Neuregulin-1 regulates LTP at CA1 hippocampal synapses through activation of dopamine D4 receptors

Neuregulin-1 (NRG-1) is genetically linked with schizophrenia, a neurodevelopmental cognitive disorder characterized by imbalances in glutamatergic and dopaminergic function. NRG-1 regulates numerous neurodevelopmental processes and, in the adult, suppresses or reverses long-term potentiation (LTP) at hippocampal glutamatergic synapses. Here we show that NRG-1 stimulates dopamine release in the hippocampus and reverses early-phase LTP via activation of D4 dopamine receptors (D4R). NRG-1 fails to depotentiate LTP in hippocampal slices treated with the antipsychotic clozapine and other more selective D4R antagonists. Moreover, LTP is not depotentiated in D4R null mice by either NRG-1 or theta-pulse stimuli. Conversely, direct D4R activation mimics NRG-1 and reduces AMPA receptor currents and surface expression. These findings demonstrate that NRG-1 mediates its unique role in counteracting LTP via dopamine signaling and opens future directions to study new aspects of NRG function. The novel functional link between NRG-1, dopamine, and glutamate has important implications for understanding how imbalances in Neuregulin-ErbB signaling can impinge on dopaminergic and glutamatergic function, neurotransmitter pathways associated with schizophrenia.     

AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons

NMDA receptor activation promotes endocytosis of AMPA receptors, which is an important mechanism underlying long-term synaptic depression. The pH-sensitive GFP variant pHluorin fused to the N terminus of GluA2 (pH-GluA2) has been used to assay NMDA-mediated AMPA receptor endocytosis and recycling. Here, we demonstrate that in somatic and dendritic regions of hippocampal neurons a large fraction of the fluorescent signal originates from intracellular pH-GluA2, and that the decline in fluorescence in response to NMDA and AMPA primarily describes an intracellular acidification, which quenches the pHluorin signal from intracellular receptor pools. Neurons expressing an endoplasmic reticulum-retained mutant of GluA2 (pH-GluA2 ΔC49) displayed a larger response to NMDA than neurons expressing wild-type pH-GluA2. A similar NMDA-elicited decline in pHluorin signal was observed by expressing cytosolic pHluorin alone without fusion to GluA2 (cyto-pHluorin). Intracellular acidification in response to NMDA was further confirmed by using the ratiometric pH indicator carboxy-SNARF-1. The NMDA-induced decline was followed by rapid recovery of the fluorescent signal from both cyto-pHluorin and pH-GluA2. The recovery was sodium-dependent and sensitive to Na⁺/H⁺-exchanger (NHE) inhibitors. Moreover, recovery was more rapid after shRNA-mediated knockdown of the GluA2 binding PDZ domain-containing protein interacting with C kinase 1 (PICK1). Interestingly, the accelerating effect of PICK1 knockdown on the fluorescence recovery was eliminated in the presence of the NHE1 inhibitor zoniporide. Our results indicate that the pH-GluA2 recycling assay is an unreliable assay for studying AMPA receptor trafficking and also suggest a role for PICK1 in regulating intracellular pH via modulation of NHE activity.     

AMPA receptor and GEF-H1/Lfc complex regulates dendritic spine development through RhoA signaling cascade

AMPA receptors (AMPA-R) are major mediators of synaptic transmission and plasticity in the developing and adult central nervous system. Activity-dependent structural plasticity mediated by dynamic changes in the morphology of spines and dendrites is also essential for the formation and tuning of neuronal circuits. RhoA and Rac1 are known to play important roles in the regulation of spine and dendrite development in response to neuronal activity. These Rho GTPases are activated by guanine nucleotide exchange factors (GEFs). In this study, we identified GEF-H1/Lfc as a component of the AMPA-R complex in the brain. GEF-H1 is enriched in the postsynaptic density and is colocalized with GluR1 at spines. GEF-H1 activity negatively regulates spine density and length through a RhoA signaling cascade. In addition, AMPA-R-dependent changes in spine development are eliminated by down-regulation of GEF-H1. Altogether, these results strongly suggest that GEF-H1 is an important mediator of AMPA-R activity-dependent structural plasticity in neurons.     

