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Cellular and circuit hyperexcitability are core features of fragile X syndrome and related autism spectrum disorder models. However, the cellular and synaptic bases of this hyperexcitability have proved elusive. We report in a mouse model of fragile X syndrome, glutamate uncaging onto individual dendritic spines yields stronger single-spine excitation than wild-type, with more silent spines. Furthermore, fewer spines are required to trigger an action potential with near-simultaneous uncaging at multiple spines. This is, in part, from increased dendritic gain due to increased intrinsic excitability, resulting from reduced hyperpolarization-activated currents, and increased NMDA receptor signaling. Using super-resolution microscopy we detect no change in dendritic spine morphology, indicating no structure-function relationship at this age. However, ultrastructural analysis shows a 3-fold increase in multiply-innervated spines, accounting for the increased single-spine glutamate currents. Thus, loss of FMRP causes abnormal synaptogenesis, leading to large numbers of poly-synaptic spines despite normal spine morphology, thus explaining the synaptic perturbations underlying circuit hyperexcitability., Fragile X syndrome and autism spectrum disorders are associated with circuit hyperexcitability, however, its cellular and synaptic bases are not well understood. Here, the authors report abnormal synaptogenesis with an increased prevalence of polysynaptic spines with normal morphology in a mouse model of fragile X.

Sensory hypersensitivity is a common and debilitating feature of neurodevelopmental disorders such as Fragile X Syndrome (FXS). How developmental changes in neuronal function culminate in network dysfunction that underlies sensory hypersensitivities is unknown. By systematically studying cellular and synaptic properties of layer 4 neurons combined with cellular and network simulations, we explored how the array of phenotypes in Fmr1-knockout (KO) mice produce circuit pathology during development. We show that many of the cellular and synaptic pathologies in Fmr1-KO mice are antagonistic, mitigating circuit dysfunction, and hence may be compensatory to the primary pathology. Overall, the layer 4 network in the Fmr1-KO exhibits significant alterations in spike output in response to thalamocortical input and distorted sensory encoding. This developmental loss of layer 4 sensory encoding precision would contribute to subsequent developmental alterations in layer 4-to-layer 2/3 connectivity and plasticity observed in Fmr1-KO mice, and circuit dysfunction underlying sensory hypersensitivity., Somatosensory hypersensitivity in Fmr-1 knockout mice is thought to arise from an increase in cortical circuit excitability. Here, the authors report that the loss of precision of sensory encoding in the Layer 4 of barrel cortex is the primary developmental circuit alteration that drives the other compensatory circuit dysfunction.

Significance The corpus callosum is a large fiber bundle which connects contralateral brain regions. After unilateral perturbations such as stroke or amputation, interhemispheric connectivity is altered and often leads to bilateral somatomotor cortical hyperactivity in patients with poor recovery. This study reports that callosal targeting of deprived layer 5 neurons is maximally potentiated in mouse primary somatosensory barrel cortex after unilateral whisker denervation. These neurons also experience an increase in excitability and spontaneous excitatory amplitudes. These results should be relevant to the cortical responses observed in human patients after unilateral nerve transection, amputation, or stroke., Central or peripheral injury causes reorganization of the brain’s connections and functions. A striking change observed after unilateral stroke or amputation is a recruitment of bilateral cortical responses to sensation or movement of the unaffected peripheral area. The mechanisms underlying this phenomenon are described in a mouse model of unilateral whisker deprivation. Stimulation of intact whiskers yields a bilateral blood-oxygen-level−dependent fMRI response in somatosensory barrel cortex. Whole-cell electrophysiology demonstrated that the intact barrel cortex selectively strengthens callosal synapses to layer 5 neurons in the deprived cortex. These synapses have larger AMPA receptor- and NMDA receptor-mediated events. These factors contribute to a maximally potentiated callosal synapse. This potentiation occludes long-term potentiation, which could be rescued, to some extent, with prior long-term depression induction. Excitability and excitation/inhibition balance were altered in a manner consistent with cell-specific callosal changes and support a shift in the overall state of the cortex. This is a demonstration of a cell-specific, synaptic mechanism underlying interhemispheric cortical reorganization.

