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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.

Injury induces synaptic, circuit, and systems reorganization. After unilateral amputation or stroke, this functional loss disrupts the interhemispheric interaction between intact and deprived somatomotor cortices to recruit deprived cortex in response to intact limb stimulation. This recruitment has been implicated in enhanced intact sensory function. In other patients, maladaptive consequences such as phantom limb pain can occur. We used unilateral whisker denervation in male and female mice to detect circuitry alterations underlying interhemispheric cortical reorganization. Enhanced synaptic strength from the intact cortex via the corpus callosum (CC) onto deep neurons in deprived primary somatosensory barrel cortex (S1BC) has previously been detected. It was hypothesized that specificity in this plasticity may depend on to which area these neurons projected. Increased connectivity to somatomotor areas such as contralateral S1BC, primary motor cortex (M1) and secondary somatosensory cortex (S2) may underlie beneficial adaptations, while increased connectivity to pain areas like anterior cingulate cortex (ACC) might underlie maladaptive pain phenotypes. Neurons from the deprived S1BC that project to intact S1BC were hyperexcitable, had stronger responses and reduced inhibitory input to CC stimulation. M1-projecting neurons also showed increases in excitability and CC input strength that was offset with enhanced inhibition. S2 and ACC-projecting neurons showed no changes in excitability or CC input. These results demonstrate that subgroups of output neurons undergo dramatic and specific plasticity after peripheral injury. The changes in S1BC-projecting neurons likely underlie enhanced reciprocal connectivity of S1BC after unilateral deprivation consistent with the model that interhemispheric takeover supports intact whisker processing.SIGNIFICANCE STATEMENTAmputation, peripheral injury, and stroke patients experience widespread alterations in neural activity after sensory loss. A hallmark of this reorganization is the recruitment of deprived cortical space which likely aids processing and thus enhances performance on intact sensory systems. Conversely, this recruitment of deprived cortical space has been hypothesized to underlie phenotypes like phantom limb pain and hinder recovery. A mouse model of unilateral denervation detected remarkable specificity in alterations in the somatomotor circuit. These changes underlie increased reciprocal connectivity between intact and deprived cortical hemispheres. This increased connectivity may help explain the enhanced intact sensory processing detected in humans.

Neurodegeneration driven by aberrant tau is a key feature of many dementias. Pathological stages of tauopathy are characterised by reduced synapse density and altered synapse function. Furthermore, changes in synaptic plasticity have been documented in the early stages of tauopathy suggesting that they may be a driver of later pathology. However, it remains unclear if synapse plasticity is specifically linked to the degeneration of neurons. This is partly because, in progressive dementias, pathology can vary widely from cell-to-cell along the prolonged disease time-course. To overcome this variability, we have taken a longitudinal experimental approach to track individual neurons through the progression of neurodegenerative tauopathy. Using repeated in vivo 2-photon imaging in rTg4510 transgenic mice, we have measured structural plasticity of presynaptic terminaux boutons and postsynaptic spines on individual axons and dendrites over long periods of time. By following individual neurons, we have measured synapse density across the neuronal population and tracked changes in synapse turnover in each neuron. We found that tauopathy drives a reduction in density of both presynaptic and postsynaptic structures and that this is partially driven by degeneration of individual axons and dendrites that are spread widely across the disease time-course. Both synaptic loss and neuronal degeneration was ameliorated by reduction in expression of the aberrant P301L transgene, but only if that reduction was initiated early in disease progression. Notably, neurite degeneration was preceded by alterations in synapse turnover that contrasted in axons and dendrites. In dendrites destined to die, there was a dramatic loss of spines in the week immediately before degeneration. In contrast, axonal degeneration was preceded by a progressive attenuation of presynaptic turnover that started many weeks before axon disappearance. Therefore, changes in synapse plasticity are harbingers of degeneration of individual neurites that occur at differing stages of tau-driven neurodegenerative disease, suggesting a cell or neurite autonomous process. Furthermore, the links between synapse plasticity and degeneration are distinct in axonal and dendritic compartments.Key findingsTauopathy driven by tau P301L in rTg4510 mice causes a progressive decrease in density of presynaptic terminaux boutons and postsynaptic dendritic spines in cortical excitatory neurons.Longitudinal imaging of individual axons and dendrites shows that there is a huge diversity of effects at varying times in different cells.Decreases in overall synapse density are driven partly, but not exclusively, by degeneration of dendrites and axons that are distributed widely across the time-course of disease.Suppression of pathological P301L tau expression can ameliorate accumulation of tau pathology, synapse loss and neurodegeneration, but only if administered early in disease progression.Neurite degeneration is preceded by aberrant structural synaptic plasticity in a cell-specific way that is markedly different in dendrites and axons.Degeneration of dendrites is immediately preceded by dramatic loss of dendritic spines.Axonal loss is characterised by a progressive attenuation of presynaptic bouton plasticity that starts months before degeneration.