Dual regulation of NR2B and NR2C expression by NMDA receptor activation in mouse cerebellar granule cell cultures

In the developing cerebellum, switching of the subunit composition of NMDA receptors occurs in granule cells from NR2B-containing receptors to NR2C-containing ones. We investigated the mechanisms underlying switching of NR2B and NR2C subunit composition in primary cultures of mouse granule cells at the physiological KCl concentration (5 mM). Granule cells extensively extended their neuritic processes 48 h after having been cultured in serum-free medium containing 5 mM KCl. Consistent with this morphological change, NR2B mRNA and NR2C mRNA were down- and up-regulated, respectively, in the granule cells. This dual regulation of the two mRNAs was abrogated by blocking excitation of granule cells with TTX. This neuronal activity–dependent regulation of NR2B and NR2C mRNAs was abolished by the addition of selective antagonists of AMPA receptors and NMDA receptors. Furthermore, the dual regulation of NR2B and NR2C mRNAs in TTX-treated cells was restored by the addition of NMDA in the presence of the AMPA receptor antagonist, but not by that of AMPA in the presence of the NMDA receptor antagonist. Importantly, the NMDA receptor activation drove the NR2B/NR2C switching of NMDA receptors in the cell-surface membrane of granule cells. This investigation demonstrates that stimulation of NMDA receptors in conjunction with the AMPA receptor–mediated excitation of granule cells plays a key role in functional subunit switching of NMDA receptors in maturing granule cells at the physiological KCl concentration.     

The neurotrophin receptor p75NTR modulates long-term depression and regulates the expression of AMPA receptor subunits in the hippocampus

Neurotrophins are involved in the modulation of synaptic transmission, including the induction of long-term potentiation (LTP) through the receptor TrkB. Because previous studies have revealed a bidirectional mode of neurotrophin action by virtue of signaling through either the neurotrophin receptor p75NTR or the Trk receptors, we tested the hypothesis that p75NTR is important for longterm depression (LTD) to occur. Although LTP was found to be unaffected in hippocampal slices of two different strains of mice carrying mutations of the p75NTR gene, hippocampal LTD was impaired in both p75NTR-deficient mouse strains. Furthermore, the expression levels of two (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits, GluR2 and GluR3, but not GluR1 or GluR4, were found to be significantly altered in the hippocampus of p75NTR-deficient mice. These results implicate p75NTR in activity-dependent synaptic plasticity and extend the concept of functional antagonism of the neurotrophin signaling system.     

Homeostatic regulation of AMPA receptor expression at single hippocampal synapses

Homeostatic synaptic response is an important measure in confining neuronal activity within a narrow physiological range. Whether or not homeostatic plasticity demonstrates synapse specificity, a key feature characteristic of Hebbian-type plasticity, is largely unknown. Here, we report that in cultured hippocampal neurons, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid subtype glutamate receptor (AMPAR) accumulation is increased selectively in chronically inhibited single synapses, whereas the neighboring normal synapses remain unaffected. This synapse-specific homeostatic regulation depends on the disparity of synaptic activity and is mediated by GluR2-lacking AMPARs and PI3-kinase signaling. These results demonstrate the existence of synaptic specificity and the crucial role of AMPAR-gated calcium in homeostatic plasticity in central neurons.     

Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

Dopamine is a powerful modulator of glutamatergic neurotransmission and NMDA receptor-dependent synaptic plasticity. Although several intracellular cascades participating in this functional dialogue have been identified over the last few decades, the molecular crosstalk between surface dopamine and glutamate NMDA receptor (NMDAR) signaling still remains poorly understood. Using a combination of single-molecule detection imaging and electrophysiology in live hippocampal neurons, we demonstrate here that dopamine D1 receptors (D1Rs) and NMDARs form dynamic surface clusters in the vicinity of glutamate synapses. Strikingly, D1R activation or D1R/NMDAR direct interaction disruption decreases the size of these clusters, increases NMDAR synaptic content through a fast lateral redistribution of the receptors, and favors long-term synaptic potentiation. Together, these data demonstrate the presence of dynamic D1R/NMDAR perisynaptic reservoirs favoring a rapid and bidirectional surface crosstalk between receptors and set the plasma membrane as the primary stage of the dopamine–glutamate interplay.Hippocampal dopaminergic neuromodulation participates in several cognitive functions including novelty detection and long-term memory storage (1, 2). As a consequence, impairments in hippocampal neuromodulatory transmission affect synaptic plasticity at glutamatergic synapses, prevent learning and memory formation, and have been proposed to be a cellular substrate for neurodevelopmental psychiatric disorders such as schizophrenia (3). In the hippocampus and cortex, pyramidal neurons express mostly dopamine D1 and D5 receptors along their dendritic tree (4–6). Their recruitment affects the trafficking and surface expression of glutamate NMDA receptors (NMDARs), two processes that are essential for excitatory neurotransmission and synaptic plasticity. Indeed, activating dopamine D1 receptors (D1Rs) promotes the surface expression and function of NMDAR and thereby favors the long-term potentiation of excitatory glutamate synapses (7–10). Reciprocally, the activation of NMDAR modulates D1R surface expression and signaling (11). The bidirectional dialogue between dopamine and glutamate NMDAR-associated signaling thus involves changes in membrane receptor content and trafficking.Although this functional interaction is usually considered as relying on intracellular protein kinase signaling cascades (7, 10, 12), physical interactions between D1R and NMDAR at the plasma membrane were recently reported to stabilize laterally diffusing surface D1R in spines, modulate D1R- and NMDAR-mediated signaling, and influence working memory (13–16). Thus, direct interactions between these receptors could contribute to the regulation of their surface distributions and play a major role in the dopamine–glutamate interplay (15, 17). In particular, because the regulation of NMDAR synaptic content involves surface diffusion processes in and out of synaptic and extrasynaptic compartments (18), the possibility emerges that dopamine might modulate NMDAR-dependent synaptic transmission by tuning NMDAR lateral dynamics through D1R–NMDAR physical interactions. To address this question and investigate the role of the D1R/NMDAR surface crosstalk in synaptic physiology, we here assessed the surface distribution and trafficking of D1 and NMDA receptors in rat hippocampal neurons using a combination of high-resolution single-nanoparticle tracking, bulk imaging, and electrophysiology.     

