Before we start: What is the question? Why is it interesting? What is the role/contribution of the different subtypes of NMDA glutamate receptor to plasticity in the brain? Why is it interesting? NMDA receptors play an important role in many forms of synaptic plasticity. NMDA receptors are important for cell survival and cell death (ischemia, excess glutamate) NMDA receptor composition changes during development and it has been proposed to be one of the main regulators of the critical period. The developmental switch between NR2A and NR2B can be modulated by experience.

Glutamate Receptors: NMDA-sensitive Tetramers with the following subunits: NR1 (always 2) 8 splice variants. Glycine binding site. The different splice variants differ in their conductances, their affinity for glycine, etc. NR2 (always 2) Glutamate binding site. Need NR1 to form functional receptors 4 genes. (NR2A, NR2B. NR2C, NR2D) Only NR2A and NR2B are expressed in the forebrain. NR2A and NR2B are developmentally regulated, with NR2B starting to decrease after a few weeks. Very little is known about the proteins that each subunit interacts with.

NMDAR Receptors: Adult distribution of NR2 Nakanishi, S., 1992., Science

Functional Properties of NR1 and NR2 Affinity for glutamate Inhibition by Protons Modulation by Glycine Potentiation by polyamines Ca2+ current Inhibition by Zn+2 Channel Kinetics Some kinetics properties (very unknown) Dendritic transport, synaptic localization, trafficking of receptors Required for release from the ER; might be important for targeting between soma and dendrites NR2B interacts with the PDZ domain of SAP-102 and of Sec8 (an exocyst protein). NR2B can also interact with RasGRF1, actuvating the ERK pathway. Calcium inlfuc and ERK pathway activation can lead to changes in cell proliferation and survival. PSD-95 binds to NR2A increasing its expression at the synapse, while at the same time depressing synaptic expression of NR2B. PDS-95 KO mice have enhanced LTP. NR2-B truncated mice do not survive. Cterminal truncationof NR2A survives, impairs synaptic but not extrasynaptic localization of NMDA receptors and reduces LTP at the CA3-CA1 synapse. Cull-candy and leszkiewicz, Science STKE, 2004

Deactivation Kinetics of NR2B subunits > Mg2+ sensitive < Mg2+ sensitive Cull-candy and leszkiewicz, Science STKE, 2004

Plasticity in the Hippocampus Studying Long Term Potentiation (LTP) and Long Term Depression (LTD) Baseline Stim. Schaffer Collateral at 0.2Hz CA1 Induction Stim. Schaffer Collateral at 100Hz, 1sec CA1 100Hz Time (min) % EPSP 100 % 200 % Post- Induction Stim. Schaffer Collateral at 0.2Hz CA1

LTP and LTD in CA1 HFS of the Schaffer collateral fibers leads to LTP in CA1. LFS of the Schaffer collateral fibers leads to LTD in CA1. These changes in synaptic strength depend on: Intracellular calcium. NMDA receptors. Simple Model: Presynaptic Postsynaptic Ca2+ HFS LFS NMDA receptor Modifed from Lisman, 1989; Bear and Malenka, 1994.

Support for this model Intracellular calcium levels “LTD” “LTP” Hippocampal slices, p 11-p22, WC Recording, Photolysis of EGTA Intracellular calcium levels “LTD” “LTP” Yang S-N. et al., 1999. J. Neurophysiol.

Support for this model Degree of NMDA receptor activation  Amount of calcium Cummings et al., 1996. Neuron Nishiyama et al., 2000. Nature

NR2A KO NR2B KO

“Genetic enhancement of learning and memory in mice” BUT… NR2B over-expression: “Genetic enhancement of learning and memory in mice” Tang et al., Nature 1999. “NR2B expression is downregulated during the period of transition between juvenile and adult, correlating with the gradual shortening of the EPSP duration through the NMDA channel. This could decrease NMDA-mediated plasticity, and perhaps explain decreased memory performance in adult animals…”

Enhanced Novel Object Recognition “Smart Mice”: Over-expression of NR2B Enhanced Novel Object Recognition Tang et al., 1999. Nature.

Selective block of NR2A % block current 2B 2A In oocytes In hippocampal slices

NR2A X LTD LTP HFS LFS

NR2B X LTD (1XLFS) LTP (1XHFS) HFS LFS

Conclusions: [Ca++] 0.1uM Ca++ (resting) too low for both calcineurin & CamIIK – stable release ~1uM Ca++ (LFS=1-5 Hz) – calcineurin: LTD >5uM Ca++ (HFS=>Hz) – calcineurin + CamIIK… CamIIK out-competes: LTP Molecular ID of NR2 NR2A necessary for LTP, not LTD. NR2B necessary for LTD, not LTP. Reduction of NR2B during development could explain increased difficulty in inducing LTD.

who or where??? “Spillover” during HFS

Time, space, co-incidence detection Induction: association cooperativity specificity Time, space, co-incidence detection NMDAR coincidence detection: glutamate & depolarization Glutamate release- local AMPAR depolarization- small ampl., passive propagation: small spread Back-propagating AP- big ampl., active propagation: wide-spread Time calcineurin – high affinity, on/off CamIIK- medium affinity, persistent on PKC- medium affinity, on/off Space Ca++ via NMDAR- local glutamate, wide-spread voltage unblock Ca++ via IP3- local glutamate (mGluR) Ca++ via VG-CC- wide-spread Ca++ AP Spill-over (2A/2B in synapse; 2B extra-synaptic) NO- trans-cellular, on/off, intermed. distance- retrograde transmitter!!

NOS Na+ channels AP

Specificity - synaptic tagging