Summary Synapse loss is a key feature of dementia, but it is unclear whether synaptic dysfunction precedes degenerative phases of the disease. Here, we show that even before any decrease in synapse density, there is abnormal turnover of cortical axonal boutons and dendritic spines in a mouse model of tauopathy-associated dementia. Strikingly, tauopathy drives a mismatch in synapse turnover; postsynaptic spines turn over more rapidly, whereas presynaptic boutons are stabilized. This imbalance between pre- and post-synaptic stability coincides with reduced synaptically driven neuronal activity in pre-degenerative stages of the disease., Graphical Abstract, Highlights • Density of cortical axonal boutons and dendritic spines is reduced early in tauopathy • Abnormalities in synaptic stability and size exist before decreases in synapse density • Turnover of dendritic spines is elevated, whereas presynaptic boutons are stabilized • Neuronal activity is reduced at stages associated with mismatched synaptic turnover, Using in vivo two-photon imaging in the rTg4510 tauopathy mouse model, Jackson et al. find that synapse stability is altered during the pre-degenerative stages of tauopathy. Mismatched abnormalities in pre- and post-synaptic turnover coincide with disrupted neuronal activity.

SummaryExperience-dependent plasticity in the adult brain has clinical potential for functional rehabilitation following central and peripheral nerve injuries. Here, plasticity induced by unilateral infraorbital (IO) nerve resection in 4-week-old rats was mapped using MRI and synaptic mechanisms were elucidated by slice electrophysiology. Functional MRI demonstrates a cortical potentiation compared to thalamus 2 weeks after IO nerve resection. Tracing thalamocortical (TC) projections with manganese-enhanced MRI revealed circuit changes in the spared layer 4 (L4) barrel cortex. Brain slice electrophysiology revealed TC input strengthening onto L4 stellate cells due to an increase in postsynaptic strength and the number of functional synapses. This work shows that the TC input is a site for robust plasticity after the end of the previously defined critical period for this input. Thus, TC inputs may represent a major site for adult plasticity in contrast to the consensus that adult plasticity mainly occurs at cortico-cortical connections.

Feedforward GABAergic inhibition sets the dendritic integration window, thereby controlling timing and output in cortical circuits. However, the manner in which feedforward inhibitory circuits emerge is unclear, despite this being a critical step for neocortical development and function. We found that sensory experience drove plasticity of the feedforward inhibitory circuit in mouse layer 4 somatosensory barrel cortex in the second postnatal week via two distinct mechanisms. First, sensory experience selectively strengthened thalamocortical-to-feedforward interneuron inputs via a presynaptic mechanism but did not regulate other inhibitory circuit components. Second, experience drove a postsynaptic mechanism in which a downregulation of a prominent thalamocortical NMDA excitatory postsynaptic potential in stellate cells regulated the final expression of functional feedforward inhibitory input. Thus, experience is required for specific, coordinated changes at thalamocortical synapses onto both inhibitory and excitatory neurons, producing a circuit plasticity that results in maturation of functional feedforward inhibition in layer 4.

Regulated exocytosis is essential for many biological processes, and many components of the protein trafficking machinery are ubiquitous. However, there are also exceptions such as SNAP-25, a neuron-specific SNARE protein, which is essential for synaptic vesicle release from presynaptic nerve terminals. In contrast, SNAP-23 is the ubiquitously-expressed SNAP-25 homologue that is critical for regulated exocytosis in non-neuronal cells. However, the role of SNAP-23 in neurons has not been elucidated. We now find that SNAP-23 is enriched in dendritic spines and colocalizes with constituents of the postsynaptic density, whereas SNAP-25 is restricted to axons. In addition, loss of SNAP-23 using genetically-altered mice or shRNA targeted to SNAP-23 leads to a dramatic decrease in NMDA receptor surface expression and NMDA receptor currents, whereas loss of SNAP-25 does not. Therefore SNAP-23 plays a unique role in the functional regulation of postsynaptic glutamate receptors.

