Mary J. Palmer

Hippocampal LTD Expression Involves a Pool of AMPARs Regulated by the NSF–GluR2 Interaction

We investigated whether the interaction between the N-ethyl-maleimide-sensitive fusion protein (NSF) and the AMPA receptor (AMPAR) subunit GluR2 is involved in synaptic plasticity in the CA1 region of the hippocampus. Blockade of the NSF-GluR2 interaction by a specific peptide (pep2m) introduced into neurons prevented homosynaptic, de novo long-term depression (LTD). Moreover, saturation of LTD prevented the pep2m-induced reduction in AMPAR-mediated excitatory postsynaptic currents (EPSCs). Minimal stimulation experiments indicated that both pep2m action and LTD were due to changes in quantal size and quantal content but were not associated with changes in AMPAR single-channel conductance or EPSC kinetics. These results suggest that there is a pool of AMPARs dependent on the NSF-GluR2 interaction and that LTD expression involves the removal of these receptors from synapses.

(RS)-2-Chloro-5-Hydroxyphenylglycine (CHPG) Activates mGlu5, but not mGlu1, Receptors Expressed in CHO Cells and Potentiates NMDA Responses in the Hippocampus

A new phenylglycine derivative, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), has been synthesized and shown to selectively activate mGlu5a receptors, compared to mGlu1 alpha receptors, when expressed in CHO cells. This selective mGlu5 receptor agonist also potentiates NMDA-induced depolarizations in rat hippocampal slices. CHPG may be a useful tool for studying the role of mGlu5 receptors in the central nervous system.

A characterisation of long‐term depression induced by metabotropic glutamate receptor activation in the rat hippocampus in vitro

1 In the CA1 region of hippocampal slices prepared from juvenile (12‐ to 18‐day‐old) rats, activation of group I metabotropic l‐glutamate (mGlu) receptors by the specific agonist (RS)‐3,5‐dihydroxyphenylglycine (DHPG) induces a form of long‐term depression (LTD) of excitatory synaptic transmission. 2 We have used a variety of electrophysiological techniques applied to CA1 neurones in hippocampal slices and from pyramidal cells in dissociated hippocampal cultures to investigate the Ca2+ dependence and locus of expression of DHPG‐induced LTD. 3 In patch‐clamp experiments from hippocampal slices, bath application of DHPG induced a depression of synaptically evoked responses that persisted for the duration of the recording (up to 2 h after commencing washout of DHPG) in 27 of 29 neurones investigated. 4 DHPG‐induced LTD was associated with an increase in both the paired‐pulse facilitation ratio and the coefficient of variation of EPSCs. 5 Using dendritic recording, there was a decrease in EPSC success rate (number of trials that elicited a detectable response) but no change in potency (mean EPSC amplitude excluding failures) associated with DHPG‐induced LTD. 6 In experiments using dissociated hippocampal cultures, application of DHPG elicited a persistent decrease in the frequency of tetrodotoxin‐resistant miniature EPSCs but no change in the amplitude of such events. 7 DHPG‐induced LTD was not blocked by intracellular application of the calcium chelator BAPTA. It was also unaffected when intracellular calcium stores were depleted by perfusion with thapsigargin. Furthermore, when synaptic transmission was blocked by perfusing with Ca2+‐free medium, DHPG application reliably induced LTD. 8 These data suggest that DHPG‐induced LTD is Ca2+ independent and is expressed presynaptically.

The potent mGlu receptor antagonist LY341495 identifies roles for both cloned and novel mGlu receptors in hippocampal synaptic plasticity

Understanding the roles of metabotropic glutamate (mGlu) receptors has been severely hampered by the lack of potent antagonists. LY341495 (2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-y l)propanoic acid) has been shown to block group II mGlu receptors in low nanomolar concentrations (Kingston, A.E., Ornstein, P.L., Wright, R.A., Johnson, B.G., Mayne, N.G., Burnett, J.P., Belagaje, R., Wu, S., Schoepp, D.D., 1998. LY341495 is a nanomolar potent and selective antagonist at group II metabotropic glutamate receptors. Neuropharmacology 37, 1-12) but can be used in higher concentrations to block all hippocampal mGlu receptors, identified so far by molecular cloning (mGlu1-5,7,8). Here we have further characterised the mGlu receptor antagonist activity of LY341495 and have used this compound to investigate roles of mGlu receptors in hippocampal long-term potentiation (LTP) and long-term depression (LTD). LY341495 competitively antagonised DHPG-stimulated PI hydrolysis in AV12-664 cells expressing either human mGlu1 or mGlu5 receptors with Ki-values of 7.0 and 7.6 microM, respectively. When tested against 10 microM L-glutamate-stimulated Ca2+ mobilisation in rat mGlu5 expressing CHO cells, it produced substantial or complete block at a concentration of 100 microM. In rat hippocampal slices, LY341495 eliminated 30 microM DHPG-stimulated PI hydrolysis and 100 microM (1S,3R)-ACPD-inhibition of forskolin-stimulated cAMP formation at concentrations of 100 and 0.03 microM, respectively. In area CA1, it antagonised DHPG-mediated potentiation of NMDA-induced depolarisations and DHPG-induced long-lasting depression of AMPA receptor-mediated synaptic transmission. LY341495 also blocked NMDA receptor-independent depotentiation and setting of a molecular switch involved in the induction of LTP; effects which have previously been shown to be blocked by the mGlu receptor antagonist (S)-MCPG. These effects may therefore be due to activation of cloned mGlu receptors. In contrast, LY341495 did not affect NMDA receptor-dependent homosynaptic LTD; an effect which may therefore be independent of cloned mGlu receptors. Finally, LY341495 failed to antagonise NMDA receptor-dependent LTP and, in area CA3, NMDA receptor-independent, mossy fibre LTP. Since in the same inputs these forms of LTP were blocked by (S)-MCPG, a novel type of mGlu receptor may be involved in their induction.

