Long-term depression-associated signaling is required for an in vitro model of NMDA receptor-dependent synapse pruning.

Activity-dependent pruning of synaptic contacts plays a critical role in shaping neuronal circuitry in response to the environment during postnatal brain development. Although there is compelling evidence that shrinkage of dendritic spines coincides with synaptic long-term depression (LTD), and that LTD is accompanied by synapse loss, whether NMDA receptor (NMDAR)-dependent LTD is a required step in the progression toward synapse pruning is still unknown. Using repeated applications of NMDA to induce LTD in dissociated rat neuronal cultures, we found that synapse density, as measured by colocalization of fluorescent markers for pre- and postsynaptic structures, was decreased irrespective of the presynaptic marker used, post-treatment recovery time, and the dendritic location of synapses. Consistent with previous studies, we found that synapse loss could occur without apparent net spine loss or cell death. Furthermore, synapse loss was unlikely to require direct contact with microglia, as the number of these cells was minimal in our culture preparations. Supporting a model by which NMDAR-LTD is required for synapse loss, the effect of NMDA on fluorescence colocalization was prevented by phosphatase and caspase inhibitors. In addition, gene transcription and protein translation also appeared to be required for loss of putative synapses. These data support the idea that NMDAR-dependent LTD is a required step in synapse pruning and contribute to our understanding of the basic mechanisms of this developmental process.

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.

NR3A-containing NMDARs promote neurotransmitter release and spike timing-dependent plasticity.

Recent evidence suggests that presynaptic-acting NMDA receptors (preNMDARs) are important for neocortical synaptic transmission and plasticity. We found that unique properties of the NR3A subunit enable preNMDARs to enhance spontaneous and evoked glutamate release and that NR3A is required for spike timing-dependent long-term depression in the juvenile mouse visual cortex. In the mature cortex, NR2B-containing preNMDARs enhanced neurotransmission in the absence of magnesium, indicating that presynaptic NMDARs may function under depolarizing conditions throughout life. Our findings indicate that NR3A relieves preNMDARs from the dual-activation requirement of ligand-binding and depolarization; the developmental removal of NR3A limits preNMDAR functionality by restoring this associative property.

Influence of the NR3A subunit on NMDA receptor functions.

Various combinations of subunits assemble to form the NMDA-type glutamate receptor (NMDAR), generating diversity in its functions. Here we review roles of the unique NMDAR subunit, NR3A, which acts in a dominant-negative manner to suppress receptor activity. NR3A-containing NMDARs display striking regional and temporal expression specificity, and, unlike most other NMDAR subtypes, they have a low conductance, are only modestly permeable to Ca(2+), and pass current at hyperpolarized potentials in the presence of magnesium. While glutamate activates triheteromeric NMDARs composed of NR1/NR2/NR3A subunits, glycine is sufficient to activate diheteromeric NR1/NR3A-containing receptors. NR3A dysfunction may contribute to neurological disorders involving NMDARs, and the subunit offers an attractive therapeutic target given its distinct pharmacological and structural properties.

Downregulation of NR3A-containing NMDARs is required for synapse maturation and memory consolidation.

NR3A is the only NMDA receptor (NMDAR) subunit that downregulates sharply prior to the onset of sensitive periods for plasticity, yet the functional importance of this transient expression remains unknown. To investigate whether removal/replacement of juvenile NR3A-containing NMDARs is involved in experience-driven synapse maturation, we used a reversible transgenic system that prolonged NR3A expression in the forebrain. We found that removal of NR3A is required to develop strong NMDAR currents, full expression of long-term synaptic plasticity, a mature synaptic organization characterized by more synapses and larger postsynaptic densities, and the ability to form long-term memories. Deficits associated with prolonged NR3A were reversible, as late-onset suppression of transgene expression rescued both synaptic and memory impairments. Our results suggest that NR3A behaves as a molecular brake to prevent the premature strengthening and stabilization of excitatory synapses and that NR3A removal might thereby initiate critical stages of synapse maturation during early postnatal neural development.

Developmental regulation of the NMDA receptor subunits, NR3A and NR1, in human prefrontal cortex.

Subunit composition of N-methyl-D-aspartate-type glutamate receptors (NMDARs) dictates their function, yet the ontogenic profiles of human NMDAR subunits from gestation to adulthood have not been determined. We examined NMDAR mRNA and protein development in human dorsolateral prefrontal cortex (DLPFC), an area in which NMDARs are critical for higher cognitive processing and NMDAR hypofunction is hypothesized in schizophrenia. Using quantitative reverse transcriptase-polymerase chain reaction and western blotting, we found NR1 expression begins low prenatally, peaks in adolescence, yet remains high throughout life, suggesting lifelong importance of NMDAR function. In contrast, NR3A levels are low during gestation, surge soon after birth, and decline progressively through adolescence and into adulthood. Because NR3A subunits uniquely attenuate NMDAR-mediated currents, limit calcium influx, and suppress dendritic spine formation, high levels during early childhood may be important for regulating neuroprotection and activity-dependent sculpting of synapses. We also examined whether subunit changes underlie reduced NMDAR activity in schizophrenia. Our results reveal normal NR1 and NR3A protein levels in DLPFC from schizophrenic patients, indicating that NMDAR hypofunction is unlikely to be maintained by gross changes in NR3A-containing NMDARs or overall NMDAR numbers. These data provide insights into NMDAR functions in the developing CNS and will contribute to designing pharmacotherapies for neurological disorders.