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.

Pharmacological manipulation of specific neural circuits to optimize therapeutic index is an unrealized goal in neurology and psychiatry. AMPA receptors are important for excitatory synaptic transmission, and their antagonists are antiepileptic. Although efficacious, AMPA-receptor antagonists, including perampanel (Fycompa), the only approved antagonist for epilepsy, induce dizziness and motor impairment. We hypothesized that blockade of forebrain AMPA receptors without blocking cerebellar AMPA receptors would be antiepileptic and devoid of motor impairment. Taking advantage of an AMPA receptor auxiliary protein, TARP γ-8, which is selectively expressed in the forebrain and modulates the pharmacological properties of AMPA receptors, we discovered that LY3130481 selectively antagonized recombinant and native AMPA receptors containing γ-8, but not γ-2 (cerebellum) or other TARP members. Two amino acid residues unique to γ-8 determined this selectivity. We also observed antagonism of AMPA receptors expressed in hippocampal, but not cerebellar, tissue from an patient with epilepsy. Corresponding to this selective activity, LY3130481 prevented multiple seizure types in rats and mice and without motor side effects. These findings demonstrate the first rationally discovered molecule targeting specific neural circuitries for therapeutic advantage.

Sensory hypersensitivity is a common and debilitating feature of neurodevelopmental disorders such as Fragile X Syndrome (FXS). However, how developmental changes in neuronal function ultimately culminate in the network dysfunction that underlies sensory hypersensitivities is not known. To address this, we studied the layer 4 barrel cortex circuit inFmr1knockout mice, a critical sensory processing circuit in this mouse model of FXS. By systematically studying cellular and synaptic properties of layer 4 neurons and combining with cellular and network simulations, we explored how the array of phenotypes inFmr1knockout produce circuit pathology during development that result in sensory processing dysfunction. We show that many of the cellular and synaptic pathologies inFmr1knockout mice are antagonistic, mitigating circuit dysfunction, and hence can be regarded as compensatory to the primary pathology. Despite this compensation, the layer 4 network in theFmr1knockout exhibits significant alterations in spike output in response to ascending thalamocortical input that we show results in impaired sensory encoding. We suggest that it is this developmental loss of layer 4 sensory encoding precision that drives subsequent developmental alterations in layer 4 – layer 2/3 connectivity and plasticity observed in theFmr1knockout, and is a critical process producing sensory hypersensitivity.

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.

