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Amyloid β (Aβ) is a central contributor to neuronal damage and cognitive impairment in Alzheimer's disease (AD). Aβ disrupts AMPA receptor-mediated synaptic plasticity, a key factor in early AD progression. Numerous studies propose that Aβ oligomers hinder synaptic plasticity, particularly long-term potentiation (LTP), by disrupting GluA1 (encoded by GRIA1) function, although the precise mechanism remains unclear. In this study, we demonstrate that Aβ mediates the accumulation of GM1 ganglioside in lipid raft domains of cultured cells, and GluA1 exhibits preferential localization in lipid rafts via direct binding to GM1. Aβ enhances the raft localization of GluA1 by increasing GM1 in these areas. Additionally, chemical LTP stimulation induces lipid raft-dependent GluA1 internalization in Aβ-treated neurons, resulting in reduced cell surface and postsynaptic expression of GluA1. Consistent with this, disrupting lipid rafts and GluA1 localization in rafts rescues Aβ-mediated suppression of hippocampal LTP. These findings unveil a novel functional deficit in GluA1 trafficking induced by Aβ, providing new insights into the mechanism underlying AD-associated cognitive dysfunction., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

In the present study, we attempted to temporally and quantitatively analyze the functional contributions of Ca 2+ -permeable (CP) α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) during long-term potentiation (LTP) expression using electrophysiological and pharmacological approaches. In hippocampal CA1 neurons, using 1-naphthyl acetyl spermine (NASPM), a CP-AMPAR antagonist, we began by demonstrating that NASPM-sensitive components, probably including the GluA1 homomer, functionally contributed to about 15% of AMPAR-mediated EPSC amplitude in basal conditions. Then, when NASPM was treated at different time points (3-30 min) after LTP induction, it was found that LTP was almost completely impaired at 3 or 10 min but maintained at 20 or 30 min, although its potentiation was reduced. Further temporal and quantitative analysis revealed that the functional expression of CP-AMPARs began increasing approximately 20 min after LTP induction and reached more than twice the basal level at 30 min. These results suggest that CP-AMPARs in the first 3-10 min of LTP might play an important role in LTP maintenance. Moreover, their decay time was also significantly increased at 30 min, suggesting that CP-AMPARs changed not only quantitatively in LTP but also qualitatively., Competing Interests: Declaration of Competing Interest The authors declare that there were no conflicts of interest., (Copyright © 2023 Elsevier Ltd and Japan Neuroscience Society. All rights reserved.)

Genetic disruption of glycosyltransferases has provided clear information on the roles of their reaction products in the body. Our group has studied the function of glycosphingolipids by genetic engineering of glycosyltransferases in cell culture and in mice, which has demonstrated both expected and unexpected results. Among these findings, aspermatogenesis in ganglioside GM2/GD2 synthase knockout mice was one of the most surprising and intriguing results. There were no sperms in testis, and multinuclear giant cells were detected instead of spermatids. Although serum levels of testosterone in the male mice were extremely low, testosterone accumulated in the interstitial tissues, including Leydig cells, and seemed not to be transferred into the seminiferous tubules or vascular cavity from Leydig cells. This was considered to be the cause of aspermatogenesis and low serum levels of testosterone. Patients with a mutant GM2/GD2 synthase gene (SPG26) showed similar clinical signs, not only in terms of the neurological aspects, but also in the male reproductive system. The mechanisms for testosterone transport by gangliosides are discussed here based on our own results and reports from other laboratories., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Changes in the shape and size of the dendritic spines are critical for synaptic transmission. These morphological changes depend on dynamic assembly of the actin cytoskeleton and occur differently in various types of neurons. However, how the actin dynamics are regulated in a neuronal cell type-specific manner remains largely unknown. We show that Fhod3, a member of the formin family proteins that mediate F-actin assembly, controls the dendritic spine morphogenesis of specific subpopulations of cerebrocortical pyramidal neurons. Fhod3 is expressed specifically in excitatory pyramidal neurons within layers II/III and V of restricted areas of the mouse cerebral cortex. Immunohistochemical and biochemical analyses revealed the accumulation of Fhod3 in postsynaptic spines. Although targeted deletion of Fhod3 in the brain did not lead to any defects in the gross or histological appearance of the brain, the dendritic spines in pyramidal neurons within presumptive Fhod3-positive areas were morphologically abnormal. In primary cultures prepared from the Fhod3-depleted cortex, defects in spine morphology were only detected in Fhod3 promoter-active cells, a small population of pyramidal neurons, and not in Fhod3 promoter-negative pyramidal neurons. Thus, Fhod3 plays a crucial role in dendritic spine morphogenesis only in a specific population of pyramidal neurons in a cell type-specific manner., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