In the hippocampus, synaptic strength between pyramidal cells is modifiable by NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) and long-term depression (LTD), both of which require coincident presynaptic and postsynaptic activity.In vivo, many pyramidal cells exhibit location-specific activity patterns and are known as “place cells.” The combination of these factors suggests that synaptic plasticity will be induced at synapses connecting place cells with overlapping firing fields, because such cells fire coincidentally when the rat is in a specific part of the environment. However, this prediction, which is important for models of how long-term synaptic plasticity can be used to encode space in the hippocampal network, has not been tested. To investigate this, action potential time series recorded simultaneously from place cells in freely moving rats were replayed concurrently into postsynaptic CA1 pyramidal cells and presynaptic inputs during perforated patch-clamp recordings from adult hippocampal slices. Place cell firing patterns induced large, pathway-specific, NMDAR-dependent LTP that was rapidly expressed within a few minutes. However, place-cell LTP was induced only if the two place cells had overlapping firing fields and if the cholinergic tone present in the hippocampus during exploration was restored by bath application of the cholinergic agonist carbachol. LTD was never observed in response to place cell firing patterns. Our findings demonstrate that spike patterns from hippocampal place cells can robustly induce NMDAR-dependent LTP, providing important evidence in support of a model in which spatial distance is encoded as the strength of synaptic connections between place cells.

SummaryPICK1 is a calcium-sensing, PDZ domain-containing protein that interacts with GluR2 and GluR3 AMPA receptor (AMPAR) subunits and regulates their trafficking. Although PICK1 has been principally implicated in long-term depression (LTD), PICK1 overexpression in CA1 pyramidal neurons causes a CaMK- and PKC-dependent potentiation of AMPAR-mediated transmission and an increase in synaptic GluR2-lacking AMPARs, mechanisms associated with NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here, we directly tested whether PICK1 participates in both hippocampal NMDAR-dependent LTP and LTD. We show that the PICK1 potentiation of AMPAR-mediated transmission is NMDAR dependent and fully occludes LTP. Conversely, blockade of PICK1 PDZ interactions or lack of PICK1 prevents LTP. These observations demonstrate an important role for PICK1 in LTP. In addition, deletion of PICK1 or blockade of PICK1 PDZ binding prevented NMDAR-dependent LTD. Thus, PICK1 plays a critical role in bidirectional NMDAR-dependent long-term synaptic plasticity in the hippocampus.

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.

Long-term synaptic plasticity is an important mechanism underlying the development of cortical circuits in a number of brain regions. In barrel cortex NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) and long-term depression (LTD) play a critical role in the development and experience-dependent plasticity of the topographical map of the rodent whiskers. However, the mechanisms underlying the induction and expression of these forms of plasticity are poorly characterised. Here we investigate the role of PKC in the regulation of synaptic strength in neonatal barrel cortex using patch-clamp recordings in brain slices. We demonstrate that PKC activity tonically maintains AMPA receptor-mediated transmission at thalamocortical synapses, and that basal transmission can be potentiated by PKC activation using postsynaptic infusion of phorbol ester. Furthermore, we show that induction of NMDAR-dependent LTP requires PKC activity. These findings demonstrate that PKC is required for the regulation of transmission at thalamocortical synapses, the major ascending sensory input to barrel cortex. Thalamocortical inputs in barrel cortex only express LTP during the first postnatal week during a critical period for experience-dependent plasticity in layer IV. Therefore, the requirement for PKC in LTP suggests an important role for this kinase in the development of the barrel cortex sensory map.

Kainate receptors (KARs) have been shown to be involved in hippocampal mossy fiber long-term potentiation (LTP); however, it is not known if KARs are involved in the induction or expression of long-term depression (LTD), the other major form of long-term synaptic plasticity. Here we describe LTD of KAR-mediated synaptic transmission (EPSC(KA) LTD) in perirhinal cortex layer II/III neurons that is distinct from LTD of AMPAR-mediated transmission, which also coexists at the same synapses. Induction of EPSC(KA) LTD requires a rise in postsynaptic Ca(2+) but is independent of NMDARs or T-type voltage-gated Ca(2+) channels; however, it requires synaptic activation of inwardly rectifying KARs and release of Ca(2+) from stores. The synaptic KARs are regulated by tonically activated mGluR5, and expression of EPSC(KA) LTD occurs via a mechanism involving mGluR5, PKC, and PICK1 PDZ domain interactions. Thus, we describe the induction and expression mechanism of a form of synaptic plasticity, EPSC(KA) LTD.

