Genetic Deletion of NR3A Accelerates Glutamatergic Synapse Maturation

Glutamatergic synapse maturation is critically dependent upon activation of NMDA-type glutamate receptors (NMDARs); however, the contributions of NR3A subunit-containing NMDARs to this process have only begun to be considered. Here we characterized the expression of NR3A in the developing mouse forebrain and examined the consequences of NR3A deletion on excitatory synapse maturation. We found that NR3A is expressed in many subcellular compartments, and during early development, NR3A subunits are particularly concentrated in the postsynaptic density (PSD). NR3A levels dramatically decline with age and are no longer enriched at PSDs in juveniles and adults. Genetic deletion of NR3A accelerates glutamatergic synaptic transmission, as measured by AMPAR-mediated postsynaptic currents recorded in hippocampal CA1. Consistent with the functional observations, we observed that the deletion of NR3A accelerated the expression of the glutamate receptor subunits NR1, NR2A, and GluR1 in the PSD in postnatal day (P) 8 mice. These data support the idea that glutamate receptors concentrate at synapses earlier in NR3A-knockout (NR3A-KO) mice. The precocious maturation of both AMPAR function and glutamate receptor expression are transient in NR3A-KO mice, as AMPAR currents and glutamate receptor protein levels are similar in NR3A-KO and wildtype mice by P16, an age when endogenous NR3A levels are normally declining. Taken together, our data support a model whereby NR3A negatively regulates the developmental stabilization of glutamate receptors involved in excitatory neurotransmission, synaptogenesis, and spine growth

Genetic deletion of NR3A accelerates glutamatergic synapse maturation.

Glutamatergic synapse maturation is critically dependent upon activation of NMDA-type glutamate receptors (NMDARs); however, the contributions of NR3A subunit-containing NMDARs to this process have only begun to be considered. Here we characterized the expression of NR3A in the developing mouse forebrain and examined the consequences of NR3A deletion on excitatory synapse maturation. We found that NR3A is expressed in many subcellular compartments, and during early development, NR3A subunits are particularly concentrated in the postsynaptic density (PSD). NR3A levels dramatically decline with age and are no longer enriched at PSDs in juveniles and adults. Genetic deletion of NR3A accelerates glutamatergic synaptic transmission, as measured by AMPAR-mediated postsynaptic currents recorded in hippocampal CA1. Consistent with the functional observations, we observed that the deletion of NR3A accelerated the expression of the glutamate receptor subunits NR1, NR2A, and GluR1 in the PSD in postnatal day (P) 8 mice. These data support the idea that glutamate receptors concentrate at synapses earlier in NR3A-knockout (NR3A-KO) mice. The precocious maturation of both AMPAR function and glutamate receptor expression are transient in NR3A-KO mice, as AMPAR currents and glutamate receptor protein levels are similar in NR3A-KO and wildtype mice by P16, an age when endogenous NR3A levels are normally declining. Taken together, our data support a model whereby NR3A negatively regulates the developmental stabilization of glutamate receptors involved in excitatory neurotransmission, synaptogenesis, and spine growth

Genetic Deletion of NR3A Accelerates Glutamatergic Synapse Maturation

Glutamatergic synapse maturation is critically dependent upon activation of NMDA-type glutamate receptors (NMDARs); however, the contributions of NR3A subunit-containing NMDARs to this process have only begun to be considered. Here we characterized the expression of NR3A in the developing mouse forebrain and examined the consequences of NR3A deletion on excitatory synapse maturation. We found that NR3A is expressed in many subcellular compartments, and during early development, NR3A subunits are particularly concentrated in the postsynaptic density (PSD). NR3A levels dramatically decline with age and are no longer enriched at PSDs in juveniles and adults. Genetic deletion of NR3A accelerates glutamatergic synaptic transmission, as measured by AMPAR-mediated postsynaptic currents recorded in hippocampal CA1. Consistent with the functional observations, we observed that the deletion of NR3A accelerated the expression of the glutamate receptor subunits NR1, NR2A, and GluR1 in the PSD in postnatal day (P) 8 mice. These data support the idea that glutamate receptors concentrate at synapses earlier in NR3A-knockout (NR3A-KO) mice. The precocious maturation of both AMPAR function and glutamate receptor expression are transient in NR3A-KO mice, as AMPAR currents and glutamate receptor protein levels are similar in NR3A-KO and wildtype mice by P16, an age when endogenous NR3A levels are normally declining. Taken together, our data support a model whereby NR3A negatively regulates the developmental stabilization of glutamate receptors involved in excitatory neurotransmission, synaptogenesis, and spine growth

Deletion of NR3A transiently accelerates expression of synapse maturation markers.

