Surface densities prewet a near-critical membrane.

Recent work has highlighted roles for thermodynamic phase behavior in diverse cellular processes. Proteins and nucleic acids can phase separate into three-dimensional liquid droplets in the cytoplasm and nucleus and the plasma membrane of animal cells appears tuned close to a two-dimensional liquid-liquid critical point. In some examples, cytoplasmic proteins aggregate at plasma membrane domains, forming structures such as the postsynaptic density and diverse signaling clusters. Here we examine the physics of these surface densities, employing minimal simulations of polymers prone to phase separation coupled to an Ising membrane surface in conjunction with a complementary Landau theory. We argue that these surface densities are a phase reminiscent of prewetting, in which a molecularly thin three-dimensional liquid forms on a usually solid surface. However, in surface densities the solid surface is replaced by a membrane with an independent propensity to phase separate. We show that proximity to criticality in the membrane dramatically increases the parameter regime in which a prewetting-like transition occurs, leading to a broad region where coexisting surface phases can form even when a bulk phase is unstable. Our simulations naturally exhibit three-surface phase coexistence even though both the membrane and the polymer bulk only display two-phase coexistence on their own. We argue that the physics of these surface densities may be shared with diverse functional structures seen in eukaryotic cells.

Diversity of synaptic protein complexes as a function of the abundance of their constituent proteins: A modeling approach.

The postsynaptic density (PSD) is a dense protein network playing a key role in information processing during learning and memory, and is also indicated in a number of neurological disorders. Efforts to characterize its detailed molecular organization are encumbered by the large variability of the abundance of its constituent proteins both spatially, in different brain areas, and temporally, during development, circadian rhythm, and also in response to various stimuli. In this study we ran large-scale stochastic simulations of protein binding events to predict the presence and distribution of PSD complexes. We simulated the interactions of seven major PSD proteins (NMDAR, AMPAR, PSD-95, SynGAP, GKAP, Shank3, Homer1) based on previously published, experimentally determined protein abundance data from 22 different brain areas and 42 patients (altogether 524 different simulations). Our results demonstrate that the relative ratio of the emerging protein complexes can be sensitive to even subtle changes in protein abundances and thus explicit simulations are invaluable to understand the relationships between protein availability and complex formation. Our observations are compatible with a scenario where larger supercomplexes are formed from available smaller binary and ternary associations of PSD proteins. Specifically, Homer1 and Shank3 self-association reactions substantially promote the emergence of very large protein complexes. The described simulations represent a first approximation to assess PSD complex abundance, and as such, use significant simplifications. Therefore, their direct biological relevance might be limited but we believe that the major qualitative findings can contribute to the understanding of the molecular features of the postsynapse.

BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3.