Unified quantitative model of AMPA receptor trafficking at synapses

Trafficking of AMPA receptors (AMPARs) plays a key role in synaptic transmission. However, a general framework integrating the two major mechanisms regulating AMPAR delivery at postsynapses (i.e., surface diffusion and internal recycling) is lacking. To this aim, we built a model based on numerical trajectories of individual AMPARs, including free diffusion in the extrasynaptic space, confinement in the synapse, and trapping at the postsynaptic density (PSD) through reversible interactions with scaffold proteins. The AMPAR/scaffold kinetic rates were adjusted by comparing computer simulations to single-particle tracking and fluorescence recovery after photobleaching experiments in primary neurons, in different conditions of synapse density and maturation. The model predicts that the steady-state AMPAR number at synapses is bidirectionally controlled by AMPAR/scaffold binding affinity and PSD size. To reveal the impact of recycling processes in basal conditions and upon synaptic potentiation or depression, spatially and temporally defined exocytic and endocytic events were introduced. The model predicts that local recycling of AMPARs close to the PSD, coupled to short-range surface diffusion, provides rapid control of AMPAR number at synapses. In contrast, because of long-range diffusion limitations, extrasynaptic recycling is intrinsically slower and less synapse-specific. Thus, by discriminating the relative contributions of AMPAR diffusion, trapping, and recycling events on spatial and temporal bases, this model provides unique insights on the dynamic regulation of synaptic strength.     

Neuroligin-1 is required for normal expression of LTP and associative fear memory in the amygdala of adult animals

Neuroligin-1 is a potent trigger for the de novo formation of synaptic connections, and it has recently been suggested that it is required for the maturation of functionally competent excitatory synapses. Despite evidence for the role of neuroligin-1 in specifying excitatory synapses, the underlying molecular mechanisms and physiological consequences that neuroligin-1 may have at mature synapses of normal adult animals remain unknown. By silencing endogenous neuroligin-1 acutely in the amygdala of live behaving animals, we have found that neuroligin-1 is required for the storage of associative fear memory. Subsequent cellular physiological studies showed that suppression of neuroligin-1 reduces NMDA receptor-mediated currents and prevents the expression of long-term potentiation without affecting basal synaptic connectivity at the thalamo-amygdala pathway. These results indicate that persistent expression of neuroligin-1 is required for the maintenance of NMDAR-mediated synaptic transmission, which enables normal development of synaptic plasticity and long-term memory in the amygdala of adult animals.     

The Rab5 guanylate exchange factor Rin1 regulates endocytosis of the EphA4 receptor in mature excitatory neurons

Ephrin signaling through Eph receptor tyrosine kinases regulates important morphogenetic events during development and synaptic plasticity in the adult brain. Although Eph-ephrin endocytosis is required for repulsive axon guidance, its role in postnatal brain and synaptic plasticity is unknown. Here, we show that Rin1, a postnatal brain-specific Rab5-GEF, is coexpressed with EphA4 in excitatory neurons and interacts with EphA4 in synaptosomal fractions. The interaction of Rin1 and EphA4 requires Rin1's SH2 domain, consistent with the view that Rin1 targets tyrosine phosphorylated receptors to Rab5 compartments. We find that Rin1 mediates EphA4 endocytosis in postnatal amygdala neurons after engagement of EphA4 with its cognate ligand ephrinB3. Rin1 was shown to suppress synaptic plasticity in the amygdala, a forebrain structure important for fear learning, possibly by internalizing synaptic receptors. We find that the EphA4 receptor is required for synaptic plasticity in the amygdala, raising the possibility that an underlying mechanism of Rin1 function in amygdala is to down-regulate EphA4 signaling by promoting its endocytosis.     

