John T. R. Isaac

Evidence for silent synapses: Implications for the expression of LTP

Recent work has suggested that some proportion of excitatory synapses on hippocampal CA1 pyramidal cells that express NMDA receptors (NMDARs) may not express functional AMPA receptors (AMPARs), thus making these synapses silent at the resting membrane potential. In agreement with this hypothesis, we demonstrate here that it is possible to stimulate synapses that yield no detectable excitatory postsynaptic currents (EPSCs) when the cell is held at -60 mV; yet at positive holding potentials (+30 to +60 mV), EPSCs can be elicited that are completely blocked by the NMDAR antagonist, D-APV. When these functionally silent synapses are subjected to an LTP induction protocol, EPSCs mediated by AMPARs appear and remain for the duration of the experiment. This conversion of silent synapses to functional synapses is blocked by D-APV. These results suggest that LTP may involve modification of AMPARs that, prior to LTP, were either not present in the postsynaptic membrane or electrophysiologically silent. This mechanism may account for several experimental results previously attributed to presynaptic changes in quantal content.

Receptor trafficking and synaptic plasticity

Long-term potentiation and long-term depression are processes that have been widely studied to understand the molecular basis of information storage in the brain. Glutamate receptors are required for the induction and expression of these forms of plasticity, and GABA (γ-aminobutyric acid) receptors are involved in their modulation. Recent insights into how these receptors are rapidly moved into and out of synaptic membranes has profound implications for our understanding of the mechanisms of long-term potentiation and long-term depression.

NSF Binding to GluR2 Regulates Synaptic Transmission

Here, we show that N-ethylmaleimide-sensitive fusion protein (NSF) interacts directly and selectively with the intracellular C-terminal domain of the GluR2 subunit of AMPA receptors. The interaction requires all three domains of NSF but occurs between residues Lys-844 and Gln-853 of rat GluR2, with Asn-851 playing a critical role. Loading of decapeptides corresponding to the NSF-binding domain of GluR2 into rat hippocampal CA1 pyramidal neurons results in a marked, progressive decrement of AMPA receptor-mediated synaptic transmission. This reduction in synaptic transmission was also observed when an anti-NSF monoclonal antibody (mAb) was loaded into CA1 neurons. These results demonstrate a previously unsuspected direct interaction in the postsynaptic neuron between two major proteins involved in synaptic transmission and suggest a rapid NSF-dependent modulation of AMPA receptor function.

The role of the GluR2 subunit in AMPA receptor function and synaptic plasticity.

The AMPA receptor (AMPAR) GluR2 subunit dictates the critical biophysical properties of the receptor, strongly influences receptor assembly and trafficking, and plays pivotal roles in a number of forms of long-term synaptic plasticity. Most neuronal AMPARs contain this critical subunit; however, in certain restricted neuronal populations and under certain physiological or pathological conditions, AMPARs that lack this subunit are expressed. There is a current surge of interest in such GluR2-lacking Ca2+-permeable AMPARs in how they affect the regulation of synaptic transmission. Here, we bring together recent data highlighting the novel and important roles of GluR2 in synaptic function and plasticity.

LTP Inhibits LTD in the Hippocampus via Regulation of GSK3β

Glycogen synthase kinase-3 (GSK3) has been implicated in major neurological disorders, but its role in normal neuronal function is largely unknown. Here we show that GSK3beta mediates an interaction between two major forms of synaptic plasticity in the brain, N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) and NMDA receptor-dependent long-term depression (LTD). In rat hippocampal slices, GSK3beta inhibitors block the induction of LTD. Furthermore, the activity of GSK3beta is enhanced during LTD via activation of PP1. Conversely, following the induction of LTP, there is inhibition of GSK3beta activity. This regulation of GSK3beta during LTP involves activation of NMDA receptors and the PI3K-Akt pathway and disrupts the ability of synapses to undergo LTD for up to 1 hr. We conclude that the regulation of GSK3beta activity provides a powerful mechanism to preserve information encoded during LTP from erasure by subsequent LTD, perhaps thereby permitting the initial consolidation of learnt information.