Brefeldin A-resistant ArfGEF 2 (BRAG2) [or Iqsec1 (IQ motif and Sec7 domain-containing protein 1)] is a guanine nucleotide exchange factor for ADP ribosylation factor 6 (Arf6), a small GTPase implicated in the membrane trafficking between the plasma membrane and endosomes. BRAG2 regulates Arf6-dependent endocytosis of AMPA receptors (AMPARs) through the direct interaction during the hippocampal long-term depression. However, the molecular mechanism by which the BRAG2-Arf6 pathway links AMPARs to the endocytic machinery remains elusive. Herein, using mouse brains of both sexes, we demonstrated that BRAG2a, an alternative isoform with a long C-terminal insert containing a proline-rich domain and type I PDZ-binding motif, was selectively localized to the excitatory postsynaptic density (PSD). Using yeast two-hybrid screening, we identified PSD-95 and endophilin 1/3 as BRAG2a-binding partners in the brain. The interaction with PSD-95 was required for synaptic targeting of BRAG2a. In cultured hippocampal neurons, stimulation of group I metabotropic glutamate receptors (mGluRs) increased the interaction of BRAG2a with endophilin 3 and concomitant Arf6 activation in a time-dependent manner. Knockdown of BRAG2 in cultured hippocampal neurons blocked the mGluR-dependent decrease in surface AMPAR levels, which was rescued by introducing wild-type BRAG2a, but not wild-type BRAG2b or BRAG2a mutants lacking the ability to activate Arf6 or to interact with endophilin 3 or PSD-95. Further postembedding immunoelectron microscopic analysis revealed the preorganized lateral distribution of BRAG2a, Arf6, and endophilin 3 for efficient endocytosis at the postsynaptic membrane. Together, the present findings unveiled a novel molecular mechanism by which BRAG2a links AMPARs to the clathrin-dependent endocytic pathway through its interaction with PSD-95 and endophilin 3.SIGNIFICANCE STATEMENT BRAG2/Iqsec1 is a GDP/GTP exchange factor for ADP ribosylation factor 6 (Arf6), a small GTPase implicated in the membrane trafficking between the plasma membrane and endosomes, and regulates Arf6-dependent endocytosis of AMPARs through direct interaction during hippocampal long-term depression, one of the mechanisms of synaptic plasticity related to learning and memory. However, the molecular mechanism by which the BRAG2-Arf6 pathway links AMPARs to the endocytic machinery remains elusive. Here, we identified isoform-specific mechanisms of BRAG2-mediated AMPAR internalization. We demonstrated that the interaction of BRAG2a isoform with PSD-95 and endophilin 3 was required for the mGluR-dependent decrease in surface AMPARs in hippocampal neurons. These results unveiled a novel molecular mechanism by which BRAG2 links AMPARs to the clathrin-mediated endocytic machinery at postsynaptic sites.

Functional consequences of mutations in postsynaptic scaffolding proteins and relevance to psychiatric disorders.

Functional studies on postsynaptic scaffolding proteins at excitatory synapses have revealed a plethora of important roles for synaptic structure and function. In addition, a convergence of recent in vivo functional evidence together with human genetics data strongly suggest that mutations in a variety of these postsynaptic scaffolding proteins may contribute to the etiology of diverse human psychiatric disorders such as schizophrenia, autism spectrum disorders, and obsessive-compulsive spectrum disorders. Here we review the most recent evidence for several key postsynaptic scaffolding protein families and explore how mouse genetics and human genetics have intersected to advance our knowledge concerning the contributions of these important players to complex brain function and dysfunction.

Neonatal Isoflurane Anesthesia or Disruption of Postsynaptic Density-95 Protein Interactions Change Dendritic Spine Densities and Cognitive Function in Juvenile Mice.