Plasma membrane insertion of the AMPA receptor GluA2 subunit is regulated by NSF binding and Q/R editing of the ion pore

The delivery of AMPA receptors to the plasma membrane is a critical step both for the synaptic delivery of these receptors and for the regulation of synaptic transmission. To directly visualize fusion events of transport vesicles containing the AMPA receptor GluA2 subunit with the plasma membrane we used pHluorin-tagged GluA2 subunits and total internal reflection fluorescence microscopy. We demonstrate that the plasma membrane insertion of GluA2 requires the NSF binding site within its intracellular cytoplasmic domain and that RNA editing of the Q/R site in the ion channel region plays a key role in GluA2 plasma membrane insertion. Finally, we show that plasma membrane insertion of heteromeric GluA2/3 receptors follows the same rules as homomeric GluA2 receptors. These results demonstrate that the plasma membrane delivery of GluA2 containing AMPA receptors is regulated by its unique structural elements.     

Gain in sensitivity and loss in temporal contrast of STDP by dopaminergic modulation at hippocampal synapses

Spike-timing-dependent plasticity (STDP) is considered a physiologically relevant form of Hebbian learning. However, behavioral learning often involves action of reinforcement or reward signals such as dopamine. Here, we examined how dopamine influences the quantitative rule of STDP at glutamatergic synapses of hippocampal neurons. The presence of 20 μM dopamine during paired pre- and postsynaptic spiking activity expanded the effective time window for timing-dependent long-term potentiation (t-LTP) to at least −45 ms, and allowed normally ineffective weak stimuli with fewer spike pairs to induce significant t-LTP. Meanwhile, dopamine did not affect the degree of t-LTP induced by normal strong stimuli with spike timing (ST) of +10 ms. Such dopamine-dependent enhancement in the sensitivity of t-LTP was completely blocked by the D1-like dopamine receptor antagonist {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390, but not by the D2-like dopamine receptor antagonist sulpiride. Surprisingly, timing-dependent long-term depression (t-LTD) at negative ST was converted into t-LTP by dopamine treatment; this conversion was also blocked by {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390. In addition, t-LTP in the presence of dopamine was completely blocked by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid, indicating that D1-like receptor-mediated modulation appears to act through the classical NMDA receptor-mediated signaling pathway that underlies STDP. These results provide a quantitative and mechanistic basis for a previously undescribed learning rule that depends on pre- and postsynaptic ST, as well as the global reward signal.     

Nogo receptor 1 regulates formation of lasting memories

Formation of lasting memories is believed to rely on structural alterations at the synaptic level. We had found that increased neuronal activity down-regulates Nogo receptor-1 (NgR1) in brain regions linked to memory formation and storage, and postulated this to be required for formation of lasting memories. We now show that mice with inducible overexpression of NgR1 in forebrain neurons have normal long-term potentiation and normal 24-h memory, but severely impaired month-long memory in both passive avoidance and swim maze tests. Blocking transgene expression normalizes these memory impairments. Nogo, Lingo-1, Troy, endogenous NgR1, and BDNF mRNA expression levels were not altered by transgene expression, suggesting that the impaired ability to form lasting memories is directly coupled to inability to down-regulate NgR1. Regulation of NgR1 may therefore serve as a key regulator of memory consolidation. Understanding the molecular underpinnings of synaptic rearrangements that carry lasting memories may facilitate development of treatments for memory dysfunction.     

Developmental switch in the kinase dependency of long-term potentiation depends on expression of GluA4 subunit-containing AMPA receptors