ATP hydrolysis is critical for many cellular processes; however, the acute requirement for ATP hydrolysis in synaptic transmission and plasticity in neurons is unknown. Here we studied the effects of postsynaptically applying the non-hydrolyzable ATP analogue adenosine 5'-[beta,gamma-methylene]triphosphate (AMP-PCP) into hippocampal CA1 pyramidal cells in hippocampal slices. The effects of this manipulation were investigated on basal transmission and on two forms of long-term synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD). AMP-PCP caused an increase in basal AMPA receptor (AMPAR)-mediated transmission, which occurred rapidly within minutes of infusing the drug. This effect was selective for AMPARs, since pharmacologically isolated NMDAR-mediated synaptic currents did not exhibit this run up. In two-pathway experiments infusion of AMP-PCP blocked the induction of both LTD and LTP. These findings show an acute and selective role for ATP hydrolysis in regulating AMPAR function both during basal transmission and long-term synaptic plasticity. Recent evidence indicates that AMPARs are selectively and acutely regulated by the ATPase N-ethylmaleimide-sensitive factor (NSF), which forms part of a multi-protein complex with AMPARs. Our data are consistent with the idea that such a mechanism that can acutely bi-directionally regulate AMPAR function at synapses and requires ATP hydrolysis is necessary for rapid activity-dependent changes in synaptic strength.

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 (gamma-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.

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.

There is intense interest in understanding the molecular mechanisms involved in long-term potentiation (LTP) in the hippocampus. Significant progress in our understanding of LTP has followed from studies of glutamate receptors, of which there are four main subtypes ( α -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), N -methyl-D-aspartate (NMDA), mGlu and kainate). This article summarizes the evidence that the kainate subtype of glutamate receptor is an important trigger for the induction of LTP at mossy fibre synapses in the CA3 region of the hippocampus. The pharmacology of the first selective kainate receptor antagonists, in particular the GLU K5 subunit selective antagonist LY382884, is described. LY382884 selectively blocks the induction of mossy fibre LTP, in response to a variety of different high-frequency stimulation protocols. This antagonist also inhibits the pronounced synaptic facilitation of mossy fibre transmission that occurs during high-frequency stimulation. These effects are attributed to the presence of presynaptic GLU K5 -subunit-containing kainate receptors at mossy fibre synapses. Differences in kainate receptor-dependent synaptic facilitation of AMPA and NMDA receptor-mediated synaptic transmission are described. These data are discussed in the context of earlier reports that glutamate receptors are not involved in mossy fibre LTP and more recent experiments using kainate receptor knockout mice, that argue for the involvement of GLU K6 but not GLU K5 kainate receptor subunits. We conclude that activation of presynaptic GLU K5 -containing kainate receptors is an important trigger for the induction of mossy fibre LTP in the hippocampus.

Glutamate receptors mediate the majority of excitatory responses in the central nervous system. The establishment and refinement of glutamatergic synaptic connections depend on the concerted actions of a-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA),N-methyl-D-aspartate (NMDA), and kainate (KA) type ionotropic glutamate receptors (iGluRs) and G-protein coupled metabotropic receptors. While a lot remains to be clarified, the most is known about the mechanisms by which the iGluR subtypes are targeted and how this is influenced by synaptic activity on both short and long time scales. Changes in their subunit compositions are also input specific and developmentally regulated. The identification of key molecular components of the postsynaptic density (PSD) and novel proteins that influence receptor targeting and clustering have started to reveal the underlying molecular mechanisms of the trafficking and targeting of iGluRs. Here we discuss the evidence that these basic mechanisms are used during developmental synaptic plasticity.

The main input to primary sensory cortex is via thalamocortical (TC) axons that form the greatest number of synapses in layer 4, but also synapse onto neurons in layer 6. The development of the TC input to layer 4 has been widely studied, but less is known about the development of the layer 6 input. Here, we show that, in neonates, the input to layer 6 is as strong as that to layer 4. Throughout the first postnatal week, there is an experience-dependent strengthening specific to layer 4, which correlates with the ability of synapses in layer 4, but not in layer 6, to undergo long-term potentiation (LTP). This strengthening consists of an increase in axon branching and the divergence of connectivity in layer 4 without a change in the strength of individual connections. We propose that experience-driven LTP stabilizes transient TC synapses in layer 4 to increase strength and divergence specifically in layer 4 over layer 6.

The development of GluR5-selective kainate receptor ligands is helping to elucidate the functions of kainate receptors in the CNS. Here we have further characterised the actions of a GluR5 selective agonist, ATPA, and a GluR5 selective antagonist, LY382884, in the CA3 region of rat hippocampal slices. In addition, we have used LY382884 to study a novel synaptic mechanism. This antagonist substantially reduces frequency facilitation of mossy fibre synaptic transmission, monitored as either AMPA or NMDA receptor-mediated EPSCs. This suggests that GluR5-containing kainate receptors on mossy fibres function as autoreceptors to facilitate the synaptic release of L-glutamate, in a frequency-dependent manner.

