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GluN2D-containing NMDA receptors-mediate synaptic currents in hippocampal interneurons and pyramidal cells in juvenile mice.

von Engelhardt J, Bocklisch C, Tönges L, Herb A, Mishina M, Monyer H - Front Cell Neurosci (2015)

Bottom Line: In contrast, much less is known about the role of GluN2D, which is expressed at low levels and is downregulated following the second postnatal week.The expression of the transgene was confined to hippocampal interneurons, most of which were parvalbumin- and/or somatostatin-positive.Electrophysiological and morphological analyses showed that GluN2D was present mainly in fast spiking basket and axo-axonic cells.

View Article: PubMed Central - PubMed

Affiliation: Synaptic Signalling and Neurodegeneration, German Cancer Research Center (DKFZ) Heidelberg, Germany ; Synaptic Signalling and Neurodegeneration, German Center for Neurodegenerative Diseases (DZNE) Bonn, Germany.

ABSTRACT
The differential regulation of the two major N-methyl-D-aspartate receptor (NMDAR) subunits GluN2A and GluN2B during development in forebrain pyramidal cells has been thoroughly investigated. In contrast, much less is known about the role of GluN2D, which is expressed at low levels and is downregulated following the second postnatal week. However, it appears that few cells, presumably interneurons, continue to express GluN2D also in juvenile mice. To investigate which hippocampal cell types express this subunit, we generated transgenic mice with EGFP-tagged GluN2D receptors. The expression of the transgene was confined to hippocampal interneurons, most of which were parvalbumin- and/or somatostatin-positive. Electrophysiological and morphological analyses showed that GluN2D was present mainly in fast spiking basket and axo-axonic cells. Based on pharmacological evidence and electrophysiological analysis of GluN2D knockout mice, we conclude that GluN2D-containing NMDARs mediate synaptic currents in hippocampal interneurons of young and juvenile mice and in CA1 pyramidal neurons of newborn mice.

No MeSH data available.


GluN2D-EGFP is expressed in the hippocampus. Visualization of EGFP-GluN2D in the hippocampus with an anti-EGFP antibody and DAB chromogen. EGFP-GluN2D-positive cells are localized in the CA1, CA2, dentate gyrus, and CA3 subregion. Most positive cells are close to or within the principal cell layer.
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Figure 1: GluN2D-EGFP is expressed in the hippocampus. Visualization of EGFP-GluN2D in the hippocampus with an anti-EGFP antibody and DAB chromogen. EGFP-GluN2D-positive cells are localized in the CA1, CA2, dentate gyrus, and CA3 subregion. Most positive cells are close to or within the principal cell layer.

Mentions: To ease the interpretation of subsequent electrophysiological data, we performed an immunocytochemical based expression analysis at the cellular level. Immunohistochemistry with a primary antibody against EGFP revealed that EGFP-GluN2D is expressed in hippocampal cells. We inferred that most of these cells were interneurons, as their cell body was located in the hilus of the dentate gyrus, the stratum oriens, and stratum radiatum of the CA3, CA2, and CA1 area (Figure 1). There were many EGFP-GluN2D-positive cells also in the CA3, CA2, and CA1 principal cell layer. These might be either pyramidal cells or interneurons located in the principal cell layer, e.g., parvalbumin (PV)-positive cells (Celio and Heizmann, 1981). We investigated which cell types express EGFP-GluN2D by double-immunohistochemistry with antibodies against EGFP and interneuron marker proteins. Since GluN2D mRNA expression decreases dramatically with development in all brain areas, including the hippocampus, we performed immunohistochemistry (and all subsequent analyses) with brains from mice of three different age groups. Co-localization of EGFP-GluN2D with interneuronal marker proteins PV, somatostatin (SOM), calretinin (CR), and calbindin (CB) was investigated in hippocampi of P3-5, P9-12, and adult mice. EGFP-GluN2D-expressing cells were found in the hippocampus of mice of all three ages. Interneuron marker proteins, however, were barely detectable in neurons of P3-5 mice, rendering a quantification of co-localization useless. PV immunoreactivity was absent in P3-5, very faint in P9-12 and strong in adult mouse hippocampi (Figure 2A), reflecting a developmental upregulation of this protein consistent with previous observations (Nitsch et al., 1990). There was an increase in the proportion of interneuronal marker protein-expressing EGFP-GluN2D-positive cells from about forty percent in hippocampi of P9-12 mice to 100% in hippocampi of adult mice (Figures 2B,C). Since many EGFP-GluN2D-positive but marker protein-negative cells in P9-12 mice had a typical interneuron location (e.g., stratum oriens or radiatum), we assume that most, if not all, are indeed interneurons in which the developmental upregulation of interneuron marker proteins had not yet occurred. The profile of interneuron marker proteins that was detected in EGFP-GluN2D-positive cells was similar in the CA1, CA3, and dentate gyrus subregion in the hippocampus of adult mice: most EGFP-GluN2D positive neurons expressed either PV or SOM and to a lesser extent CB. Some fluorescent cells also expressed CR. One can infer that there is co-expression of PV and SOM in EGFP-GluN2D-positive neurons, given that PV and SOM are expressed in ca. 80 and 50% of fluorescent cells, respectively (Figure 2C).