The AMPA-receptor subunit GluA4 is expressed transiently in CA1 pyramidal neurons at the time synaptic connectivity is forming, but its physiological significance is unknown. Here we show that GluA4 expression is sufficient to alter the signaling requirements of long-term potentiation (LTP) and can fully explain the switch in the LTP kinase dependency from PKA to Ca2⁺/calmodulin-dependent protein kinase II during synapse maturation. At immature synapses, activation of PKA leads to a robust potentiation of AMPA-receptor function via the mobilization of GluA4. Analysis of GluA4-deficient mice indicates that this mechanism is critical for neonatal PKA-dependent LTP. Furthermore, lentiviral expression of GluA4 in CA1 neurons conferred a PKA-dependent synaptic potentiation and LTP regardless of the developmental stage. Thus, GluA4 defines the signaling requirements for LTP and silent synapse activation during a critical period of synapse development.Activity-dependent plasticity at immature glutamatergic synapses is thought to underlie fine tuning of the synaptic circuitry and optimize the network for its adult functions. The synaptic mechanisms of plasticity at immature contacts differ from those in the adult because of developmental alterations in the expression of several molecules that are critical in mediating and modulating synaptic transmission. For example, in area CA1 of the hippocampus, the signaling cascades necessary for long-term potentiation (LTP) are altered during the first weeks of postnatal life, corresponding to the time of formation and maturation of glutamatergic synapses. In the neonate, LTP is dependent mainly on the activation of PKA, but later in development LTP requires the activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) together with other kinases (1, 2). In parallel, expression of the AMPA-receptor subunit GluA4 in the hippocampal pyramidal neurons is strongly down-regulated and replaced by other subunits, including GluA1 (3, 4).Both GluA4 and GluA1 and a splice variant of GluA2, GluA2_L, contain a long intracellular C-terminal domain (CTD) that is thought to be involved in activity-dependent synaptic incorporation of AMPA receptors (5–8, but also see ref. 9). Spontaneous synaptic activity and consequent activity-dependent PKA phosphorylation is sufficient to drive recombinant GluA4, but not GluA1, into synapses (4, 10), suggesting that the switch in the subunit composition of AMPA receptors may explain some of the developmental changes in the mechanisms of LTP. However, the exact role of the developmentally restricted expression of GluA4 in synaptic transmission and plasticity remains unknown.Here we show that GluA4 expression is sufficient to alter the signaling mechanisms underlying LTP and to confer PKA-dependent postsynaptic potentiation. Thus, the expression of GluA4 can explain fully the developmental switch in the LTP kinase dependency in CA1 pyramidal neurons.     

GABAB receptor deficiency causes failure of neuronal homeostasis in hippocampal networks

Stabilization of neuronal activity by homeostatic control systems is fundamental for proper functioning of neural circuits. Failure in neuronal homeostasis has been hypothesized to underlie common pathophysiological mechanisms in a variety of brain disorders. However, the key molecules regulating homeostasis in central mammalian neural circuits remain obscure. Here, we show that selective inactivation of GABA_B, but not GABA_A, receptors impairs firing rate homeostasis by disrupting synaptic homeostatic plasticity in hippocampal networks. Pharmacological GABA_B receptor (GABA_BR) blockade or genetic deletion of the GB_1a receptor subunit disrupts homeostatic regulation of synaptic vesicle release. GABA_BRs mediate adaptive presynaptic enhancement to neuronal inactivity by two principle mechanisms: First, neuronal silencing promotes syntaxin-1 switch from a closed to an open conformation to accelerate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly, and second, it boosts spike-evoked presynaptic calcium flux. In both cases, neuronal inactivity removes tonic block imposed by the presynaptic, GB_1a-containing receptors on syntaxin-1 opening and calcium entry to enhance probability of vesicle fusion. We identified the GB_1a intracellular domain essential for the presynaptic homeostatic response by tuning intermolecular interactions among the receptor, syntaxin-1, and the Ca_V2.2 channel. The presynaptic adaptations were accompanied by scaling of excitatory quantal amplitude via the postsynaptic, GB_1b-containing receptors. Thus, GABA_BRs sense chronic perturbations in GABA levels and transduce it to homeostatic changes in synaptic strength. Our results reveal a novel role for GABA_BR as a key regulator of population firing stability and propose that disruption of homeostatic synaptic plasticity may underlie seizure''s persistence in the absence of functional GABA_BRs.Neural circuits achieve an ongoing balance between plasticity and stability to enable adaptations to constantly changing environments while maintaining neuronal activity within a stable regime. Hebbian-like plasticity, reflected by persistent changes in synaptic and intrinsic properties, is crucial for refinement of neural circuits and information storage; however, alone it is unlikely to account for the stable functioning of neural networks (1). In the last 2 decades, major progress has been made toward understanding the homeostatic negative feedback systems underlying restoration of a baseline neuronal function after prolonged activity perturbations (2–4). Homeostatic processes may counteract the instability by adjusting intrinsic neuronal excitability, inhibition-to-excitation balance, and synaptic strength via postsynaptic or presynaptic modifications (5, 6) through a profound molecular reorganization of synaptic proteins (7, 8). These stabilizing mechanisms have been collectively termed homeostatic plasticity. Homeostatic mechanisms enable invariant firing rates and patterns of neural networks composed from intrinsically unstable activity patterns of individual neurons (9).However, nervous systems are not always capable of maintaining constant output. Although some mutations, genetic knockouts, or pharmacologic perturbations induce a compensatory response that restores network firing properties around a predefined “set point” (10), the others remain uncompensated, or their compensation leads to pathological function (11). The inability of neural networks to compensate for a perturbation may result in epilepsy and various types of psychiatric disorders (12). Therefore, determining under which conditions activity-dependent regulation fails to compensate for a perturbation and identifying the key regulatory molecules of neuronal homeostasis is critical for understanding the function and malfunction of central neural circuits.In this work, we explored the mechanisms underlying the failure in stabilizing hippocampal network activity by combining long-term extracellular spike recordings by multielectrode arrays (MEAs), intracellular patch-clamp recordings of synaptic responses, imaging of synaptic vesicle exocytosis, and calcium dynamics, together with FRET-based analysis of intermolecular interactions at individual synapses. Our results demonstrate that metabotropic, G protein-coupled receptors for GABA, GABA_BRs, are essential for firing rate homeostasis in hippocampal networks. We explored the mechanisms by which GABA_BRs gate homeostatic synaptic plasticity. Our study raises the possibility that persistence of epileptic seizures in GABA_BR-deficient mice (13–15) is directly linked to impairments in a homeostatic control system.     