The mechanisms involved in mossy fiber LTP in the hippocampus are not well established. In the present study, we show that the kainate receptor antagonist LY382884 (10 μM) is selective for presynaptic kainate receptors in the CA3 region of the hippocampus. At a concentration at which it blocks mossy fiber LTP, LY382884 selectively blocks the synaptic activation of a presynaptic kainate receptor that facilitates AMPA receptor-mediated synaptic transmission. Following the induction of mossy fiber LTP, there is a complete loss of the presynaptic kainate receptor-mediated facilitation of synaptic transmission. These results identify a central role for the presynaptic kainate receptor in the induction of mossy fiber LTP. In addition, these results suggest that the pathway by which kainate receptors facilitate glutamate release is utilized for the expression of mossy fiber LTP.

Sensory experience during an early critical period guides the development of thalamocortical circuits in many cortical areas. This process has been hypothesized to involve long-term potentiation (LTP) and long-term depression (LTD) at thalamocortical synapses. Here, we show that thalamocortical synapses in rat barrel cortex can express LTD, and that LTD is most readily induced during a developmental period that is similar to the critical period for thalamocortical plasticity in vivo. Thalamocortical LTD is homosynaptic and dependent on activation of N-methyl-D-aspartate (NMDA) receptors. The age-related decline of LTD is not due to changes in inhibition nor to changes in NMDA receptor voltage dependence. Minimal stimulation experiments indicate that, unlike thalamocortical LTP, thalamocortical LTD is not associated with a significant change in failure rate. The existence of LTD and its developmental time course suggest that LTD, like LTP, may contribute to the refinement of thalamocortical inputs in vivo.

Despite decades of study, the mechanisms by which synapses express the increase in strength during long-term potentiation (LTP) remain an area of intense interest. Here, we have studied how AMPA receptor subunit composition changes during the early phases of hippocampal LTP in CA1 pyramidal neurons. We studied LTP at silent synapses that initially lack AMPA receptors, but contain NMDA receptors. We show that strongly inwardly rectifying AMPA receptors are initially incorporated at silent synapses during LTP and are then subsequently replaced by non-rectifying AMPA receptors. These findings suggest that silent synapses initially incorporate GluA2-lacking, calcium-permeable AMPA receptors during LTP that are then replaced by GluA2-containing calcium-impermeable receptors. We also show that LTP consolidation at CA1 synapses requires a rise in intracellular calcium concentration during the early phase of expression, indicating that calcium influx through the GluA2-lacking AMPA receptors drives their replacement by GluA2-containing receptors during LTP consolidation. Taken together with previous studies in hippocampus and in other brain regions, these findings suggest that a common mechanism for the expression of activity-dependent glutamatergic synaptic plasticity involves the regulation of GluA2-subunit composition and highlights a critical role for silent synapses in this process.

The subcellular locations of synapses on pyramidal neurons strongly influences dendritic integration and synaptic plasticity. Despite this, there is little quantitative data on spatial distributions of specific types of synaptic input. Here we use array tomography (AT), a high-resolution optical microscopy method, to examine thalamocortical (TC) input onto layer 5 pyramidal neurons. We first verified the ability of AT to identify synapses using parallel electron microscopic analysis of TC synapses in layer 4. We then use large-scale array tomography (LSAT) to measure TC synapse distribution on L5 pyramidal neurons in a 1.00 × 0.83 × 0.21 mm(3) volume of mouse somatosensory cortex. We found that TC synapses primarily target basal dendrites in layer 5, but also make a considerable input to proximal apical dendrites in L4, consistent with previous work. Our analysis further suggests that TC inputs are biased toward certain branches and, within branches, synapses show significant clustering with an excess of TC synapse nearest neighbors within 5-15 μm compared to a random distribution. Thus, we show that AT is a sensitive and quantitative method to map specific types of synaptic input on the dendrites of entire neurons. We anticipate that this technique will be of wide utility for mapping functionally-relevant anatomical connectivity in neural circuits.