GluN2D-containing NMDA receptors-mediate synaptic currents in hippocampal interneurons and pyramidal cells in juvenile mice.

von Engelhardt J, Bocklisch C, Tönges L, Herb A, Mishina M, Monyer H - Front Cell Neurosci (2015)

GluN2D-EGFP is expressed in the hippocampus. Visualization of EGFP-GluN2D in the hippocampus with an anti-EGFP antibody and DAB chromogen. EGFP-GluN2D-positive cells are localized in the CA1, CA2, dentate gyrus, and CA3 subregion. Most positive cells are close to or within the principal cell layer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4373385&req=5

Figure 1: GluN2D-EGFP is expressed in the hippocampus. Visualization of EGFP-GluN2D in the hippocampus with an anti-EGFP antibody and DAB chromogen. EGFP-GluN2D-positive cells are localized in the CA1, CA2, dentate gyrus, and CA3 subregion. Most positive cells are close to or within the principal cell layer.
Mentions: To ease the interpretation of subsequent electrophysiological data, we performed an immunocytochemical based expression analysis at the cellular level. Immunohistochemistry with a primary antibody against EGFP revealed that EGFP-GluN2D is expressed in hippocampal cells. We inferred that most of these cells were interneurons, as their cell body was located in the hilus of the dentate gyrus, the stratum oriens, and stratum radiatum of the CA3, CA2, and CA1 area (Figure 1). There were many EGFP-GluN2D-positive cells also in the CA3, CA2, and CA1 principal cell layer. These might be either pyramidal cells or interneurons located in the principal cell layer, e.g., parvalbumin (PV)-positive cells (Celio and Heizmann, 1981). We investigated which cell types express EGFP-GluN2D by double-immunohistochemistry with antibodies against EGFP and interneuron marker proteins. Since GluN2D mRNA expression decreases dramatically with development in all brain areas, including the hippocampus, we performed immunohistochemistry (and all subsequent analyses) with brains from mice of three different age groups. Co-localization of EGFP-GluN2D with interneuronal marker proteins PV, somatostatin (SOM), calretinin (CR), and calbindin (CB) was investigated in hippocampi of P3-5, P9-12, and adult mice. EGFP-GluN2D-expressing cells were found in the hippocampus of mice of all three ages. Interneuron marker proteins, however, were barely detectable in neurons of P3-5 mice, rendering a quantification of co-localization useless. PV immunoreactivity was absent in P3-5, very faint in P9-12 and strong in adult mouse hippocampi (Figure 2A), reflecting a developmental upregulation of this protein consistent with previous observations (Nitsch et al., 1990). There was an increase in the proportion of interneuronal marker protein-expressing EGFP-GluN2D-positive cells from about forty percent in hippocampi of P9-12 mice to 100% in hippocampi of adult mice (Figures 2B,C). Since many EGFP-GluN2D-positive but marker protein-negative cells in P9-12 mice had a typical interneuron location (e.g., stratum oriens or radiatum), we assume that most, if not all, are indeed interneurons in which the developmental upregulation of interneuron marker proteins had not yet occurred. The profile of interneuron marker proteins that was detected in EGFP-GluN2D-positive cells was similar in the CA1, CA3, and dentate gyrus subregion in the hippocampus of adult mice: most EGFP-GluN2D positive neurons expressed either PV or SOM and to a lesser extent CB. Some fluorescent cells also expressed CR. One can infer that there is co-expression of PV and SOM in EGFP-GluN2D-positive neurons, given that PV and SOM are expressed in ca. 80 and 50% of fluorescent cells, respectively (Figure 2C).

Bottom Line: In contrast, much less is known about the role of GluN2D, which is expressed at low levels and is downregulated following the second postnatal week.The expression of the transgene was confined to hippocampal interneurons, most of which were parvalbumin- and/or somatostatin-positive.Electrophysiological and morphological analyses showed that GluN2D was present mainly in fast spiking basket and axo-axonic cells.

View Article: PubMed Central - PubMed

Affiliation: Synaptic Signalling and Neurodegeneration, German Cancer Research Center (DKFZ) Heidelberg, Germany ; Synaptic Signalling and Neurodegeneration, German Center for Neurodegenerative Diseases (DZNE) Bonn, Germany.

ABSTRACT
The differential regulation of the two major N-methyl-D-aspartate receptor (NMDAR) subunits GluN2A and GluN2B during development in forebrain pyramidal cells has been thoroughly investigated. In contrast, much less is known about the role of GluN2D, which is expressed at low levels and is downregulated following the second postnatal week. However, it appears that few cells, presumably interneurons, continue to express GluN2D also in juvenile mice. To investigate which hippocampal cell types express this subunit, we generated transgenic mice with EGFP-tagged GluN2D receptors. The expression of the transgene was confined to hippocampal interneurons, most of which were parvalbumin- and/or somatostatin-positive. Electrophysiological and morphological analyses showed that GluN2D was present mainly in fast spiking basket and axo-axonic cells. Based on pharmacological evidence and electrophysiological analysis of GluN2D knockout mice, we conclude that GluN2D-containing NMDARs mediate synaptic currents in hippocampal interneurons of young and juvenile mice and in CA1 pyramidal neurons of newborn mice.

No MeSH data available.