Molecular lock regulates binding of glycine to a primitive NMDA receptor

The earliest metazoan ancestors of humans include the ctenophore Mnemiopsis leidyi. The genome of this comb jelly encodes homologs of vertebrate ionotropic glutamate receptors (iGluRs) that are distantly related to glycine-activated NMDA receptors and that bind glycine with unusually high affinity. Using ligand-binding domain (LBD) mutants for electrophysiological analysis, we demonstrate that perturbing a ctenophore-specific interdomain Arg-Glu salt bridge that is notably absent from vertebrate AMPA, kainate, and NMDA iGluRs greatly increases the rate of recovery from desensitization, while biochemical analysis reveals a large decrease in affinity for glycine. X-ray crystallographic analysis details rearrangements in the binding pocket stemming from the mutations, and molecular dynamics simulations suggest that the interdomain salt bridge acts as a steric barrier regulating ligand binding and that the free energy required to access open conformations in the glycine-bound LBD is largely responsible for differences in ligand affinity among the LBD variants.Glutamate receptor ion channels (iGluRs) are membrane proteins that mediate excitatory synaptic transmission in the brain by detecting release of the amino acid glutamate from nerve terminals (1). In combination with GluN2 subunits, which bind glutamate, NMDA subtype iGluRs use glycine as a coagonist, which binds to GluN1, GluN3A, and GluN3B subunits (2–6). NMDA receptors play key roles in synaptic plasticity and memory formation, and mutations of NMDA receptor genes underlie a diverse set of neurological and psychiatric diseases (7). Like all iGluRs, NMDA receptors are assembled from modular subunits containing amino terminal and S1S2 ligand binding domains (LBDs), which can be genetically isolated and expressed as soluble proteins for biochemical and structural analysis (4, 8–10). The LBDs of both the glutamate and glycine binding subunits are clamshell-shaped proteins of molecular mass around 30 kDa in which two lobes are connected by a hinge formed by antiparallel β-strands; in the activated state, ligands are trapped in a cavity formed when the clamshell closes. Strikingly, the volume of the ligand binding cavity for the GluN1, GluN3A, and GluN3B subunits is just large enough to accommodate glycine, whereas iGluR glutamate binding subunits have cavities that are four to five times larger and bind both glutamate and up to six or seven water molecules (4, 10–13).We recently reported the discovery of glycine-activated iGluRs from the comb jelly Mnemiopsis leidyi and the sea gooseberry Pleurobrachia bachei, candidates for earliest lineage metazoans, for which ML032222a and PbGluR3 glycine complex crystal structures reveal a salt bridge at the perimeter of the ligand binding cleft (14). This salt bridge links the upper and lower lobes of the LBD in the closed cleft glycine-bound conformation. Ctenophore iGluR subunits bind glycine with such high affinity that the ligand cannot be removed by exhaustive dialysis, suggesting an unusually stable ligand-bound closed-cleft conformation, perhaps stabilized by the interdomain salt bridge. Prior electrophysiological and crystallographic studies on vertebrate AMPA and kainate subtype iGluRs revealed that the stability of the closed cleft conformation is determined not only by contacts of the LBD with the neurotransmitter ligand but also by contacts formed between the upper and lower lobes of the clamshell assembly that occur only in the ligand-bound closed-cleft conformation (15, 16). Comparison of crystal structures of ctenophore iGluR LBDs with those of vertebrate NMDA receptor GluN1 and GluN3 subunits that also bind glycine, but for which apo proteins can be prepared without difficulty (4, 10), reveals that the salt bridge is unique to ctenophore iGluRs, further suggesting that it might underlie the high stability of the glycine complex.To investigate this, we prepared ML032222a mutant proteins and analyzed their ligand binding properties using electrophysiological, biochemical, and crystallographic techniques. To gain further insight into how these mutants perturb large-scale LBD dynamics, we computed conformational free energy landscapes for the apo state and glycine complexes of wild-type (WT) ML032222a and the R703K and E423S mutants, which weaken and break the interdomain salt bridge, respectively. This analysis reveals that, similar to vertebrate GluN1 and GluN3 glycine binding subunits, the apo state for ML032222a can access closed cleft conformations, although it is more stable in slightly open conformations. The R703K and E423S mutants destabilize closed cleft conformations for the glycine complex. Conformational dynamics inferred from the free energy landscapes suggest that the interdomain salt bridge is positioned at the most likely point of ligand entry to (and exit from) the binding pocket and thus acts as a steric barrier regulating the binding and dissociation of glycine.     