Despite extensive investigation, it remains unclear whether presynaptic and/or postsynaptic modifications are primarily responsible for the expression of long-term potentiation (LTP) in the CA1 region of the hippocampus. Here we address this issue by using techniques that maximize the likelihood of stimulating a single axon and thereby presumably a single synapse before and after the induction of LTP. Several basic properties of synaptic transmission were examined including the probability of neurotransmitter release (Pr), the quantal size (q), and the so-called potency, which is defined as the average size of the synaptic response when release of transmitter does occur. LTP was routinely associated with an increase in potency, whereas increases in Pr alone were not observed. LTP was also reliably induced when baseline Pr was high, indicating that synapses with high Pr can express LTP. These results suggest that the mechanism for the expression of LTP involves an increase in q and is difficult to explain by an increase in Pr alone.

In cerebral cortex there is a developmental switch from NR2B- to NR2A-containing NMDA receptors (NMDARs) driven by activity and sensory experience. This subunit switch alters NMDAR function, influences synaptic plasticity, and its dysregulation is associated with neurological disorders. However, the mechanisms driving the subunit switch are not known. Here, we show in hippocampal CA1 pyramidal neurons that the NR2B to NR2A switch driven acutely by activity requires activation of NMDARs and mGluR5, involves PLC, Ca2+ release from IP3R-dependent stores, and PKC activity. In mGluR5 knockout mice the developmental NR2B-NR2A switch in CA1 is deficient. Moreover, in visual cortex of mGluR5 knockout mice, the NR2B-NR2A switch evoked in vivo by visual experience is absent. Thus, we establish that mGluR5 and NMDARs are required for the activity-dependent NR2B-NR2A switch and play a critical role in experience-dependent regulation of NMDAR subunit composition in vivo. In cerebral cortex there is a developmental switch from NR2B- to NR2A-containing NMDA receptors (NMDARs) driven by activity and sensory experience. This subunit switch alters NMDAR function, influences synaptic plasticity, and its dysregulation is associated with neurological disorders. However, the mechanisms driving the subunit switch are not known. Here, we show in hippocampal CA1 pyramidal neurons that the NR2B to NR2A switch driven acutely by activity requires activation of NMDARs and mGluR5, involves PLC, Ca2+ release from IP3R-dependent stores, and PKC activity. In mGluR5 knockout mice the developmental NR2B-NR2A switch in CA1 is deficient. Moreover, in visual cortex of mGluR5 knockout mice, the NR2B-NR2A switch evoked in vivo by visual experience is absent. Thus, we establish that mGluR5 and NMDARs are required for the activity-dependent NR2B-NR2A switch and play a critical role in experience-dependent regulation of NMDAR subunit composition in vivo.

SummaryN-methyl-D-aspartate (NMDA) receptors (NMDARs) play a central role in development, synaptic plasticity, and neurological disease. NMDAR subunit composition defines their biophysical properties and downstream signaling. Casein kinase 2 (CK2) phosphorylates the NR2B subunit within its PDZ-binding domain; however, the consequences for NMDAR localization and function are unclear. Here we show that CK2 phosphorylation of NR2B regulates synaptic NR2B and NR2A in response to activity. We find that CK2 phosphorylates NR2B, but not NR2A, to drive NR2B-endocytosis and remove NR2B from synapses resulting in an increase in synaptic NR2A expression. During development there is an activity-dependent switch from NR2B to NR2A at cortical synapses. We observe an increase in CK2 expression and NR2B phosphorylation over this same critical period and show that the acute activity-dependent switch in NR2 subunit composition at developing hippocampal synapses requires CK2 activity. Thus, CK2 plays a central role in determining the NR2 subunit content of synaptic NMDARs.

Trafficking of AMPA receptors (AMPARs) is important for many forms of synaptic plasticity. However, the link between activity and resulting synaptic alterations is not fully understood. We identified a direct interaction between N-ethylmaleimide-sensitive fusion protein (NSF), an ATPase involved in membrane fusion events and stabilization of surface AMPARs, and Polo-like kinase- 2 (Plk2), an activity-inducible kinase that homeostatically decreases excitatory synapse number and strength. Plk2 disrupted the interaction of NSF with the GluA2 subunit of AMPARs, promoting extensive loss of surface GluA2 in rat hippocampal neurons, greater association of GluA2 with adaptor proteins PICK1 and GRIP1, and decreased synaptic AMPAR current. Plk2 engagement of NSF, but not Plk2 kinase activity, was required for this mechanism and occurred through a motif in the Plk2 protein that was independent of the canonical polo box interaction sites. These data reveal that heightened synaptic activity, acting through Plk2, leads to homeostatic decreases in surface AMPAR expression via the direct dissociation of NSF from GluA2.