Traditionally, neurotransmitters are associated with a fast, or phasic, type of action on neurons in the central nervous system (CNS). However, accumulating evidence indicates that γ-aminobutyric acid (GABA) and glutamate can also have a continual, or tonic, influence on these cells. Here, in voltage- and current-clamp recordings in rat brain slices, we identify three types of tonically active receptors in a single CNS structure, the thalamic reticular nucleus (TRN). Thus, TRN contains constitutively active GABAA receptors (GABAA Rs), which are located on TRN neurons and generate a persistent outward Cl(-) current. When TRN neurons are depolarized, blockade of this current increases their action potential output in response to current injection. Furthermore, TRN contains tonically active GluN2B-containing N-methyl-D-aspartate receptors (NMDARs). These are located on reticuloreticular GABAergic terminals in TRN and generate a persistent facilitation of vesicular GABA release from these terminals. In addition, TRN contains tonically active metabotropic glutamate type 2 receptors (mGlu2Rs). These are located on glutamatergic cortical terminals in TRN and generate a persistent reduction of vesicular glutamate release from these terminals. Although tonically active GABAA Rs, NMDARs and mGlu2Rs operate through different mechanisms, we propose that the continual and combined activity of these three receptor types ultimately serves to hyperpolarize TRN neurons, which will differentially affect the output of these cells depending upon the current state of their membrane potential. Thus, when TRN cells are relatively depolarized, their firing in single-spike tonic mode will be reduced, whereas when these cells are relatively hyperpolarized, their ability to fire in multispike burst mode will be facilitated.

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.

Summary Background Ionotropic glutamate receptors (iGluRs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the central nervous system. Based on both molecular and pharmacological criteria, iGluRs have been divided into two major classes, the non-NMDA class, which includes both AMPA and kainate subtypes of receptors, and the NMDA class. One evolutionarily conserved feature of iGluRs is their desensitization in the continued presence of glutamate. Thus, when in a desensitized state, iGluRs can be bound to glutamate, yet the channel remains closed. However, the relevance of desensitization to nervous system function has remained enigmatic. Results Here, we report the identification and characterization of a novel polypeptide (con-ikot-ikot) from the venom of a predatory marine snail Conus striatus that specifically disrupts the desensitization of AMPA receptors (AMPARs). The stoichiometry of con-ikot-ikot appears reminiscent of the proposed subunit organization of AMPARs, i.e., a dimer of dimers, suggesting that it acts as a molecular four-legged clamp that holds the AMPAR channel open. Application of con-ikot-ikot to hippocampal slices caused a large and rapid increase in resting AMPAR-mediated current leading to neuronal death. Conclusions Our findings provide insight into the mechanisms that regulate receptor desensitization and demonstrate that in the arms race between prey and predators, evolution has selected for a toxin that blocks AMPAR desensitization, thus revealing the fundamental importance of desensitization for regulating neural function.

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.

Transmission through the thalamus activates circuits involving the GABAergic neurons of the thalamic reticular nucleus (TRN). TRN cells receive excitatory inputs from thalamocortical and corticothalamic cells and send inhibitory projections to thalamocortical cells. The inhibitory output of TRN neurons largely depends on the level of excitatory drive to these cells but may also be partly under the control of mechanisms intrinsic to the TRN. We examined two such possible mechanisms, short-term plasticity at glutamatergic synapses in the TRN and intra-TRN inhibition. In rat brain slices, responses of TRN neurons to brief trains of stimuli applied to glutamatergic inputs were recorded in voltage- or current-clamp mode. In voltage clamp, TRN cells showed no change in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated excitatory postsynaptic current amplitudes to stimulation at non-gamma frequencies (30 Hz), simulating background activity, but exhibited short-term depression in these amplitudes to stimulation at gamma frequencies (30 Hz), simulating sensory transmission. In current clamp, TRN cells increased their spike outputs in burst and tonic firing modes to increasing stimulus-train frequencies. These increases in spike output were most likely due to temporal summation of excitatory postsynaptic potentials. However, the frequency-dependent increase in tonic firing was attenuated at gamma stimulus frequencies, indicating that the synaptic depression selectively observed in this frequency range acts to suppress TRN cell output. In contrast, intra-TRN inhibition reduced spike output selectively at non-gamma stimulus frequencies. Thus, our data indicate that two intrinsic mechanisms play a role in controlling the tonic spike output of TRN neurons and these mechanisms are differentially related to two physiologically meaningful stimulus frequency ranges.