The AMPA-type glutamate receptor (AMPAR) is a homotetrameric or heterotetrameric ion channel composed of various combinations of four subunits (GluA1-4), and its abundance in the synapse determines the strength of synaptic activity. The formation of oligomers in the endoplasmatic reticulum (ER) is crucial for AMPAR subunits' ER-exit and translocation to the cell membrane. Although N-glycosylation on different AMPAR subunits has been shown to regulate the ER-exit of hetero-oligomers, its role in the ER-exit of homo-oligomers remains unclear. In this study, we investigated the role of N-glycans at GluA1N63/N363 and GluA2N370 in ER-exit under the homo-oligomeric expression conditions, whose mutants are known to show low cell surface expressions. In contrast to the N-glycosylation site mutant GluA1N63Q, the cell surface expression levels of GluA1N363Q and GluA2N370Q increased in a time-dependent manner. Unlike wild-type (WT) GluA1, GluA2WT rescued surface GluA2N370Q expression. Additionally, the expression of GluA1N63Q reduced the cell surface expression level of GluA1WT. In conclusion, our findings suggest that these N-glycans have distinct roles in the ER-exit of GluA1 and GluA2 homo-oligomers; N-glycan at GluA1N63 is a prerequisite for GluA1 ER-exit, whereas N-glycans at GluA1N363 and GluA2N370 control the ER-exit rate.

The number and subunit compositions of AMPA receptors (AMPARs), hetero- or homotetramers composed of four subunits GluA1-4, in the synapse is carefully tuned to sustain basic synaptic activity. This enables stimulation-induced synaptic plasticity, which is central to learning and memory. The AMPAR tetramers have been widely believed to be stable from their formation in the endoplasmic reticulum until their proteolytic decomposition. However, by observing GluA1 and GluA2 at the level of single molecules, we find that the homo- and heterotetramers are metastable, instantaneously falling apart into monomers, dimers, or trimers (in 100 and 200 ms, respectively), which readily form tetramers again. In the dendritic plasma membrane, GluA1 and GluA2 monomers and dimers are far more mobile than tetramers and enter and exit from the synaptic regions. We conclude that AMPAR turnover by lateral diffusion, essential for sustaining synaptic function, is largely done by monomers of AMPAR subunits, rather than preformed tetramers.

Learning depends on experience-dependent modification of synaptic efficacy and neuronal connectivity in the brain. We provide direct evidence for physiological roles of the recycling endosome protein GRASP1 in glutamatergic synapse function and animal behavior. Mice lacking GRASP1 showed abnormal excitatory synapse number, synaptic plasticity, and hippocampal-dependent learning and memory due to a failure in learning-induced synaptic AMPAR incorporation. We identified two GRASP1 point mutations from intellectual disability (ID) patients that showed convergent disruptive effects on AMPAR recycling and glutamate uncaging-induced structural and functional plasticity. Wild-type GRASP1, but not ID mutants, rescued spine loss in hippocampal CA1 neurons in Grasp1 knockout mice. Together, these results demonstrate a requirement for normal recycling endosome function in AMPAR-dependent synaptic function and neuronal connectivity in vivo, and suggest a potential role for GRASP1 in the pathophysiology of human cognitive disorders., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Methods of cell biology and electrophysiology using dissociated primary cultured neurons allow in vitro study of molecular functions; however, analysis of intact neuronal circuitry is often preferable. To investigate exogenous genes, viral vectors are most commonly injected using a pipette that is inserted from the top of the cortex. Although there are few reports that describe the success rate of injection in detail, it is sometimes difficult to locate the pipette tip accurately within the CA1 pyramidal cell layer because the pyramidal layer is only 0.1 mm thick. In the present study, we have developed a system to inject viral vectors accurately into the mouse hippocampal CA1 pyramidal cell layer using a stereotaxic injection system with simultaneous electrophysiological monitoring of theta oscillation. The pipette tip was positioned reliably based on integrated values of the theta oscillation in the hippocampal CA1 pyramidal cell layer. This approach allows accurate injection of solutions and provides an efficient method of gene transfer using viral vectors into the hippocampus, which can be a useful tool for studies involving the molecular mechanisms of neuronal functions.

