Different NMDA receptor subtypes mediate induction of long-term potentiation and two forms of short-term potentiation at CA1 synapses in rat hippocampus in vitro.

Potentiation at synapses between CA3 and the CA1 pyramidal neurons comprises both transient and sustained phases, commonly referred to as short-term potentiation (STP or transient LTP) and long-term potentiation (LTP), respectively. Here, we utilized four subtype-selective N-methyl-d-aspartate receptor (NMDAR) antagonists to investigate whether the induction of STP and LTP is dependent on the activation of different NMDAR subtypes. We find that the induction of LTP involves the activation of NMDARs containing both the GluN2A and the GluN2B subunits. Surprisingly, however, we find that STP can be separated into two components, the major form of which involves activation of NMDARs containing both GluN2B and GluN2D subunits. These data demonstrate that synaptic potentiation at CA1 synapses is more complex than is commonly thought, an observation that has major implications for understanding the role of NMDARs in cognition.

Characterisation of an mGlu8 receptor-selective agonist and antagonist in the lateral and medial perforant path inputs to the dentate gyrus.

Since its characterisation in 2001, the mGlu8-selective agonist DCPG has been widely used to explore the potential functional role of this group III mGlu receptor within the central nervous system. This research has implicated mGlu8 receptors in a number of disease states and conditions such as epilepsy and anxiety, suggesting that mGlu8-selective ligands may hold important therapeutic potential. However, there is evidence that DCPG exerts off-target effects at higher concentrations, limiting its use as an mGlu8-selective agonist. Here, we have used field recordings in rat hippocampal slices to investigate the effects of DCPG in the lateral perforant path (LPP), a pathway known to express high levels of mGlu8. We show that DCPG does inhibit excitatory transmission in this pathway, but produces a biphasic concentration-response curve suggesting activation of two distinct receptor types. The putative mGlu8-selective antagonist MDCPG antagonises the high, but not the low, potency component of this concentration-response curve. In addition, higher concentrations of DCPG also depress excitatory transmission in the medial perforant path (MPP), a pathway expressing very low levels of mGlu8 receptors. Experiments in slices from mice lacking mGlu8 receptors indicate that concentrations of DCPG >1 µM produce large non-selective effects in both the LPP and MPP. Further experiments in slices from mGlu2, 4 and 7 knock-out mice, as well as in an mGlu2-deficient substrain of Wistar rat, reveal that these non-selective effects are mediated primarily by mGlu2 receptors. Taken together, our results confirm the mGlu8-selectivity of DCPG at submicromolar concentrations, but suggest that care must be taken when employing higher concentrations of the agonist, which may additionally activate mGlu2 receptors, especially at synapses where their expression is high. MDCPG may be a useful tool in determining whether observable DCPG effects are attributable to mGlu8, versus mGlu2, receptor activation.

The Jak/STAT pathway is involved in synaptic plasticity.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is involved in many cellular processes, including cell growth and differentiation, immune functions and cancer. It is activated by various cytokines, growth factors, and protein tyrosine kinases (PTKs) and regulates the transcription of many genes. Of the four JAK isoforms and seven STAT isoforms known, JAK2 and STAT3 are highly expressed in the brain where they are present in the postsynaptic density (PSD). Here, we demonstrate a new neuronal function for the JAK/STAT pathway. Using a variety of complementary approaches, we show that the JAK/STAT pathway plays an essential role in the induction of NMDA-receptor dependent long-term depression (NMDAR-LTD) in the hippocampus. Therefore, in addition to established roles in cytokine signaling, the JAK/STAT pathway is involved in synaptic plasticity in the brain.

Tyrosine dephosphorylation regulates AMPAR internalisation in mGluR-LTD.