BACKGROUND: Experimental evidence shows postnatal exposure to anesthesia negatively affects brain development. The PDZ2 domain, mediating protein-protein interactions of the postsynaptic density-95 protein, serves as a molecular target for several inhaled anesthetics. The authors hypothesized that early postnatal disruption of postsynaptic density-95 PDZ2 domain interactions has persistent effects on dendritic spines and cognitive function. METHODS: One-week-old mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active postsynaptic density-95 wild-type PDZ2 peptide along with their respective controls. A subset of these mice also received 4 mg/kg of the nitric oxide donor molsidomine. Hippocampal spine density, long-term potentiation, novel object recognition memory, and fear learning and memory were evaluated in mice. RESULTS: Exposure of 7-day-old mice to isoflurane or postsynaptic density-95 wild-type PDZ2 peptide relative to controls causes: (1) a long-term decrease in mushroom spines at 7 weeks (mean ± SD [spines per micrometer]): control (0.8 ± 0.2) versus isoflurane (0.4 ± 0.2), P < 0.0001, and PDZ2MUT (0.7 ± 0.2) versus PDZ2WT (0.4 ± 0.2), P < 0.001; (2) deficits in object recognition at 6 weeks (mean ± SD [recognition index]): naïve (70 ± 8) versus isoflurane (55 ± 14), P = 0.010, and control (65 ± 13) versus isoflurane (55 ± 14), P = 0.045, and PDZ2MUT (64 ±11) versus PDZ2WT (53 ± 18), P = 0.045; and (3) deficits in fear learning at 7 weeks and memory at 8 weeks (mean ± SD [% freezing duration]): Learning, control (69 ± 12) versus isoflurane (52 ± 13), P < 0.0001, and PDZ2MUT (65 ± 14) versus PDZ2WT (55 ± 14) P = 0.011, and Memory, control (80 ± 17) versus isoflurane (56 ± 23), P < 0.0001 and PDZ2MUT (73 ± 18) versus PDZ2WT (44 ± 19) P < 0.0001. Impairment in long-term potentiation has fully recovered here at 7 weeks (mean ± SD [% baseline]): control (140 ± 3) versus isoflurane (137 ± 8), P = 0.560, and PDZ2MUT (136 ± 17) versus PDZ2WT (128 ± 11), P = 0.512. The isoflurane induced decrease in mushroom spines was preventable by introduction of a nitric oxide donor. CONCLUSIONS: Early disruption of PDZ2 domain-mediated protein-protein interactions mimics isoflurane in decreasing mushroom spine density and causing learning and memory deficits in mice. Prevention of the decrease in mushroom spine density with a nitric oxide donor supports a role for neuronal nitric oxide synthase pathway in mediating this cellular change associated with cognitive impairment.

Modality-specific thalamocortical inputs instruct the identity of postsynaptic L4 neurons.

During development, thalamocortical (TC) input has a critical role in the spatial delineation and patterning of cortical areas, yet the underlying cellular and molecular mechanisms that drive cortical neuron differentiation are poorly understood. In the primary (S1) and secondary (S2) somatosensory cortex, layer 4 (L4) neurons receive mutually exclusive input originating from two thalamic nuclei: the ventrobasalis (VB), which conveys tactile input, and the posterior nucleus (Po), which conveys modulatory and nociceptive input. Recently, we have shown that L4 neuron identity is not fully committed postnatally, implying a capacity for TC input to influence differentiation during cortical circuit assembly. Here we investigate whether the cell-type-specific molecular and functional identity of L4 neurons is instructed by the origin of their TC input. Genetic ablation of the VB at birth resulted in an anatomical and functional rewiring of Po projections onto L4 neurons in S1. This induced acquisition of Po input led to a respecification of postsynaptic L4 neurons, which developed functional molecular features of Po-target neurons while repressing VB-target traits. Respecified L4 neurons were able to respond both to touch and to noxious stimuli, in sharp contrast to the normal segregation of these sensory modalities in distinct cortical circuits. These findings reveal a behaviourally relevant TC-input-type-specific control over the molecular and functional differentiation of postsynaptic L4 neurons and cognate intracortical circuits, which instructs the development of modality-specific neuronal and circuit properties during corticogenesis.

Functional organization of postsynaptic glutamate receptors.

Glutamate receptors are the most abundant excitatory neurotransmitter receptors in the brain, responsible for mediating the vast majority of excitatory transmission in neuronal networks. The AMPA- and NMDA-type ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate the fast synaptic responses, while metabotropic glutamate receptors (mGluRs) are coupled to downstream signaling cascades that act on much slower timescales. These functionally distinct receptor sub-types are co-expressed at individual synapses, allowing for the precise temporal modulation of postsynaptic excitability and plasticity. Intriguingly, these receptors are differentially distributed with respect to the presynaptic release site. While iGluRs are enriched in the core of the synapse directly opposing the release site, mGluRs reside preferentially at the border of the synapse. As such, to understand the differential contribution of these receptors to synaptic transmission, it is important to not only consider their signaling properties, but also the mechanisms that control the spatial segregation of these receptor types within synapses. In this review, we will focus on the mechanisms that control the organization of glutamate receptors at the postsynaptic membrane with respect to the release site, and discuss how this organization could regulate synapse physiology.