Background Aneurysm formation after stereotactic irradiation of skull base tumors is rare. The formation and rupture of an internal carotid artery (ICA) aneurysm in a patient with skull base Ewing sarcoma/primitive neuroectodermal tumor (PNET), who underwent surgery followed by multiple courses of intensity-modulated radiation therapy (IMRT) and chemotherapy, is described. Case Description A 25-year-old man presented with a sinonasal tumor with intraorbital and intracranial growth. At that time cerebral angiography did not reveal any vascular abnormalities. The lesion was resected subtotally. Histopathologic diagnosis was Ewing sarcoma/PNET. The patient underwent multiple courses of chemotherapy and three courses of IMRT at 3, 28, and 42 months after initial surgery. The total biologically effective dose delivered to the right ICA was 220.2 Gy. Seven months after the third IMRT, the patient experienced profound nasal bleeding that resulted in hypovolemic shock. Angiography revealed a ruptured right C4-C5 aneurysm and irregular stenotic changes of the ICA. Lifesaving endovascular trapping of the right ICA was done. The patient recovered well after surgery but died due to tumor recurrence 6 months later. Conclusion Excessive irradiation of the ICA may occasionally result in aneurysm formation, which should be borne in mind during stereotactic irradiation of malignant skull base tumors.

For the diagnosing modality called MRE (magnetic resonance elastography), the displacement vector of a wave propagating in a human tissue can be measured. The average of the local wavelength from this measured data could be an index for the diagnosing, because the local wave length becomes larger when the tissue is stiffer. By assuming that the local form of the wave is given approximately as multiple complex plane waves, we identify the real part of the complex linear phase of the strongest plane wave of this multiple complex plane waves, by first applying the FBI transform (Fourier-Bros-Iagolnitzer transform) with an appropriate size of Gaussian window and then taking the maximum of the modulus of the transform with respect to the Fourier variable. The real part of the linear phase is nothing but the real inner product of the wave vector and the position vector. Similarly the imaginary part of the linear phase describes the attenuation of the wave and it is given as a real inner product of a real vector and the position vector. This vector can also be recovered by our method. We also apply these methods to design some denoising and filtering for noisy MRE data.

Objective: Notch3 is critically important for the structure and myogenic response of distal arteries, particularly of cerebral arteries. However, signaling pathways acting downstream of Notch3 remain largely unknown., Methods and Results: Transcriptome analysis using tail arteries of Notch3-null mice identified a core set of 17 novel Notch3-regulated genes confirmed in tail or brain arteries. Postnatal deletion of RBP-Jκ in smooth muscle cells recapitulated the structural, functional, and molecular defects of brain arteries induced by Notch3 deficiency. Transient in vivo blockade of the Notch pathway with γ-secretase inhibitors uncovered, in addition to Notch3, 6 immediate responders, including the voltage-gated potassium channel Kv1.5, which opposes to myogenic constriction of brain arteries, and the glutamate receptor-interacting protein 2 (Grip2) with no previously established role in the cerebrovasculature. We identified a vascular smooth muscle cell isoform of Grip2. We showed that Notch3-RBP-Jκ specifically regulates this isoform. Finally, we found that cerebral arteries of Grip2 mutant mice, which express an N-terminally truncated Grip2 protein, exhibited selective attenuation of pressure-induced contraction., Conclusions: Our data provide insight into how Notch3 signals in the brain arteries, establishing the postnatal requirement of smooth muscle RBP-Jκ in this context. Notch3-regulated transcriptome provides potential for modulating myogenic response in the cerebrovasculature.