Long-term depression (LTD) can be induced at hippocampal CA1 synapses by activation of either NMDA receptors (NMDARs) or group I metabotropic glutamate receptors (mGluRs), using their selective agonists NMDA and (RS)-3,5-dihydroxyphenylglycine (DHPG), respectively. Recent studies revealed that DHPG-LTD is dependent on activation of postsynaptic protein tyrosine phosphatases (PTPs), which transiently dephosphorylate tyrosine residues in AMPA receptors (AMPARs). Here we show that while both endogenous GluR2 and GluR3 AMPAR subunits are tyrosine phosphorylated at basal activity, only GluR2 is dephosphorylated in DHPG-LTD. The tyrosine dephosphorylation of GluR2 does not occur in NMDA-LTD. Conversely, while NMDA-LTD is associated with the dephosphorylation of GluR1-serine-845, DHPG-LTD does not alter the phosphorylation of this site. The increased AMPAR endocytosis in DHPG-LTD is PTP-dependent and involves tyrosine dephosphorylation of cell surface AMPARs. Together, these results indicate that the subunit selective tyrosine dephosphorylation of surface GluR2 regulates AMPAR internalisation in DHPG-LTD but not in NMDA-LTD in the hippocampus.

An analysis of the stimulus requirements for setting the molecular switch reveals a lower threshold for metaplasticity than synaptic plasticity.

The requirements for the synaptic activation of metabotropic glutamate (mGlu) receptors and for the induction of metaplasticity in the hippocampus are not known. In the present study, we have investigated the synaptic activation of mGlu5 receptors and the setting of the molecular switch, a form of metaplasticity, at CA1 synapses in the mouse hippocampus. We find that as few as eight stimuli (delivered at 100Hz) are sufficient to set the molecular switch, since a subsequent tetanus delivered to the same input is able to induce long-term potentiation (LTP) in the presence of the mGlu receptor antagonist MCPG ((S)-alpha-methyl-4-carboxyphenylglycine). In addition, we find that the molecular switch can be activated over a wide frequency range. When 10 shocks were delivered the threshold frequency was 4Hz. The ability of 10 shocks (delivered at 100Hz) to set the molecular switch was lost in the mGlu5 knockout. These data show that mGlu5 receptors can be activated synaptically and metaplasticity can be induced by relatively few stimuli. Indeed, metaplasticity was induced by stimuli that were subthreshold for the induction of LTP per se. Thus, metaplasticity has a lower threshold than the synaptic plasticity that it regulates.

Differential roles of NR2A and NR2B-containing NMDA receptors in LTP and LTD in the CA1 region of two-week old rat hippocampus.

The role of NMDA receptors in the induction of long-term potentiation (LTP) and long-term depression (LTD) is well established but which particular NR2 subunits are involved in these plasticity processes is still a matter of controversy. We have studied the effects of subtype selective NMDA receptor antagonists on LTP induced by high frequency stimulation (100 Hz for 1s) and LTD induced by low frequency stimulation (1 Hz for 15 min) in the CA1 region of hippocampal slices from 14 day old Wistar rats. Against recombinant receptors in HEK293 cells NVP-AAM077 (NVP) was approximately 14-fold selective for NR2A vs NR2B receptors, whilst Ro 25-6981 (Ro) was highly selective for NR2B receptors. On NMDA receptor-mediated EPSCs from Schaffer collaterals in CA1 neurones, NVP and Ro both reduced the amplitude but differentially affected the time constant of decay. The data are compatible with the selective effect of NVP (0.1 microM) and Ro (4 microM) on native NR2A and NBR2B receptors, respectively. NVP reduced both LTP and LTD whereas Ro reduced only LTP. Thus, LTP was reduced by 63% at 0.1 microM NVP and almost completely at 0.4 microM whereas 5 microM Ro reduced LTP by 45%. These data are consistent with a role for both NR2A and NR2B in the induction of LTP, under our experimental conditions. In comparison, LTD was unaffected by Ro (5 microM) even in the presence of a glutamate uptake inhibitor threo-beta-benzylaspartic acid (TBOA) to increase the concentration of glutamate at NR2B containing receptors. NVP (0.2-0.4 microM), however, produced a concentration dependent inhibition of LTD which was complete at 0.4 microM. The lack of effect of 0.1 microM NVP on LTD contrasts with its marked effect on LTP and raises the possibility that different NVP-sensitive NR2 subunit-containing NMDA receptors are required for LTP and LTD in this preparation.

The regulation of hippocampal LTP by the molecular switch, a form of metaplasticity, requires mGlu5 receptors.

