NMDARs are glutamate-gated ion channels and are enriched at excitatory synapses, where they are strategically positioned to play a crucial role in regulation of synaptic function. A unique feature of NMDARs is their high permeability to Ca2+. Ca2+ influx through NMDARs is essential for synaptogenesis, plasticity of neural circuitry, and higher cognitive functions, such as learning and memory. Emerging evidence reveals that PKA signaling represents a fundamental mechanism by which NMDAR-mediated Ca2+ influx is modulated in neurons. We discovered that serine 1166 on the NMDAR subunit GluN2B is a direct target of PKA relevant to NMDAR Ca2+ permeability and Ca2+ signaling in spines3. We further showed that adverse experience in the form of forced swim, but not exposure to fox urine, elicits striking phosphorylation of Ser1166 in vivo. Our data identify a novel molecular and functional target of PKA essential to NMDAR Ca2+ signaling at synapses that is regulated by the stress. Whereas the impact of PKA-induced phosphorylation of Ser1166 on NMDAR function and spine signaling is well-established, its impact on NMDAR-dependent synaptic plasticity and cognition is, as yet, unclear. To address this issue, we generated a novel mouse in which we knocked in GluN2B bearing a single point mutation, S1166A by means of CRISPR technology. The overall objective of the proposed research is to characterize the S1166A KI mouse and examine the impact of loss of phosphorylation at Ser1166 on synaptic plasticity and cognition. The overall hypothesis driving this research is that impaired Ca2+ permeability associated with the S1166A mutation will lead to impaired NMDAR-dependent synaptic plasticity and deficits in hippocampal-based behaviors. We will test this hypothesis in the following Aims: 1. Examine the impact of loss of Ser1166 on NMDAR-dependent synaptic plasticity at CA1 synapses and hippocampal-based learning in S1166A KI mice. Experiments will examine 1) the role phosphorylation of Ser1166 in transient incorporation of CP-AMPARs and the ability of strategies which promote incorporation of CP-AMPARs to rescue synaptic plasticity; 2) the impact of loss of Ser1166 on hippocampal-based visual and spatial learning in KI mice; 3) the ability of overexpression of WT GluN2B delivered directly into the CA1 of living mice via the lentiviral expression system to rescue impaired cognition. 2. Examine the impact of stress-induced phosphorylation of GluN2B at Ser1166 on NMDAR-dependent synaptic plasticity and cognition in WT mice. Experiments will examine 1) the impact of stress in the form of forced swim on TBS- vs. HFS-LTP in WT mice; 2) the impact of stress on visual and spatial learning in WT mice; 3) the ability of norepinephrine administered ip to mimic, and ?-adrenergic blockers to inhibit, the impact of stress on cognition in WT mice; 4) assess the ability of optogenetic activation of noradrenergic projections from the locus coeruleus to the CA1 to lower the threshold and/or increase the magnitude of LTP in WT mice.
NMDA receptors mediate excitatory transmission and are critical to synaptogenesis, formation of neural circuitry and higher cognitive functions such as learning and memory. Emerging evidence reveals that PKA signaling represents a fundamental mechanism by which NMDAR-mediated Ca2+ influx is modulated in neurons. The overall objectives of the proposed research are to characterize the GluN2B S1166A knock-in mouse, and to determine the impact of loss of this single residue on synaptic plasticity and cognition. This groundbreaking work will lay a foundation for future studies to further understand the mechanisms underlying the dysfunction of cognition in stroke, Alzheimer?s disease and other neurological disorders in which cognition is impaired.