<p>(<b>A</b>) Representative immunoblots from NR3A-KO compared to WT controls show increased PSD levels of (<b>B</b>) NR1, (<b>C</b>) NR2A, and (<b>D</b>) GluR1 at P8 that return to WT levels by P16 and adult ages. (<b>E</b>) NR2B expression is unchanged in the NR3A-KO. (<b>B, C, D, E</b>) Data are averaged means of immunoreactive values relative to protein loads (µg) and presented as percent of control values. NR1 values for age P8 are re-plotted here from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042327#pone.0042327-Roberts1" target="_blank">[14]</a> for comparative purposes. Error bars represent SEM. n = 7–10. Significance from control: * <i>p</i><0.05.</p

Developmental reduction in NR3A levels and PSD targeting.

<p>(<b>A</b>) Representative immunoblots of biochemical fractionation from P8, P16, and >P40 forebrain. (<b>B</b>) NR3A protein levels in the PNS, SPM, and PSD fractions from mice at P8, P16, and >P40. NR3A levels decrease during development in all fractions. Data are averaged means of immunoreactive (IR) values relative to protein loads (µg). (<b>C</b>) Averaged data normalized to initial homogenate values (total receptor protein) to highlight the shift in NR3A expression away from the PSD fraction. Error bars represent SEM. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Genetic deletion of NR3A accelerates the expression of AMPAR-mediated currents.

<p>(<b>A</b>) AMPA input-output (I–O) curve demonstrating the amplitude of synaptic AMPAR currents recorded at −80 mV in CA1 pyramidal neurons from P8 NR3A-KO and WT controls (WT, n = 12 neurons; NR3A-KO, n = 12 neurons). RMANOVA revealed a significant effect of genotype on AMPAR current amplitude across stimulation intensities (F_{(1, 10)} = 9.557, <i>p</i> = 0.005). (<b>B</b>) Synaptic AMPA currents recorded at P16–17 are similar between WT and NR3A-KO mice (WT, n = 13 neurons; NR3A-KO, n = 11 neurons). In contrast to AMPA currents recorded at P8, there is no significant effect of genotype on synaptic AMPAR currents at this age (RMANOVA, <i>p</i> = 0.99). Data are averaged means of NR3A-KO and control values. Error bars represent SEM. Significance from control: * <i>p</i><0.05.</p

Glutamate receptor subunits NR1, NR2A, NR2B, and GluR1 are highly enriched in PSDs of postnatal mice.

<p>Relative protein levels of (<b>A</b>) NR1, (<b>B</b>) NR2A, (<b>C</b>) NR2B, and (<b>D</b>) GluR1 in mouse forebrain. Protein data are averaged means of immunoreactive (IR) values relative to total protein loads (µg). n = 5–9. Error bars represent SEM. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Subcellular fractionation of presynaptic and postsynaptic proteins in forebrain neurons.

<p>(<b>A</b>) Schematic illustration of the enrichment process, highlighting the key fractions examined in this study (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042327#s2" target="_blank">Methods</a> for details). The PNS fraction obtained from the homogenate is subjected to a series of centrifugation steps to isolate pre- and postsynaptic plasma membranes (SPM), including layering on a sucrose gradient, and finally extracting with detergent to obtain the postsynaptic densities (PSD). (<b>B</b>) Representative immunoblots show NR1, PSD-95, and synaptophysin (Syp) expression in fractions from P16 mouse forebrain, with 5 or 10 µg total protein loaded in each lane as indicated. (<b>C</b>) Quantification of fraction fold-enrichment made with sample values normalized to PNS fractions. Error bars represent SEM. PNS, postnuclear supernatant; CYT, cytosol; LM, light membranes; P2, crude synaptosomes; P3, lysed synaptic membranes; SPM, purified synaptic plasma membranes; TSF, Triton-soluble fraction; PSD, postsynaptic densities.</p