Structural basis for PDZ domain interactions in the post-synaptic density scaffolding protein Shank3.

The Shank proteins are crucial scaffolding elements of the post-synaptic density (PSD). One of the best-characterized domains in Shank is the PDZ domain, which binds to C-terminal segments of several other PSD proteins. We carried out a detailed structural analysis of Shank3 PDZ domain-peptide complexes, to understand determinants of binding affinity towards different ligand proteins. Ligand peptides from four different proteins were cocrystallized with the Shank3 PDZ domain, and binding affinities were determined calorimetrically. In addition to conserved class I interactions between the first and third C-terminal peptide residue and Shank3, side chain interactions of other residues in the peptide with the PDZ domain are important factors in defining affinity. Structural conservation suggests that the binding specificities of the PDZ domains from different Shanks are similar. Two conserved buried water molecules in PDZ domains may affect correct local folding of ligand recognition determinants. The solution structure of a tandem Shank3 construct containing the SH3 and PDZ domains showed that the two domains are close to each other, which could be of relevance, when recognizing and binding full target proteins. The SH3 domain did not affect the affinity of the PDZ domain towards short target peptides, and the schizophrenia-linked Shank3 mutation R536W in the linker between the domains had no effect on the structure or peptide interactions of the Shank3 SH3-PDZ unit. Our data show the spatial arrangement of two adjacent Shank domains and pinpoint affinity determinants for short PDZ domain ligands with limited sequence homology.

Roles of afadin in functional differentiations of hippocampal mossy fiber synapse.

A hippocampal mossy fiber synapse has a complex structure and is implicated in learning and memory. In this synapse, the mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions and wrap around a multiply-branched spine, forming synaptic junctions. We have recently shown using transmission electron microscopy, immunoelectron microscopy and serial block face-scanning electron microscopy that atypical puncta adherentia junctions are formed in the afadin-deficient mossy fiber synapse and that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities and the density of synaptic vesicles docked to active zones are decreased in the afadin-deficient synapse. We investigated here the roles of afadin in the functional differentiations of the mossy fiber synapse using the afadin-deficient mice. The electrophysiological studies showed that both the release probability of glutamate and the postsynaptic responsiveness to glutamate were markedly reduced, but not completely lost, in the afadin-deficient mossy fiber synapse, whereas neither long-term potentiation nor long-term depression was affected. These results indicate that afadin plays roles in the functional differentiations of the presynapse and the postsynapse of the hippocampal mossy fiber synapse.

Protein Crowding within the Postsynaptic Density Can Impede the Escape of Membrane Proteins.

Mechanisms regulating lateral diffusion and positioning of glutamate receptors within the postsynaptic density (PSD) determine excitatory synaptic strength. Scaffold proteins in the PSD are abundant receptor binding partners, yet electron microscopy suggests that the PSD is highly crowded, potentially restricting the diffusion of receptors regardless of binding. However, the contribution of macromolecular crowding to receptor retention remains poorly understood. We combined experimental and computational approaches to test the effect of synaptic crowding on receptor movement and positioning in Sprague Dawley rat hippocampal neurons. We modeled AMPA receptor diffusion in synapses where the distribution of scaffold proteins was determined from photoactivated localization microscopy experiments, and receptor-scaffold association and dissociation rates were adjusted to fit single-molecule tracking and fluorescence recovery measurements. Simulations predicted that variation of receptor size strongly influences the fractional synaptic area the receptor may traverse, and the proportion that may exchange in and out of the synapse. To test the model experimentally, we designed a set of novel transmembrane (TM) probes. A single-pass TM protein with one PDZ binding motif concentrated in the synapse as do AMPARs yet was more mobile there than the much larger AMPAR. Furthermore, either the single binding motif or an increase in cytoplasmic bulk through addition of a single GFP slowed synaptic movement of a small TM protein. These results suggest that both crowding and binding limit escape of AMPARs from the synapse. Moreover, tight protein packing within the PSD may modulate the synaptic dwell time of many TM proteins important for synaptic function. SIGNIFICANCE STATEMENT: Small alterations to the distribution within synapses of key transmembrane proteins, such as receptors, can dramatically change synaptic strength. Indeed, many diseases are thought to unbalance neural circuit function in this manner. Processes that regulate this in healthy synapses are unclear, however. By combining computer simulations with imaging methods that examined protein dynamics at multiple scales in space and time, we showed that both steric effects and protein-protein binding each regulate the mobility of receptors in the synapse. Our findings extend our knowledge of the synapse as a crowded environment that counteracts molecular diffusion, and support the idea that both molecular collisions and biochemical binding can be involved in the regulation of neural circuit performance.