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.

Feedforward inhibitory GABAergic transmission is critical for mature cortical circuit function; in the neonate, however, GABA is depolarizing and believed to have a different role. Here we show that the GABAA receptor-mediated conductance is depolarizing in excitatory (stellate) cells in neonatal (postnatal day [P]3-5) layer IV barrel cortex, but GABAergic transmission at this age is not engaged by thalamocortical input in the feedforward circuit and has no detectable circuit function. However, recruitment occurs at P6-7 as a result of coordinated increases in thalamic drive to fast-spiking interneurons, fast-spiking interneuron-stellate cell connectivity and hyperpolarization of the GABAA receptor-mediated response. Thus, GABAergic circuits are not engaged by thalamocortical input in the neonate, but are poised for a remarkably coordinated development of feedforward inhibition at the end of the first postnatal week, which has profound effects on circuit function at this critical time in development.

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.

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.

NR2C-containing N-methyl-D-aspartate (NMDA) receptors are highly expressed in cerebellar granule cells where they mediate the majority of current in the adult. NMDA receptors composed of NR1/NR2C exhibit a low conductance and reduced sensitivity to Mg(2+), compared with the more commonly studied NR2A- and NR2B-containing receptors. Despite these interesting features, very little is known about the regulation of NR2C function. Here we investigate the role of phosphorylation of NR2C in regulating NMDA receptor trafficking and ion channel properties. We identify a phosphorylation site, serine 1244 (Ser(1244)), near the extreme COOH terminus of NR2C, which is phosphorylated by both cAMP-dependent protein kinase and protein kinase C. This residue is located adjacent to the consensus PDZ ligand, a region that regulates protein-protein interactions and receptor trafficking in NR2A and NR2B. We show that Ser(1244) on NR2C is phosphorylated in vitro, in heterologous cells, and in neurons. Moreover, we demonstrate for the first time that NR2C interacts with the PSD-95 family of PDZ domain-containing proteins but that phosphorylation of Ser(1244) does not influence this PDZ interaction. Furthermore, Ser(1244) phosphorylation does not regulate surface expression of NR1/NR2C receptors. However, we find that this site does regulate the kinetics of the ion channel: a phosphomimetic mutation at Ser(1244) accelerates both the rise and decay of NMDA-evoked currents in excised patches from HEK-293 cells. Therefore, phosphorylation of Ser(1244) does not regulate trafficking but unexpectedly affects ion channel function, suggesting that phosphorylation of Ser(1244) on NR2C may be important in defining the functional properties of NMDA receptor-mediated currents in the cerebellum.

Developing neuronal networks acquire the ability to precisely time events, a key feature required for information processing. In the barrel cortex, encoding of information requires a high-precision temporal code with a resolution of ∼5 ms; however, it is not known what process drives the maturation in timing precision. Here, we report that long-term potentiation (LTP) at thalamocortical synapses in the neonatal layer IV barrel cortex produces a dramatic improvement in the timing of neuronal output and synaptic input. LTP strongly reduces the latency and variability of synaptically evoked action potentials, improving the fidelity of timing to within that predicted to be required for adult sensory processing. Such changes in timing also occur during development in the neonate. LTP also reduces the summation of EPSPs shortening the window for coincidence detection for synaptic input. In contrast to these reliable effects, LTP produced only a modest and variable change in synaptic efficacy. Thus, our findings suggest that the primary role of this form of neonatal LTP is for the acquisition of timing precision and the refinement of coincidence detection, rather than an increase in synaptic strength. Therefore, neonatal thalamocortical LTP may be a critical prerequisite for the maturation of information processing in the barrel cortex.

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.