cAMP/PKA signaling and RIM1alpha mediate presynaptic LTP in the lateral amygdala

NMDA receptor-dependent long-term potentiation (LTP) of glutamatergic synaptic transmission in sensory pathways from auditory thalamus or cortex to the lateral amygdala (LA) underlies the acquisition of auditory fear conditioning. Whereas the mechanisms of postsynaptic LTP at thalamo–LA synapses are well understood, much less is known about the sequence of events mediating presynaptic NMDA receptor-dependent LTP at cortico–LA synapses. Here, we show that presynaptic cortico–LA LTP can be entirely accounted for by a persistent increase in the vesicular release probability. At the molecular level, we found that signaling via the cAMP/PKA pathway is necessary and sufficient for LTP induction. Moreover, by using mice lacking the active-zone protein and PKA target RIM1α (RIM1α^−/−), we demonstrate that RIM1α is required for both chemically and synaptically induced presynaptic LTP. Further analysis of cortico–LA synaptic transmission in RIM1α^−/− mice revealed a deficit in Ca²⁺-release coupling leading to a lower baseline release probability. Our results reveal the molecular mechanisms underlying the induction of presynaptic LTP at cortico–LA synapses and indicate that RIM1α-dependent LTP may involve changes in Ca²⁺-release coupling.     

Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD

Proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) domains play key roles in the assembly and regulation of cellular signaling pathways and represent putative targets for new pharmacotherapeutics. Here we describe the first small-molecule inhibitor (FSC231) of the PDZ domain in protein interacting with C kinase 1 (PICK1) identified by a screening of ~44,000 compounds in a fluorescent polarization assay. The inhibitor bound the PICK1 PDZ domain with an affinity similar to that observed for endogenous peptide ligands (K_i~10.1 μM). Mutational analysis, together with computational docking of the compound in simulations starting from the PDZ domain structure, identified the binding mode of FSC231. The specificity of FSC231 for the PICK1 PDZ domain was supported by the lack of binding to PDZ domains of postsynaptic density protein 95 (PSD-95) and glutamate receptor interacting protein 1 (GRIP1). Pretreatment of cultured hippocampal neurons with FSC231 inhibited coimmunopreciptation of the AMPA receptor GluR2 subunit with PICK1. In agreement with inhibiting the role of PICK1 in GluR2 trafficking, FSC231 accelerated recycling of pHluorin-tagged GluR2 in hippocampal neurons after internalization in response to NMDA receptor activation. FSC231 blocked the expression of both long-term depression and long-term potentiation in hippocampal CA1 neurons from acute slices, consistent with inhibition of the bidirectional function of PICK1 in synaptic plasticity. Given the proposed role of the PICK1/AMPA receptor interaction in neuropathic pain, excitotoxicity, and cocaine addiction, FSC231 might serve as a lead in the future development of new therapeutics against these conditions.     

