The Role Of Extrasynaptic NMDA Receptors In Induction Of Long-term Potentiation In Adult Mouse Hippocampal CA1 Neurons

Synapse is the key part of the neurons where the neurons communicate with one another and transfer all kinds of electrical or chemical signals. N-methyl-D-aspartate receptors (NMDA receptors, NMDARs), as the ion channel-type glutamate receptors, are vital for mammal neuron system. NMDA receptors are divided into synaptic NMDARs (S-NMDARs) and extrasynaptic NMDARs (ES-NMDARs) according to the location of the cellular membrane. However, the investigations about the physiological roles of different NMDAR pools on neuronal cellular membrane have never been done due to existence of the technical difficulties in selective activation of S- or ES-NMDARs. In 2002, it was the first time that Hardingham and his colleagues activated ES-NMDARs selectively in culture system. Their study showed that the S- and ES-NMDARs have opposite effects upon intracellular signaling, cAMP response element binding protein (CREB) gene expression and cellular survival & apoptosis: Calcium influx via the activated S-NMDARs induces CREB activity and brain derived neurotrophic factor (BDNF) gene expression to be up-regulated which promotes cellular survival; on the contrary, calcium influx through ES-NMDARs down-regulates CREB activity and BDNF expression which leads to apoptosis. However, the role of ES-NMDARs in long-term potentiation (LTP) remains unclear yet. In this research, we investigated the role of ES-NMDARs in LTP induction in adult mice hippocampal CA1 area from three objectives as follows:(1) Build the methods of recording selectively ES-NMDARs mediated excitatory postsynaptic current (ES-NMDA-EPSC) or ES-NMDARs mediated excitatory postsynaptic potential (ES-NMDA-EPSP) in adult mice hippocampal slices using whole-cell patch clamp and extracellular recording techniques respectively;(2) Elucidate the role of ES-NMDARs in LTP induction;(3) Study the effect of different subunits of S- and ES-NMDARs on LTP induction.Main methods and results:1. Selectively record the ES-NMDA-EPSCs:(1) Prepared hippocampal acute slices (400μM) from healthy C57BL/6J mice (7-8 weeks old, male). (2) Made visually guided whole-cell patch clamp recordings of ES-NMDA-EPSCs. In hippocampal CA1 area, healthy pyramidal neurons which have smooth surface and good perception were patched. Non-NMDA receptors antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and Gamma-Aminobutyric acid receptor A (GABAA) antagonist picrotoxin were bath applied, holding the cells at +40 mV, and S-NMDA-EPSC was obtained accordingly.(3) 5 Hz for 16 sec was delivered to activate S-NMDARs, which were then blocked by pretreated MK-801 (20μM) within artificial cerebrospinal fluid (ACSF).(4) 30 min after wash out of MK-801, a short train HFS (100 Hz for 10 msec) was delivered to activate ES-NMDARs, and ES-NMDA-EPSC was recorded accordingly.2. The role of S- and ES-NMDARs in LTP induction:After gaining the stable baseline for filed EPSP (fEPSP) in hippocampal CA1 area by using field potential recording technique, 5 Hz was delivered for 16 sec to activate S-NMDARs which were then blocked by pretreated MK-801 (20μM) within artificial cerebrospinal fluid (ACSF). Then in 30-50 min posterior to wash-out of MK-801, three trains of high-frequency stimulations (100 Hz/1 sec, 15 sec interval) were applied to selectively activate ES-NMDARs, as the result LTP induction failed; And similar to ES-NMDARs, delivering of 5 Hz to selectively activate S-NMDARs failed to induce LTP in extracellular recording pattern.3. The effects of different subunits of NMDARs on LTP induction: We studied the roles of different subunits of NMDA receptors in LTP induction by using NR2A subunit or NR2B subunit antagonist. LTP could be induced by selectively activating either ES-NMDARs or S-NMDARs when the NR2B subunits were blocked; However, LTP couldn't be induced by selectively activating whether extrasynaptic NMDARs or S-NMDARs when the NR2A subunits were blocked.Main conclusions:(1) NR2B-containing NMDARs, regardless of where they are located in synaptic area or extrasynaptic area, make a negative control over LTP induction.(2) The balance between NR2A and NR2B is associated with LTP induction.In the present study, we established selective activation of S-NMDARs and ES-NMDARs in slices by combination of neuropharmacological methods and electrophysiological stimulation pattern. This protocol provides a good experimental pathway to study the physiological and pathological functions of NMDARs. In the study, it is the first time that the effects of ES-NMDARs upon LTP induction are investigated.