Food restriction induces synaptic incorporation of calcium-permeable AMPA receptors in nucleus accumbens.

Chronic food restriction potentiates behavioral and cellular responses to drugs of abuse and D-1 dopamine receptor agonists administered systemically or locally in the nucleus accumbens (NAc). However, the alterations in NAc synaptic transmission underlying these effects are incompletely understood. AMPA receptor trafficking is a major mechanism for regulating synaptic strength, and previous studies have shown that both sucrose and d-amphetamine rapidly alter the abundance of AMPA receptor subunits in the NAc postsynaptic density (PSD) in a manner that differs between food-restricted and ad libitum fed rats. In this study we examined whether food restriction, in the absence of reward stimulus challenge, alters AMPAR subunit abundance in the NAc PSD. Food restriction was found to increase surface expression and, specifically, PSD abundance, of GluA1 but not GluA2, suggesting synaptic incorporation of GluA2-lacking Ca2+-permeable AMPARs (CP-AMPARs). Naspm, an antagonist of CP-AMPARs, decreased the amplitude of evoked EPSCs in NAc shell, and blocked the enhanced locomotor response to local microinjection of the D-1 receptor agonist, SKF-82958, in food-restricted, but not ad libitum fed, subjects. Although microinjection of the D-2 receptor agonist, quinpirole, also induced greater locomotor activation in food-restricted than ad libitum fed rats, this effect was not decreased by Naspm. Taken together, the present findings are consistent with the synaptic incorporation of CP-AMPARs in D-1 receptor-expressing medium spiny neurons in NAc as a mechanistic underpinning of the enhanced responsiveness of food-restricted rats to natural rewards and drugs of abuse.

The MuSK activator agrin has a separate role essential for postnatal maintenance of neuromuscular synapses.

The motoneural control of skeletal muscle contraction requires the neuromuscular junction (NMJ), a midmuscle synapse between the motor nerve and myotube. The formation and maintenance of NMJs are orchestrated by the muscle-specific receptor tyrosine kinase (MuSK). Motor neuron-derived agrin activates MuSK via binding to MuSK's coreceptor Lrp4, and genetic defects in agrin underlie a congenital myasthenic syndrome (an NMJ disorder). However, MuSK-dependent postsynaptic differentiation of NMJs occurs in the absence of a motor neuron, indicating a need for nerve/agrin-independent MuSK activation. We previously identified the muscle protein Dok-7 as an essential activator of MuSK. Although NMJ formation requires agrin under physiological conditions, it is dispensable for NMJ formation experimentally in the absence of the neurotransmitter acetylcholine, which inhibits postsynaptic specialization. Thus, it was hypothesized that MuSK needs agrin together with Lrp4 and Dok-7 to achieve sufficient activation to surmount inhibition by acetylcholine. Here, we show that forced expression of Dok-7 in muscle enhanced MuSK activation in mice lacking agrin or Lrp4 and restored midmuscle NMJ formation in agrin-deficient mice, but not in Lrp4-deficient mice, probably due to the loss of Lrp4-dependent presynaptic differentiation. However, these NMJs in agrin-deficient mice rapidly disappeared after birth, and postsynaptic specializations emerged ectopically throughout myotubes whereas exogenous Dok-7-mediated MuSK activation was maintained. These findings demonstrate that the MuSK activator agrin plays another role essential for the postnatal maintenance, but not for embryonic formation, of NMJs and also for the postnatal, but not prenatal, midmuscle localization of postsynaptic specializations, providing physiological and pathophysiological insight into NMJ homeostasis.

