NMDA receptor subunits GluRε1, GluRε3 and GluRζ1 are enriched at the mossy fibre–granule cell synapse in the adult mouse cerebellum

Abstract

Cerebellar N-methyl-D-aspartate (NMDA) receptors are concentrated in the granular layer and are involved in motor coordination and the induction of long-term potentiation at mossy fibre-granule cell synapses. In the present study, we used immunohistochemistry to examine the distribution of NMDA receptor subunits in the adult mouse cerebellum. We found that appropriate pepsin pretreatment of sections greatly enhanced the sensitivity and specificity of immunohistochemical detection. As a result, intense immunolabelling for GluRepsilon1 (NR2A), GluRepsilon3 (NR2C), and GluRzeta1 (NR1) all appeared in synaptic glomeruli of the granular layer. Double immunofluorescence showed that these subunits were colocalized in individual synaptic glomeruli. Within the glomerulus, NMDA receptor subunits were located between centrally-located huge mossy fibre terminals and peripherally-located tiny Golgi axon terminals. By immunoelectron microscopy, all three subunits were detected at the postsynaptic junction in granule cell dendrites, forming synapses with mossy fibre terminals. Consistent with the known functional localization, GluRepsilon1, GluRepsilon3, and GluRzeta1 are, thus, anatomically concentrated at the mossy fibre-granule cell synapse. By contrast, immunohistochemical signals were very low in Purkinje cell somata and dendrites in the molecular layer. The lack of GluRzeta1 immunolabelling in Purkinje cells was unexpected because the cells express GluRzeta1 mRNA at high levels and high levels of GluRzeta1 protein in the molecular layer were revealed by immunoblot. As Purkinje cells are exceptionally lacking GluRepsilon expression, the discrepant result may provide in vivo evidence suggesting the importance of accompanying GluRepsilon subunits in synaptic localization of GluRzeta1.

Ontogeny of postsynaptic density proteins at glutamatergic synapses

“…Mouse NR2A and NR2B antibodies (affinity-purified; C-terminal, i.e., recognizes intracellular domain) were characterized in general and in hippocampus in Watanabe et al (1998) and for immunogold in Yamada et al (2001); these NR2A (amino acids 1126-1408) and NR2B (amino acids 1353-1432) antibodies were made from equivalent mouse subunits, GluRɛ1 and GluRɛ2. The latter antibodies were used to quantify NR2A and NR2B immunogold labeling in synapses, but were not used for immunoblots because our supply of these antibodies was low.…”

NMDA Receptor GluRϵ/NR2 Subunits Are Essential for Postsynaptic Localization and Protein Stability of GluRζ1/NR1 Subunit

“…Procedures for immunization and antibody purification were reported previously . We also used rabbit anti-GluR1-C2 (Yamada et al, 2001), rat monoclonal anti-GluR1-N (GluR1-pan) (Yamada et al, 2001), rabbit antiGluR⑀1C , rabbit anti-GluR⑀2C , rabbit anti-GluR⑀3C (Yamada et al, 2001), rabbit and guinea pig anti-postsynaptic density (PSD)-95 (Fukaya and Watanabe, 2000), guinea pig anti-vesicular glutamate transporter VGluT1 (Miyazaki et al, 2003), rabbit anti-vesicular GABA transporter VGAT (Miyazaki et al, 2003), rabbit anti-synaptophysin (Fukaya and Watanabe, 2000), and goat anti-calreticulin (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. All antibodies were used at the concentration of 0.5-1 g/ml, unless noted otherwise.…”

“…6 A). The total GluR1 content, as assessed using the GluR1-pan antibody raised against the N-terminal region (GluR1-N antibody) (Yamada et al, 2001), was three times lower in the cerebellum than in the cerebrum: the cerebellum/cerebrum ratio was calculated as 1.00 Ϯ 0.10/3.55 Ϯ 0.43. Compared with the total GluR1 subunit, the ratio was further reduced for GluR1-C2 (1.00 Ϯ 0.02/11.76 Ϯ 0.50), whereas it was comparable for GluR1-C2Ј (1.00 Ϯ 0.22/3.44 Ϯ 0.41).…”

“…Before conventional immunohistochemical incubation, paraffin sections were subjected to pepsin pretreatment, which is essential for immunohistochemical detection of NMDA receptor subunits and the PSD-95/synapse-associated protein (SAP)-90 protein family Fukaya and Watanabe, 2000;Yamada et al, 2001;Oshima et al, 2002). After treatment with 1 mg/ml pepsin (Dako, Carpinteria, CA) in 0.2N HCl at 37°C for 10 min, sections were immunoreacted successively with rabbit or guinea pig primary antibodies overnight, biotinylated secondary antibodies for 1 hr, and streptoavidin-peroxidase for 30 min, using a Histofine SAB-PO(R) kit (Nichirei, Tokyo, Japan).…”

Phospholipase C β4 in the Medial Septum Controls Cholinergic Theta Oscillations and Anxiety Behaviors

“…The specificity of PLC-␤4 and glutamic acid decarboxylase (GAD) antibodies has been shown previously (Yamada et al, 2001;Nakamura et al, 2004). In the present study, we produced goat antibodies to PV and mGluR1a using the same fusion proteins as had been used for production of rabbit and guinea pig antibodies (Tanaka et al, 2000;Nakamura et al, 2004), and also to rat choline transporter 1 using C-terminal 50 aa sequence fused to glutathione S-transferase (Narushima et al, 2007).…”