Intriguingly, the relative amount of GluD2 was almost comparable to that of GluD1 in the postsynaptic density fraction prepared from the cerebral cortex and hippocampus. In contrast, GluD2 was overwhelmingly predominant in the cerebellum. Thus, we have determined the relative extracerebellar expression of GluD1 and GluD2 at regional, neuronal, and synaptic levels. These data provide a molecularanatomical basis for possible competitive and cooperative interactions of GluD family members at synapses in various brain regions.

GluD1 knockout mice with a pure C57BL/6N background show impaired fear memory, social interaction, and enhanced depressive-like behavior

“…Behavioral analyses under a pure C57BL/6N genetic background suggest that GluD1 plays critical roles in contextual fear memory, sociability, and depressive-like behavior. We originally developed the Grid1 +/flox mouse in which exon 4 of Grid1 gene was flanked by loxP sequences [70]. Grid1 flox/flox mice under a C57BL/6N genetic background allow us to delete the Grid1 gene in a brain region-specific manner using region-specific Cre mice or Creexpressing virus injections.…”

“…GluD1-KO mice were generated using the C57BL/6N ES cell line, RENKA [67] and maintained in a pure C57BL/6N background [70]. Briefly, exon 4 of the Grid1 gene and a Pgk promoter-driven neomycin-resistance cassette were flanked by loxP sequences (Grid1 flox ).…”

Probing the ionotropic activity of glutamate GluD2 receptor in HEK cells with genetically-engineered photopharmacology

“…They are considered as glutamate receptors solely based on their strong sequence and structure homology with AMPA, NMDA and kainate receptors ( Lomeli et al, 1993 ; Araki et al, 1993 ; Schmid and Hollmann, 2008 ; Elegheert et al, 2016 ; Burada et al, 2020a ; Burada et al, 2020b ). GluD receptors are widely expressed throughout the brain, GluD1 predominantly in the forebrain, while GluD2 is highly enriched in cerebellar Purkinje cells (PCs) ( Konno et al, 2014 ; Hepp et al, 2015 ; Nakamoto et al, 2020b ). Both GluD1 and GluD2 play a role in the formation, stabilization, function and plasticity of synapses through their interaction with members of the cerebellin (Cbln) family ( Fossati et al, 2019 ; Tao et al, 2018 ; Matsuda et al, 2010 ; Kakegawa et al, 2008 ).…”

Ultrastructural localization of glutamate delta 1 (GluD1) receptor immunoreactivity in the mouse and monkey striatum

“…GluD1, and its family member GluD2, are categorized as ionotropic receptors, but do not exhibit the typical fast ligand‐gated ion flow (Kakegawa et al, 2011). In the cerebellar cortex, GluD2 is responsible for the development, maintenance, and regeneration of parallel fiber synapses on Purkinje cells (Berridge et al, 2018; Hirano, 2012; Ichikawa, Sakimura, & Watanabe, 2016; Kashiwabuchi et al, 1995; Nakamoto, Konno, et al, 2020; Pernice et al, 2019; Yuzaki, 2011). In line with these observations, recent studies showing that ablation of GluD1 results in changes in dendritic spine density and morphology of pyramidal neurons within the medial prefrontal cortex and CA1 region of the hippocampus, support a similar role of GluD1 in maintaining synaptic integrity in forebrain nuclei (Gupta et al, 2015).…”

“…Our current understanding of GluD1 function in the CNS is hampered by the limited knowledge of its subcellular localization within the mammalian brain. Although some electron microscopic data of GluD1 localization have been reported for the mouse cerebellar cortex and hippocampal formation (Hepp et al, 2015; Konno et al, 2014; Nakamoto, Konno, et al, 2020), such information is lacking for the striatum. In a recent study, Liu et al (2020) demonstrated strong GluD1 expression within the mouse dorsal striatum and provided evidence for a GluD1‐mediated regulation of the thalamostriatal projection from the parafascicular nucleus (Pf).…”