The role of metabotropic glutamate (mGlu) receptors in long-term potentiation (LTP) in the hippocampus is controversial. In the present study, we have used mice in which the mGlu1, mGlu5 or mGlu7 receptor has been deleted, by homologous recombination, to study the role of these receptor subtypes in LTP at CA1 synapses. We investigated the effects of the knockouts on both LTP and the molecular switch, a form of metaplasticity that renders LTP insensitive to the actions of the mGlu receptor antagonist MCPG ((S)-alpha-methyl-4-carboxyphenylglycine). We find that LTP is readily induced in the three knockouts and in an mGlu1 and mGlu5 double knockout. In addition, the molecular switch operates normally in either the mGlu1 or mGlu7 knockout. In contrast, the molecular switch is completely non-functional in the mGlu5 knockout, such that MCPG invariably blocks the induction of additional LTP in an input where LTP has already been induced. The effect of the mGlu5 receptor knockout was replicated in wildtype mouse slices perfused with the specific mGlu5 receptor antagonist MPEP (2-methyl-6-(phenylethynyl)-pyridine). In addition, the mGlu5 selective agonist CHPG ((RS)-2-chloro-5-hydroxyphenylglycine) sets the molecular switch. These data demonstrate that the operation of the molecular switch requires activation of mGlu5 receptors.

Interactions between NMDA receptors and mGlu5 receptors expressed in HEK293 cells.

1 Ca2+ imaging was used to investigate interactions between responses induced by N-methyl-D-aspartate (NMDA; 15 microm) and (RS)-3,5-dihydroxyphenyl-glycine (DHPG; 30 microm) in human embryonic kidney (HEK) 293 cells, transiently transfected with rat recombinant NR1a, NR2A and mGlu5a cDNA. 2 Responses to NMDA were reversibly depressed by DHPG from 244+/-14 to 194+/-12% of baseline. Treatment with thapsigargin (1 microm, 10 min) prevented this effect. 3 After thapsigargin pretreatment, repeated applications of NMDA showed a gradual rundown in amplitude over a period of several hours, and were unaffected by DHPG. 4 Continuous perfusion with staurosporine (0.1 microm), after thapsigargin pretreatment, converted the run-down to a small increase in NMDA responses to 123+/-6 % of baseline. DHPG induced a further and sustained potentiation of NMDA responses to 174+/-12% of the initial baseline. 5 The protein tyrosine kinase (PTK) inhibitors genistein (50 microm) and 3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2; 1 microm) inhibited the staurosporine- and DHPG-induced potentiation of NMDA responses. 6 The protein phosphatase (PTP) inhibitors orthovanadate (100 microm) and phenyl arsine oxide (PAO, 1 microm) facilitated the staurosporine-evoked potentiation of NMDA responses and occluded DHPG-induced potentiation. 7 In conclusion, complex interactions can be demonstrated between mGlu5 and NMDA receptors expressed in HEK293 cells. There is a negative inhibitory influence of Ca2+ release and PKC activation. Inhibition of these processes reveals a tonic, mGlu5 receptor and PTK-dependent potentiation of NMDA receptors that can be augmented by either stimulating mGlu5 receptors or by inhibiting PTPs.

Altered short-term synaptic plasticity in mice lacking the metabotropic glutamate receptor mGlu7.

Eight subtypes of metabotropic glutamate (mGlu) receptors have been identified of which two, mGlu5 and mGlu7, are highly expressed at synapses made between CA3 and CA1 pyramidal neurons in the hippocampus. This input, the Schaffer collateral-commissural pathway, displays robust long-term potentiation (LTP), a process believed to utilise molecular mechanisms that are key processes involved in the synaptic basis of learning and memory. To investigate the possible function in LTP of mGlu7 receptors, a subtype for which no specific antagonists exist, we generated a mouse lacking this receptor, by homologous recombination. We found that LTP could be induced in mGlu7-/- mice and that once the potentiation had reached a stable level there was no difference in the magnitude of LTP between mGlu7-/- mice and their littermate controls. However, the initial decremental phase of LTP, known as short-term potentiation (STP), was greatly attenuated in the mGlu7-/- mouse. In addition, there was less frequency facilitation during, and less post-tetanic potentiation following, a high frequency train in the mGlu7-/- mouse. These results show that the absence of mGlu7 receptors results in alterations in short-term synaptic plasticity in the hippocampus.