NMDA receptor–BK channel coupling regulates synaptic plasticity in the barrel cortex

Postsynaptic N-methyl-D-aspartate receptors (NMDARs) are crucial mediators of synaptic plasticity due to their ability to act as coincidence detectors of presynaptic and postsynaptic neuronal activity. However, NMDARs exist within the molecular context of a variety of postsynaptic signaling proteins, which can fine-tune their function. Here, we describe a form of NMDAR suppression by large-conductance Ca²⁺- and voltage-gated K⁺ (BK) channels in the basal dendrites of a subset of barrel cortex layer 5 pyramidal neurons. We show that NMDAR activation increases intracellular Ca²⁺ in the vicinity of BK channels, thus activating K⁺ efflux and strong negative feedback inhibition. We further show that neurons exhibiting such NMDAR–BK coupling serve as high-pass filters for incoming synaptic inputs, precluding the induction of spike timing–dependent plasticity. Together, these data suggest that NMDAR-localized BK channels regulate synaptic integration and provide input-specific synaptic diversity to a thalamocortical circuit.

Glutamate is the primary excitatory chemical transmitter in the mammalian central nervous system (CNS), where it is essential for neuronal viability, network function, and behavioral responses (1). Glutamate activates a variety of pre- and postsynaptic receptors, including ionotropic receptors (iGluRs) that form ligand-gated cation-permeable ion channels. The iGluR superfamily includes α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), kainate receptors, and N-methyl-D-aspartate receptors (NMDARs), all of which form tetrameric assemblies that are expressed throughout the CNS (2).NMDARs exhibit high sensitivity to glutamate (apparent half maximal effective concentration in the micromolar range) and a voltage-dependent block by Mg²⁺ (3, 4), slow gating kinetics (5), and high permeability to Ca²⁺ (6, 7) (for a review, see ref. 8). Together, these characteristics confer postsynaptic NMDARs with the ability to detect and decode coincidental activity of pre- and postsynaptic neurons: presynaptic glutamate release brings about the occupation of the agonist-binding site and AMPAR-driven postsynaptic depolarization, removing the voltage-dependent Mg²⁺ block. The coincidence of these two events leads to NMDAR activation and a Ca²⁺ influx through the channel (8, 9), which initiates several forms of synaptic plasticity (10, 11).Large-conductance Ca²⁺- and voltage-gated K⁺ (BK) channels are opened by a combination of membrane depolarization and relatively high levels of intracellular Ca²⁺ (12, 13). In CNS neurons, such micromolar Ca²⁺ increases are usually restricted to the immediate vicinity of Ca²⁺ sources, including voltage-gated Ca²⁺ channels (VGCCs) (14–16) and ryanodine receptors (RyRs) (17, 18). In addition, Ca²⁺ influx through nonselective cation-permeable channels, including NMDARs, has also been shown to activate BK channels in granule cells from the olfactory bulb and dentate gyrus (19–21). In these neurons, Ca²⁺ entry through NMDARs opens BK channels in somatic and perisomatic regions, causing the repolarization of the surrounding plasma membrane and subsequent closure of NMDARs. Because BK channel activation blunts NMDAR-mediated excitatory responses, it provides a negative feedback mechanism that modulates the excitability of these neurons (19, 20). Thus, the same characteristics that make NMDARs key components in excitatory synaptic transmission and plasticity can paradoxically give rise to an inhibitory response when NMDARs are located in the proximity of BK channels. However, it is unclear whether functional NMDAR–BK coupling is relevant at dendrites and dendritic spines.The barrel field area in the primary somatosensory cortex, also known as the barrel cortex (BC), processes information from peripheral sensory receptors for onward transmission to cortical and subcortical brain regions (22, 23). Sensory information is received in the BC from different nuclei of the thalamus. Among these nuclei, the ventral posterior medial nucleus, ventrobasal nucleus, and posterior medial nucleus are known to directly innervate layer 5 pyramidal neurons (BC-L5PNs) (24–27). In basal dendrites of BC-L5PN, the coactivation of neighboring dendritic inputs can initiate NMDAR-mediated dendritically restricted spikes characterized by large Ca²⁺ transients and long-lasting depolarizations (28–30), providing the appropriate environment for BK activation.To determine whether functional NMDAR–BK coupling plays a role in synaptic transmission, and potentially synaptic plasticity, we investigated the thalamocortical synapses at basal dendrites of BC-L5PNs. We found that the suppression of NMDAR activity by BK channels occurs in the basal dendrites of about 40% of BC-L5PNs, where NMDAR activation triggers strong negative feedback inhibition by delivering Ca²⁺ to nearby BK channels. This inhibition regulates the amplitude of postsynaptic responses and increases the threshold for the induction of synaptic plasticity. Our findings thus unveil a calibration mechanism that can decode the amount and frequency of afferent synaptic inputs by selectively attenuating synaptic plasticity and providing input-specific synaptic diversity to a thalamocortical circuit.