Modulation of AMPA receptor unitary conductance by synaptic activity

Activity-dependent alteration in synaptic strength is a fundamental property of the vertebrate central nervous system and is thought to underlie learning and memory. The most extensively studied model of activity-dependent synaptic plasticity is long-term potentiation (LTP) of glutamate-responsive (glutamatergic) synapses, a widespread phenomenon involving multiple mechanisms. The best characterized form of LTP occurs in the CA1 region of the hippocampus, in which LTP is initiated by transient activation of NMDA (N-methyl-D-aspartate) receptors and is expressed as a persistent increase in synaptic transmission through AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptors. This increase is due, at least in part, to a postsynaptic modification of AMPA-receptor function; this modification could be caused by an increase in the number of receptors, their open probability, their kinetics or their single-channel conductance. Here we show that the induction of LTP in the CA1 region of the hippocampus is often associated with an increase in single-channel conductance of AMPA receptors. This shows that elementary channel properties can be rapidly modified by synaptic activity and provides an insight into one molecular mechanism by which glutamatergic synapses can alter their strength.

Silent Synapses during Development of Thalamocortical Inputs

During development, activity-dependent mechanisms are thought to contribute to the refinement of topographical projections from the thalamus to the cortex. Because activity-dependent increases in synaptic strength may contribute to the stabilization of synaptic connections, we have explored the mechanisms of long-term potentiation (LTP) at thalamocortical synapses in rat somatosensory (barrel) cortex. During early postnatal development (postnatal days 2-5), we find that a significant proportion of thalamocortical synapses are functionally silent and that these are converted to functional synapses during LTP. Silent synapses disappear by postnatal day 8-9, the exact time at which the susceptibility of these synapses to LTP is lost. These findings suggest that the activity-dependent conversion of silent to functional synapses due to correlated pre- and postsynaptic activity may contribute to the early development and refinement of thalamocortical inputs to cortex.

Transient incorporation of native GluR2-lacking AMPA receptors during hippocampal long-term potentiation

Postnatal glutamatergic principal neuron synapses are typically presumed to express only calcium-impermeable (CI), GluR2-containing AMPARs under physiological conditions. Here, however, we demonstrate that long-term potentiation (LTP) in CA1 hippocampal pyramidal neurons causes rapid incorporation of GluR2-lacking calcium-permeable (CP)-AMPARs: CP-AMPARs are present transiently, being replaced by GluR2-containing AMPARs ∼25 min after LTP induction. Thus, CP-AMPARs are physiologically expressed at CA1 pyramidal cell synapses during LTP, and may be required for LTP consolidation.

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.

Developmental and activity- dependent regulation of kainate receptors at thalamocortical synapses

Most of the fast excitatory synaptic transmission in the mammalian brain is mediated by ionotrophic glutamate receptors, of which there are three subtypes: AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate), NMDA (N -methyl-D-aspartate) and kainate. Although kainate-receptor subunits (GluR5–7, KA1 and 2) are widely expressed in the mammalian central nervous system,, little is known about their function. The development of pharmacological agents that distinguish between AMPA and kainate receptors has now allowed the functions of kainate receptors to be investigated,. The modulation of synaptic transmission by kainate receptors,, and their synaptic activation,,,,,, in a variety of brain regions have been reported. The expression of kainate receptor subunits is developmentally regulated, but their role in plasticity and development is unknown. Here we show that developing thalamocortical synapses express postsynaptic kainate receptors as well as AMPA receptors; however, the two receptor subtypes do not co-localize. During the critical period for experience-dependent plasticity, the kainate-receptor contribution to transmission decreases; a similar decrease occurs when long-term potentiation is induced in vitro. This indicates that during development there is activity-dependent regulation of the expression of kainate receptors at thalamocortical synapses.