Neonatal hippocampus exhibits distinct patterns of network activity that are dependent on the interaction between inhibitory and excitatory transmission. Kainate receptors are ideally positioned to regulate this activity by virtue of their ability to regulate presynaptic function in GABAergic interneurones. Indeed, kainate receptors are highly expressed in neonatal hippocampal interneurones, yet the role and mechanisms by which they might regulate neonatal circuitry are unexplored. To address this we investigated the kainate receptor-dependent regulation of GABAergic transmission onto neonatal CA1 pyramidal neurones. Kainate receptor activation produced two distinct opposing effects, a very large increase in the frequency of spontaneous IPSCs, and a robust depression of evoked GABAergic transmission. The up-regulation of spontaneous transmission was due to activation of somatodendritic and axonal receptors while the depression of evoked transmission could be fully accounted for by a direct regulation of GABA release by kainate receptors located at the terminals. None of the effects of kainate receptor agonists were sensitive to GABAB receptor antagonists, nor was there any postsynaptic kainate receptor-dependent effects observed in CA1 pyramidal cells that could account for our findings. Our data demonstrate that kainate receptors profoundly regulate neonatal CA1 GABAergic circuitry by two distinct opposing mechanisms, and indicate that these two effects are mediated by functionally distinct populations of receptors. Thus kainate receptors are strategically located to play a critical role in shaping early hippocampal network activity and by virtue of this have a key role in hippocampal development.

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.

PICK1 (protein interacting with C kinase-1) regulates the surface expression of the AMPA receptor (AMPAR) GluR2 subunit, however, the functional consequences of this interaction are not well understood. Previous work has suggested that PICK1 promotes the internalization of AMPARs. However, we found that when PICK1 is virally expressed in the CA1 region of hippocampal slices, it causes an increase in AMPAR-mediated EPSC amplitude. This effect is associated with increased AMPAR rectification and sensitivity to polyamine toxin. These effects are blocked by PKC or calcium/calmodulin-dependent protein kinase II inhibitors, indicating that the virally expressed PICK1 signals through an endogenous kinase cascade. In contrast, blockade of interactions with GluR2 at the N-ethylmaleimide-sensitive factor site did not cause a change in subunit composition, suggesting that the effects of PICK1 are not simply a nonspecific consequence of removing AMPARs from the surface. Immunocytochemical and biochemical analyses in dissociated cultured hippocampal neurons show that PICK1 causes a decrease in endogenous GluR2 surface expression but no change in GluR1 surface levels. To address the physiological role of PICK1, we virally expressed C-terminal GluR2 peptides. Blockade of endogenous PICK1 PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain interactions produced opposite effects on synaptic strength and AMPAR rectification to those observed with PICK1 expression. This demonstrates that AMPAR subunit composition is physiologically regulated through a mechanism involving PICK1 PDZ domain interactions. These findings suggest that PICK1 acts to downregulate the GluR2 content of AMPARs at hippocampal CA1 synapses, thereby increasing synaptic strength at resting membrane potentials.

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.

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.

NMDAR-dependent long-term depression involves the activation of protein phosphatase 1 (PP1) and 2B (calcineurin) and the subsequent dephosphorylation of synaptic proteins. In this issue of Neuron, Morishita et al. (2001) provide evidence that precise targeting of PP1 to synaptic substrates is critical for the expression of LTD.

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.

We investigated the role of PDZ proteins (GRIP, ABP, and PICK1) interacting with the C-terminal GluR2 by infusing a ct-GluR2 peptide ("pep2-SVKI") into CA1 pyramidal neurons in hippocampal slices using whole-cell recordings. Pep2-SVKI, but not a control or PICK1 selective peptide, caused AMPAR-mediated EPSC amplitude to increase in approximately one-third of control neurons and in most neurons following the prior induction of LTD. Pep2-SVKI also blocked LTD; however, this occurred in all neurons. A PKC inhibitor prevented these effects of pep2-SVKI on synaptic transmission and LTD. We propose a model in which the maintenance of LTD involves the binding of AMPARs to PDZ proteins to prevent their reinsertion. We also present evidence that PKC regulates AMPAR reinsertion during dedepression.