The thalamocortical (TC) input to layer IV provides the major pathway for ascending sensory information to the mammalian sensory cortex. During development there is a dramatic refinement of this input that underlies the maturation of the topographical map in layer IV. Over the last ten years our understanding of the mechanisms of the developmental and experience-driven changes in synaptic function at TC synapses has been greatly advanced. Here we describe these studies that point to a key role for NMDA receptor-dependent synaptic plasticity, a role for kainate receptors and for a rapid maturation in GABAergic inhibition. The expression mechanisms of some of the forms of neonatal synaptic plasticity are novel and, in combination with other mechanisms, produce a layer IV circuit that exhibits functional properties necessary for mature sensory processing.

During the first week of life, there is a shift from kainate to AMPA receptor-mediated thalamocortical transmission in layer IV barrel cortex. However, the mechanisms underlying this change and the differential properties of AMPA and kainate receptor-mediated transmission remain essentially unexplored. To investigate this, we studied the quantal properties of AMPA and kainate receptor-mediated transmission using strontium-evoked miniature EPSCs. AMPA and kainate receptor-mediated transmission exhibited very different quantal properties but were never coactivated by a single quantum of transmitter, indicating complete segregation to different synapses within the thalamocortical input. Nonstationary fluctuation analysis showed that synaptic AMPA receptors exhibited a range of single-channel conductance (γ) and a strong negative correlation between γ and functional channel number, indicating that these two parameters are reciprocally regulated at thalamocortical synapses. We obtained the first estimate of γ for synaptic kainate receptors (

The glutamate receptor subtypes AMPA and kainate are involved in synaptic transmission and synaptic plasticity in the CNS. Recently there has been considerable interest in understanding the molecular regulation of these receptors by proteins that directly bind to AMPA and kainate receptor subunits. Amongst the first interaction partners to be discovered were NSF, ABP, GRIP and PICK1, which bind the AMPA receptor subunit GLUA2. We have studied the functional roles of the interactions of these proteins in regulating AMPA receptor-mediated synaptic transmission and synaptic plasticity in the hippocampus. We have also started to investigate the functions of PICK1 and GRIP on kainate receptor-mediated synaptic transmission in this region. In this article we reflect upon this work, which has led to some new ideas about how AMPA and kainate receptors are regulated at synapses.

We identified four PDZ domain-containing proteins, syntenin, PICK1, GRIP, and PSD95, as interactors with the kainate receptor (KAR) subunits GluR5(2b,) GluR5(2c), and GluR6. Of these, we show that both GRIP and PICK1 interactions are required to maintain KAR-mediated synaptic function at mossy fiber-CA3 synapses. In addition, PKC alpha can phosphorylate ct-GluR5(2b) at residues S880 and S886, and PKC activity is required to maintain KAR-mediated synaptic responses. We propose that PICK1 targets PKC alpha to phosphorylate KARs, causing their stabilization at the synapse by an interaction with GRIP. Importantly, this mechanism is not involved in the constitutive recycling of AMPA receptors since blockade of PDZ interactions can simultaneously increase AMPAR- and decrease KAR-mediated synaptic transmission at the same population of synapses.

1. The molecular properties of synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors are an important factor determining excitatory synaptic transmission in the brain. Changes in the number (N) or single-channel conductance (gamma) of functional AMPA receptors may underlie synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD). These parameters have been estimated using non-stationary fluctuation analysis (NSFA). 2. The validity of NSFA for studying the channel properties of synaptic AMPA receptors was assessed using a cable model with dendritic spines and a microscopic kinetic description of AMPA receptors. Electrotonic, geometric and kinetic parameters were altered in order to determine their effects on estimates of the underlying gamma. 3. Estimates of gamma were very sensitive to the access resistance of the recording (R(A)) and the mean open time of AMPA channels. Estimates of gamma were less sensitive to the distance between the electrode and the synaptic site, the electrotonic properties of dendritic structures, recording electrode capacitance and background noise. Estimates of gamma were insensitive to changes in spine morphology, synaptic glutamate concentration and the peak open probability (P(o)) of AMPA receptors. 4. The results obtained using the model agree with biological data, obtained from 91 dendritic recordings from rat CA1 pyramidal cells. A correlation analysis showed that R(A) resulted in a slowing of the decay time constant of excitatory postsynaptic currents (EPSCs) by approximately 150 %, from an estimated value of 3.1 ms. R(A) also greatly attenuated the absolute estimate of gamma by approximately 50-70 %. 5. When other parameters remain constant, the model demonstrates that NSFA of dendritic recordings can readily discriminate between changes in gamma vs. changes in N or P(o). Neither background noise nor asynchronous activation of multiple synapses prevented reliable discrimination between changes in gamma and changes in either N or P(o). 6. The model (available online) can be used to predict how changes in the different properties of AMPA receptors may influence synaptic transmission and plasticity.