The matrix protein Hikaru genki localizes to cholinergic synaptic clefts and regulates postsynaptic organization in the Drosophila brain.

The synaptic cleft, a crucial space involved in neurotransmission, is filled with extracellular matrix that serves as a scaffold for synaptic differentiation. However, little is known about the proteins present in the matrix and their functions in synaptogenesis, especially in the CNS. Here, we report that Hikaru genki (Hig), a secreted protein with an Ig motif and complement control protein domains, localizes specifically to the synaptic clefts of cholinergic synapses in the Drosophila CNS. The data indicate that this specific localization is achieved by capture of secreted Hig in synaptic clefts, even when it is ectopically expressed in glia. In the absence of Hig, the cytoskeletal scaffold protein DLG accumulated abnormally in cholinergic postsynapses, and the synaptic distribution of acetylcholine receptor (AchR) subunits Dα6 and Dα7 significantly decreased. hig mutant flies consistently exhibited resistance to the AchR agonist spinosad, which causes lethality by specifically activating the Dα6 subunit, suggesting that loss of Hig compromises the cholinergic synaptic activity mediated by Dα6. These results indicate that Hig is a specific component of the synaptic cleft matrix of cholinergic synapses and regulates their postsynaptic organization in the CNS.

PICK1 mediates synaptic recruitment of AMPA receptors at neurexin-induced postsynaptic sites.

In the CNS, synapse formation and maturation play crucial roles in the construction and consolidation of neuronal circuits. Neurexin and neuroligin localize on the opposite sides of synaptic membrane and interact with each other to promote the assembly and specialization of synapses. However, the excitatory synapses induced by the neurexin-neuroligin complex are initially immature synapses that lack AMPA receptors. Previously, PICK1 (protein interacting with C kinase 1) was shown to cluster and regulate the synaptic localization of AMPA receptors. Here, we report that during synaptogenesis induced by neurexin in cultured neurons from rat hippocampus, PICK1 recruited AMPA receptors to immature postsynaptic sites. This synaptic recruitment of AMPA receptors depended on the interaction between GluA2 and PICK1, and on the lipid-binding ability of PICK1, but not the interaction between PICK1 and neuroligin. Last, our results demonstrated that the recruitment of GluA2 to synapses could be prevented by ICA69 (islet cell autoantigen 69 kDa), a key binding partner of PICK1. Our study showed that PICK1, being negatively regulated by ICA69, could facilitate synapse maturation.

Pansynaptic enlargement at adult cortical connections strengthened by experience.

Behavioral experience alters the strength of neuronal connections in adult neocortex. These changes in synaptic strength are thought to be central to experience-dependent plasticity, learning, and memory. However, it is not known how changes in synaptic transmission between neurons become persistent, thereby enabling the storage of previous experience. A long-standing hypothesis is that altered synaptic strength is maintained by structural modifications to synapses. However, the extent of synaptic modifications and the changes in neurotransmission that the modifications support remain unclear. To address these questions, we recorded from pairs of synaptically connected layer 2/3 pyramidal neurons in the barrel cortex and imaged their contacts with high-resolution confocal microscopy after altering sensory experience by whisker trimming. Excitatory connections strengthened by experience exhibited larger axonal varicosities, dendritic spines, and interposed contact zones. Electron microscopy showed that contact zone size was strongly correlated with postsynaptic density area. Therefore, our findings indicate that whole synapses are larger at strengthened connections. Synaptic transmission was both stronger and more reliable following experience-dependent synapse enlargement. Hence, sensory experience modified both presynaptic and postsynaptic function. Our findings suggest that the enlargement of synaptic contacts is an integral part of long-lasting strengthening of cortical connections and, hence, of information storage in the neocortex.