High affinity glutamate transport plays an important role in maintaining a low extracellular glutamate concentration in the CNS. Excitotoxicity due to a loss of glutamate transporter function has been implicated in disease processes such as stroke and amyotrophic lateral sclerosis (ALS). We studied the effects of glutamate transport inhibitors on thalamocortical synapses at developing (postnatal day 3–8) layer IV neurons in the barrel cortex using the thalamocortical slice preparation and whole-cell recordings. Inhibition of glutamate transport by D,L-threo-β-hydroxyaspartate (THA), a combination of THA and dihydrokainate (DHK), or by L-trans-pyrrolidine-2,4-dicarboxylate (tPDC), caused a reversible blockade of AMPA and kainate receptor-mediated dual component excitatory postsynaptic currents (AMPA/KA EPSCs). This effect was not blocked by cyclothiazide (CTZ) indicating that is was not due to desensitisation of AMPARs. Under conditions in which NMDA receptors were unblocked the transport inhibitors caused the massive activation of NMDA receptors leading to the rapid loss of recordings. Previous studies using these transport inhibitors on brain slices from older animals reported no or only modest effects on synaptic transmission. Therefore the data in the present study suggest that neurons in the developing neocortex are particularly sensitive to glutamate transporter function. Furthermore the effects of transport inhibition are dependent upon whether neurons are sufficiently depolarised to relieve the voltage-dependent block of NMDA receptors.

Synapses are anatomical specializations at the interface of two neurons. They have fascinated researchers ever since Sherrington coined the term ‘synapse’ in 1879. Studies of synapses have provided amazing insight into their function and were boosted by the discovery, now more than 30 years ago, that synapses can undergo long-term activity-dependent plasticity. Over the last two decades much effort has been devoted to the study of long-term synaptic plasticity as a candidate mechanism underlying learning and memory. More recently dysfunctional synaptic plasticity has been implicated in several brain diseases, such as depression, addiction, dementia and anxiety disorders. Here we report on an international meeting that took place in Geneva in July 2008 that brought together scientists who study synapses and disease mechanisms. Brain diseases constitute a major burden to society. In Europe, alone, it is estimated that direct and indirect costs of brain diseases represent more than 350 billion euros per year. When broken down into individual diseases the most costly are depression (105 billion euros, a sum equal to the cost of all cardiovascular diseases), addiction (56 billion even without including nicotine abuse), dementia (54 billion) and anxiety disorders (40 billion), followed by autism and schizophrenia. The Synaptic Basis of Disease meeting, which was organized as a satellite meeting to the July 2008 FENS meeting in Geneva (Fig. 1), was convened to bring together leading researchers working on basic synaptic mechanisms with those working on neurological disorders. Indeed, many labs at the meeting have programs working on both aspects. The main focus of the meeting was to generate discussion amongst some of the leading experimentalists working on synaptic and disease mechanisms. Figure 1 The poster for the symposium

Lack of AMPA receptor desensitization during basal synaptic transmission in the hippocampal slice. Excitatory postsynaptic currents in the CA1 region of rat hippocampal slices are mediated primarily by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in response to synaptically released glutamate. Outside-out patches from pyramidal cells in this region have shown that AMPA receptors are desensitized by short (1 ms) pulses of glutamate. We have taken a number of approaches to ask whether synaptic receptors desensitize in response to synaptically released glutamate in the slice. Recordings with paired pulses and minimal stimulation conditions that are presumably activating only a single release site do not show evidence for desensitization. Furthermore, cyclothiazide, a drug that blocks desensitization, does not alter paired-pulse ratios even under conditions of high probability of release, which should maximize desensitization. These results suggest that synaptic receptors do not desensitize in response to synaptically released glutamate during basal synaptic transmission.