Kainate (KA) receptor-mediated excitatory postsynaptic currents (EPSCs) exhibit slow kinetics at the great majority of synapses. However, native or heterologously expressed KA receptors exhibit rapid kinetics in response to agonist application. One possibility to explain this discrepancy is that KA receptors are extrasynaptic and sense glutamate diffusing from the synaptic cleft. We investigated this by studying the effect of three manipulations that change glutamate clearance on evoked KA EPSCs at thalamocortical synapses. First, we used high-frequency stimulation to increase extrasynaptic glutamate levels. This caused an apparent increase in the relative contribution of the KA EPSC to transmission and slowed the decay kinetics. However, scaling and summing the EPSC evoked at low frequency reproduced this, demonstrating that the effect was due to postsynaptic summation of KA EPSCs. Second, we applied inhibitors of high-affinity glutamate transport. This caused a depression in both AMPA and KA EPSC amplitude due to the activation of a presynaptic glutamatergic autoreceptor. However, transport inhibitors had no selective effect on the amplitude or kinetics of the KA EPSC. Third, to increase glutamate clearance, we raised temperature during recordings. This shortened the decay of both the AMPA and KA components and increased their amplitudes, but this effect was the same for both. Therefore these data provide evidence against glutamate diffusion out of the synaptic cleft as the mechanism for the slow kinetics of KA EPSCs. Other possibilities such as interactions of KA receptors with other proteins or novel properties of native synaptic heteromeric receptors are required to explain the slow kinetics.

Synaptic plasticity is the process by which the brain alters the strength of its synaptic connections, a fundamental function of the brain that enables individuals to learn from experience. The study of synaptic plasticity often involves the application of standard in vitro electrophysiological techniques to hippocampal slice preparations. This unit discusses many of the special considerations that are applicable for the optimal study of synaptic plasticity in this system. Most of these principles also apply to the study of synaptic plasticity in other brain slice preparations. Curr. Protoc. Neurosci. 54:6.13.1-6.13.26. © 2011 by John Wiley & Sons, Inc. Keywords: LTP; LTD; hippocampus; synaptic plasticity; glutamate receptors; organotypic slice; WinLTP

Most of the fast excitatory synaptic transmission in the mammalian brain is mediated by ionotrophic glutamate receptors, of which there are three subtypes: AMPA (alpha-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 colocalize. 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.

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 (alpha-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.

We have taken a number of different experimental approaches to address whether long-term potentiation (LTP) in hippocampal CA1 pyramidal cells is due primarily to presynaptic or postsynaptic modifications. Examination of miniature EPSCs or EPSCs evoked using minimal stimulation indicate that quantal size increasing during LTP. The conversion of silent to functional synapses may contribute to the LTP-induced changes in mEPSC frequency and failure rate that previously have been attributed to an increase in the probability if transmitter release.

Summary Recent work has suggested that some proportion of excitatory synapses on hippocampal CA1 pyramidal cells that express NMOA receptors (NMDARa) may not express functional AMPA receptors (AMPARs), thus making these synapses silent at the resting membrane potential. In agreement with this hypothesis, we demonstrete here that it is pQssible 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 NMOAR 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.

To investigate the role of phosphatidylinositol 3 kinase (PI3 kinase) in hippocampal synaptic plasticity, we used whole-cell patch clamp recordings from rat CA1 neurons to determine the effects of PI3 kinase inhibitors on long-term depression (LTD). PI3 kinase blockade caused a loss of synapse specificity of LTD that was dependent on the co-activation of NMDA-type glutamate receptors (NMDARs) and metabotropic glutamate receptors (mGluRs), and involved release of Ca(2+) from intracellular stores. These findings suggest that the synapse specificity of hippocampal LTD may not be an intrinsic property of this form of homosynaptic plasticity, but rather that it can be regulated by PI3 kinase.