BDNF-dependent recycling facilitates TrkB translocation to postsynaptic density during LTP via a Rab11-dependent pathway.

Brain-derived neurotrophic factor (BDNF) plays an important role in the activity-dependent regulation of synaptic structure and function via tropomyosin related kinase B (TrkB) receptor activation. However, whether BDNF could regulate TrkB levels at synapse during long-term potentiation (LTP) is still unknown. We show in cultured rat hippocampal neurons that chemical LTP (cLTP) stimuli selectively promote endocytic recycling of BDNF-dependent full-length TrkB (TrkB-FL) receptors, but not isoform T1 (TrkB.T1) receptors, via a Rab11-dependent pathway. Moreover, neuronal-activity-enhanced TrkB-FL recycling could facilitate receptor translocation to postsynaptic density and enhance BDNF-induced extracellular signal-regulated kinase and phosphatidylinositol 3-kinase activation and rat hippocampal neuron survival. Finally, we found that cLTP could stimulate the switch of Rab11 from an inactive to an active form and that GTP-bound Rab11 could enhance the interaction between TrkB-FL and PSD-95. Therefore, the recycling endosome could serve as a reserve pool to supply TrkB-FL receptors for LTP maintenance. These findings provide a mechanistic link between Rab11-dependent endocytic recycling and TrkB modulation of synaptic plasticity.

Subsynaptic AMPA receptor distribution is acutely regulated by actin-driven reorganization of the postsynaptic density.

AMPA receptors (AMPARs) mediate synaptic transmission and plasticity during learning, development, and disease. Mechanisms determining subsynaptic receptor position are poorly understood but are key determinants of quantal size. We used a series of live-cell, high-resolution imaging approaches to measure protein organization within single postsynaptic densities in rat hippocampal neurons. By photobleaching receptors in synapse subdomains, we found that most AMPARs do not freely diffuse within the synapse, indicating they are embedded in a matrix that determines their subsynaptic position. However, time lapse analysis revealed that synaptic AMPARs are continuously repositioned in concert with plasticity of this scaffold matrix rather than simply by free diffusion. Using a fluorescence correlation analysis, we found that across the lateral extent of single PSDs, component proteins were differentially distributed, and this distribution was continually adjusted by actin treadmilling. The C-terminal PDZ ligand of GluA1 did not regulate its mobility or distribution in the synapse. However, glutamate receptor activation promoted subsynaptic mobility. Strikingly, subsynaptic immobility of both AMPARs and scaffold molecules remained essentially intact even after loss of actin filaments. We conclude that receptors are actively repositioned at the synapse by treadmilling of the actin cytoskeleton, an influence which is transmitted only indirectly to receptors via the pliable and surprisingly dynamic internal structure of the PSD.

TNiK is required for postsynaptic and nuclear signaling pathways and cognitive function.

Traf2 and NcK interacting kinase (TNiK) contains serine-threonine kinase and scaffold domains and has been implicated in cell proliferation and glutamate receptor regulation in vitro. Here we report its role in vivo using mice carrying a knock-out mutation. TNiK binds protein complexes in the synapse linking it to the NMDA receptor (NMDAR) via AKAP9. NMDAR and metabotropic receptors bidirectionally regulate TNiK phosphorylation and TNiK is required for AMPA expression and synaptic function. TNiK also organizes nuclear complexes and in the absence of TNiK, there was a marked elevation in GSK3ß and phosphorylation levels of its cognate phosphorylation sites on NeuroD1 with alterations in Wnt pathway signaling. We observed impairments in dentate gyrus neurogenesis in TNiK knock-out mice and cognitive testing using the touchscreen apparatus revealed impairments in pattern separation on a test of spatial discrimination. Object-location paired associate learning, which is dependent on glutamatergic signaling, was also impaired. Additionally, TNiK knock-out mice displayed hyperlocomotor behavior that could be rapidly reversed by GSK3ß inhibitors, indicating the potential for pharmacological rescue of a behavioral phenotype. These data establish TNiK as a critical regulator of cognitive functions and suggest it may play a regulatory role in diseases impacting on its interacting proteins and complexes.