There is considerable controversy surrounding the mechanism of expression of long-term potentiation of AMPA receptor-mediated synaptic transmission in the CA1 region of the hippocampus, a process thought to be important for learning and memory in the mammalian CNS. We have re-examined the expression mechanism of this form of synaptic plasticity using whole-cell dendritic recordings, minimal stimulation to activate one or a few synapses, and failures analysis. Dendritic recordings provide improved resolution of small synaptic events, as compared to previous studies using somatic recordings, because there is less dendritic filtering of signals. We find that long-term potentiation (LTP) is associated with changes in the size of synaptic responses when they occur (potency) in all cells and this is accompanied by significant decreases in failure rate in approximately 60% of the experiments. This suggests that in some cells an increase in quantal amplitude is the sole expression mechanism for LTP and, in the cells where failure rate decreased, there is an additional mechanism causing a change in quantal content.

Transmission of sensory information through the dorsal thalamus involves two types of modality-related nuclei, first order and higher order, between which there are thought to be no intrathalamic interactions. We now show that within the somatosensory thalamus, cells in one nucleus, the ventrobasal complex, can influence activity in another nucleus, the medial division of the posterior complex. Stimulation of ventrobasal complex cells evoked inhibitory postsynaptic currents in cells of the medial division of the posterior complex. These currents exhibited the reversal potential and pharmacology of a GABAA receptor-mediated chloride conductance, indicating that they result from the activation of a disynaptic pathway involving the GABAergic cells of the thalamic reticular nucleus. These findings provide the first direct evidence for intrathalamic interactions between dorsal thalamic nuclei.

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.

Genetic causes, or predisposition, are increasingly accepted to be part of the ethiopathogenesis of many neuropsychiatric diseases. While genes can be studied in any type of cells, their physiological function in human brain cells is difficult to evaluate, particularly in living subjects. As a first step towards the characterisation of human inducible pluripotent stem cell (iPSC)-derived neurons from autism spectrum disorder (ASD) patients, we used gene expression and functional studies to define the regional identity of the typical forebrain differentiation, demonstrate expression patterns of genes of interest in ASD and understand the properties of ‘control’ iPSC-derived neurons (iCell-Neurons™), with a focus on receptors and ion channels that play a central role in synaptic physio-pathology. The gene expression profile of the iCell-Neurons™ closely resembled that observed in neonatal prefrontal cortex tissues. Functional studies, performed mainly using calcium flux assays, demonstrated the presence of ionotropic glutamate (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-d-aspartate) and gamma-aminobutyric acid type A receptors. Voltage-gated sodium and calcium channels were also identified using similar techniques. Overall, the results reported here suggest that iCell-Neurons™ are a good cellular model of a relatively immature forebrain human neuron population that can be used both as a control in comparison to patients cells, and as host cells in which mutations, insertions and deletions can be used in order to study the molecular mechanisms of ASD and other neurological disorders in an isogenic cellular background.

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.

Cerebellar motor coordination and cerebellar Purkinje cell synaptic function require metabotropic glutamate receptor 1 (mGluR1, Grm1). We used an unbiased proteomic approach to identify protein partners for mGluR1 in cerebellum and discovered glutamate receptor δ2 (GluRδ2, Grid2, GluΔ2) and protein kinase Cγ (PKCγ) as major interactors. We also found canonical transient receptor potential 3 (TRPC3), which is also needed for mGluR1-dependent slow EPSCs and motor coordination and associates with mGluR1, GluRδ2, and PKCγ. Mutation of GluRδ2 changes subcellular fractionation of mGluR1 and TRPC3 to increase their surface expression. Fitting with this, mGluR1-evoked inward currents are increased in GluRδ2 mutant mice. Moreover, loss of GluRδ2 disrupts the time course of mGluR1-dependent synaptic transmission at parallel fiber–Purkinje cells synapses. Thus, GluRδ2 is part of the mGluR1 signaling complex needed for cerebellar synaptic function and motor coordination, explaining the shared cerebellar motor phenotype that manifests in mutants of the mGluR1 and GluRδ2 signaling pathways.