Synaptic Activation of Presynaptic Glutamate Transporter Currents in Nerve Terminals

Glutamate uptake by high-affinity transporters is responsible for limiting the activation of postsynaptic receptors and maintaining low levels of ambient glutamate. The reuptake process generates membrane currents, which can be activated by synaptically released glutamate in glial cells and some postsynaptic neurons. However, less is known about presynaptic transporter currents because the small size of synaptic boutons precludes direct recordings. Here, we have recorded from two giant nerve terminals: bipolar cell synaptic terminals in goldfish retina and the calyx of Held in rat auditory brainstem. Exocytosis was evoked by brief depolarizations and measured as an increase in membrane capacitance. In isolated bipolar cell terminals, exocytosis was associated with an anion (NO3- or Cl-) current. The current peaked 2.8 msec after the start of the depolarization and decayed with a mean time constant of 8.5 msec. It was inhibited by the nontransportable glutamate transporter antagonist sc-threo-β-benzyloxyaspartate (TBOA) but was insensitive to the GLT1/EAAT2 subtype-selective antagonist dihydrokainate and was affected by extracellular pH buffering. A TBOA-sensitive anion current was also evoked by application of exogenous glutamate to bipolar cell terminals. The large single-channel conductance, derived from noise analysis, and previous immunolocalization studies suggest that synaptically released glutamate activates EAAT5-type transporters in bipolar cell terminals. In contrast, neither exocytosis nor exogenous glutamate evoked a transporter current in the calyx of Held. Glutamate transporter currents with rapid kinetics are therefore identified and characterized in bipolar cell terminals, providing a valuable system for investigating the function and modulation of presynaptic glutamate transporters.

Presynaptic mechanisms involved in the expression of STP and LTP at CA1 synapses in the hippocampus

The study of long-term potentiation (LTP) has for many years been the centre of a raging debate as to whether the process is expressed by presynaptic or postsynaptic mechanisms. Here we present evidence that two forms of synaptic plasticity at CA3-CA1 synapses in the hippocampus are expressed by presynaptic changes. One form is short-term potentiation (STP) and the other a neonatal form of early-LTP (E-LTP). We review recent experimental data that suggests that this latter form of LTP involves an increase in the probability of neurotransmitter release (Pr). We describe how this is caused by the rapid down-regulation of a high affinity kainate receptor, which otherwise responds to ambient levels of l-glutamate by depressing Pr.

A novel, competitive mGlu5 receptor antagonist (LY344545) blocks DHPG-induced potentiation of NMDA responses but not the induction of LTP in rat hippocampal slices