Regulation of glutamate release by heteromeric nicotinic receptors in layer V of the secondary motor region (Fr2) in the dorsomedial shoulder of prefrontal cortex in mouse.

We studied how nicotinic acetylcholine receptors (nAChRs) regulate glutamate release in the secondary motor area (Fr2) of the dorsomedial murine prefrontal cortex, in the presence of steady agonist levels. Fr2 mediates response to behavioral situations that require immediate attention and is a candidate for generating seizures in the frontal epilepsies caused by mutant nAChRs. Morphological analysis showed a peculiar chemoarchitecture and laminar distribution of pyramidal cells and interneurons. Tonic application of 5 µM nicotine on Layer V pyramidal neurons strongly increased the frequency of spontaneous glutamatergic excitatory postsynaptic currents. The effect was inhibited by 1 µM dihydro-ß-erythroidine (which blocks α4-containing nAChRs) but not by 10 nM methyllicaconitine (which blocks α7-containing receptors). Excitatory postsynaptic currents s were also stimulated by 5-iodo-3-[2(S)-azetidinylmethoxy]pyridine, selective for ß2-containing receptors, in a dihydro-ß-erythroidine -sensitive way. We next studied the association of α4 with different populations of glutamatergic terminals, by using as markers the vesicular glutamate transporter type (VGLUT) 1 for corticocortical synapses and VGLUT2 for thalamocortical projecting fibers. Immunoblots showed higher expression of α4 in Fr2, as compared with the somatosensory cortex. Immunofluorescence showed intense VGLUT1 staining throughout the cortical layers, whereas VGLUT2 immunoreactivity displayed a more distinct laminar distribution. In Layer V, colocalization of α4 nAChR subunit with both VGLUT1 and VGLUT2 was considerably stronger in Fr2 than in somatosensory cortex. Thus, in Fr2, α4ß2 nAChRs are expressed in both intrinsic and extrinsic glutamatergic terminals and give a major contribution to control glutamate release in Layer V, in the presence of tonic agonist levels.

The effect of chronic thienorphine administration on long-term potentiation and synaptic structure in rat hippocampus.

Thienorphine is a new nonselective partial agonist of opioid receptors, which is currently under a Phase II clinical trial in China as a new treatment for opioid dependence. In this study, we compared the effect of thienorphine with morphine on long-term potentiation (LTP) in the lateral perforant path (LPP)-granule cell synapse of the rat dentate gyrus (DG). Furthermore, the effect of thienorphine on the synaptic structure of the CA1 hippocampal region and the expression of synaptophysin was investigated. Results indicated interesting differences between thienorphine and morphine on the modulation of hippocampal synaptic plasticity. Chronic thienorphine treatment facilitated LTP in the LPP-DG cell synapses more than chronic morphine treatment. Morphometric measurement and analysis showed that chronic thienorphine administration decreased the length of the active zone and reduced the thickness of CA1 postsynaptic densities compared with the saline group (control), but were elevated compared with the morphine group. Furthermore, the expression of hippocampal synaptophysin was increased with chronic thienorphine administration but reduced with chronic morphine treatment. Taken together, our study clearly demonstrates that chronic thienorphine treatment enhances LTP, modulates hippocampal synaptic structure, and increases the expression of hippocampal synaptophysin. Therefore, further study is warranted to investigate thienorphine as a new treatment for opioid dependence.