We have investigated the pharmacological properties of LY344545, a structurally related epimer of the broad spectrum competitive metabotropic glutamate receptor antagonist, LY341495. We have found that LY344545 also antagonizes competitively nearly all mGlu receptor subtypes, but with a wide spectrum of activity. The order of potency for the human receptor isoforms was mGlu5a (IC50 of 5.5±0.6 μM)>mGlu2=mGlu3>mGlu1α=mGlu7>mGlu6=mGlu8. No significant mGlu4 receptor antagonist activity was detected at the highest concentration used (100 μM). 100 μM LY344545 displaced 50±5% of [3H]‐CGP39653 binding, but less than 30% of [3H]‐kainate or [3H]‐AMPA in radioligand binding assays. LY344545 antagonized L‐glutamate stimulated Ca2+ release in CHO cells transfected with mGlu receptors in a concentration dependent manner with a 10 fold higher affinity for the rat mGlu5a receptor (Ki=2.1±0.6 μM) compared to the rat mGlu1α receptor (Ki=20.5±2.1 μM). 50 μM (1S, 3R)‐ACPD‐induced Ca2+ rises in hippocampal CA1 neurones were also antagonized (IC50=6.8±0.7 μM). LY344545 antagonized 10 μM (S)‐3,5‐DHPG‐induced potentiation of NMDA depolarizations in CA1 neurones (EC50=10.6±1.0 μM). At higher concentrations (100 μM), LY344545 was an NMDA receptor antagonist. LY344545 also blocked the induction, but not the expression, of LTP at CA3 to CA1 synapses with an IC50>300 μM. This effect is consistent with its weak activity at NMDA receptors. These results demonstrate that the binding of ligands to mGlu receptor subtypes is critically dependent on the spatial orientation of the same molecular substituents within a given chemical pharmacophore. The identification of LY344545 as the first competitive antagonist to show selectivity towards mGlu5 receptors supports the potential to design more selective and potent competitive antagonists of this receptor. These results further indicate that mGlu receptor‐mediated potentiation of NMDA responses is not essential for the induction of LTP.

Multiple, Developmentally Regulated Expression Mechanisms of Long-Term Potentiation at CA1 Synapses

Long-term potentiation (LTP) of AMPA receptor-mediated synaptic transmission at hippocampal CA1 synapses has been extensively studied, but the mechanisms responsible for its expression remain unresolved. We tested a hypothesis that there are multiple, developmentally regulated expression mechanisms by directly comparing LTP in hippocampal slices obtained from rats of two ages. At postnatal day 12 (P12), LTP was fully accounted for by an increase in potency (mean amplitude of responses excluding failures). This was associated with either an increase in AMPA receptor single-channel conductance (γ) or no change in γ, suggesting an increase in the number of AMPA receptors. At P6, LTP was explained by an additional two mechanisms. In the majority of neurons, LTP was associated with an increase in success rate and a decrease in paired-pulse facilitation. In the remaining neurons, LTP was attributable to an increase in potency. However, in contrast to P12 neurons, the potency increase was associated with a decrease in γ, suggesting the insertion of receptors with lower γ. We conclude that there are multiple expression mechanisms for LTP at CA1 synapses that are developmentally regulated. These findings suggest that a single class of synapse uses a number of different molecular mechanisms to produce long-term changes in synaptic strength.

Bi-directional modulation of AMPA receptor unitary conductance by synaptic activity

BackgroundKnowledge of how synapses alter their efficiency of communication is central to the understanding of learning and memory. The most extensively studied forms of synaptic plasticity are long-term potentiation (LTP) and its counterpart long-term depression (LTD) of AMPA receptor-mediated synaptic transmission. In the CA1 region of the hippocampus, it has been shown that LTP often involves a rapid increase in the unitary conductance of AMPA receptor channels. However, LTP can also occur in the absence of any alteration in AMPA receptor unitary conductance. In the present study we have used whole-cell dendritic recording, failures analysis and non-stationary fluctuation analysis to investigate the mechanism of depotentiation of LTP.ResultsWe find that when LTP involves an increase in unitary conductance, subsequent depotentiation invariably involves the return of unitary conductance to pre-LTP values. In contrast, when LTP does not involve a change in unitary conductance then depotentiation also occurs in the absence of any change in unitary conductance, indicating a reduction in the number of activated receptors as the most likely mechanism.ConclusionsThese data show that unitary conductance can be bi-directionally modified by synaptic activity. Furthermore, there are at least two distinct mechanisms to restore synaptic strength from a potentiated state, which depend upon the mechanism of the previous potentiation.

Activation of mGlu receptors induces LTD without affecting postsynaptic sensitivity of CA1 neurons in rat hippocampal slices

Two forms of long‐term depression (LTD) of excitatory synaptic transmission have been identified in the mammalian CNS, which are induced by the synaptic activation of N‐methyl‐d‐aspartate (NMDA) and metabotropic glutamate (mGlu) receptors, respectively. The mGlu receptor‐dependent form of LTD can be activated by application of 3,5‐dihydroxyphenylglycine (DHPG), a group I selective mGlu receptor agonist. DHPG‐induced LTD is increasingly being used to investigate the mechanisms of mGlu receptor‐dependent LTD. However, recent experiments have argued for both a pre‐ and postsynaptic locus of expression of DHPG‐induced LTD. In the present study we report that DHPG‐induced LTD is not associated with changes in the sensitivity of CA1 neurons to bath applied AMPA. Furthermore, in contrast to homosynaptic LTD, DHPG‐induced LTD is also not associated with changes in sensitivity to focally uncaged l‐glutamate. These data do not support the notion that DHPG‐induced LTD requires a modification of AMPA receptors, such as their internalisation, but are compatible with a presynaptic mechanism of expression.