We measured the angular dependence of central and off-axis detectors in a 2D ionization chamber array, MatriXX, and applied correction factors (CFs) to improve the accuracy of composite dose verification of IMRT and VMAT. The MatriXX doses were measured with a 10° step for gantry angles (θ) of 0°-180°, and a 1° step for lateral angles of 90°-110° in a phantom, with a 30 × 10 cm2 field for 6 MV and 10 MV photons. The MatriXX doses were also calculated under the same conditions by the Monte Carlo (MC) algorithm. The CFs for the angular dependence of MatriXX were obtained as a function of θ from the ratios of MatriXX-measured doses to MC-calculated doses, and normalized at θ = 0°. The corrected MatriXX were validated with different fields, various simple plans, and clinical treatment plans. The dose distributions were compared with those of MC calculations and film. The absolute doses were also compared with ionization chamber and MC-calculated doses. The angular dependence of MatriXX showed over-responses of up to 6% and 4% at θ = 90° and under-responses of up to 15% and 11% at 92°, and 8% and 5% at 180° for 6 MV and 10 MV photons, respectively. At 92°, the CFs for the off-axis detectors were larger by up to 7% and 6% than those for the central detectors for 6 MV and 10 MV photons, respectively, and were within 2.5% at other gantry angles. For simple plans, MatriXX doses with angular correction were within 2% of those measured with the ionization chamber at the central axis and off-axis. For clinical treatment plans, MatriXX with angular correction agreed well with dose distributions calculated by the treatment planning system (TPS) for gamma evaluation at 3% and 3 mm. The angular dependence corrections of MatriXX were useful in improving the measurement accuracy of composite dose verification of IMRT and VMAT.

To evaluate the involvement of trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) GluR2 and GluR3 subunits in an acute inflammatory orofacial pain, we analyzed nocifensive behavior, phosphorylated extracellular signal-regulated kinase (pERK) and Fos expression in Vi/Vc, Vc and C1/C2 in GluR2 delta7 knock-in (KI), GluR3 delta7 KI mice and wild-type mice. We also studied Vc neuronal activity to address the hypothesis that trafficking of GluR2 and GluR3 subunits plays an important role in Vi/Vc, Vc and C1/C2 neuronal activity associated with orofacial inflammation in these mice. Late nocifensive behavior was significantly depressed in GluR2 delta7 KI and GluR3 delta7 KI mice. In addition, the number of pERK-immunoreactive (IR) cells was significantly decreased bilaterally in the Vi/Vc, Vc and C1/C2 in GluR2 delta7 KI and GluR3 delta7 KI mice compared to wild-type mice at 40 min after formalin injection, and was also significantly smaller in GluR3 delta7 KI compared to GluR2 delta7 KI mice. The number of Fos protein-IR cells in the ipsilateral Vi/Vc, Vc and C1/C2 was also significantly smaller in GluR2 delta7 KI and GluR3 delta7 KI mice compared to wild-type mice 40 min after formalin injection. Nociceptive neurons functionally identified as wide dynamic range neurons in the Vc, where pERK- and Fos protein-IR cell expression was prominent, showed significantly lower spontaneous activity in GluR2 delta7 KI and GluR3 delta7 KI mice than wild-type mice following formalin injection. These findings suggest that GluR2 and GluR3 trafficking is involved in the enhancement of Vi/Vc, Vc and C1/C2 nociceptive neuronal excitabilities at 16-60 min following formalin injection, resulting in orofacial inflammatory pain.

From a genetic screen for Drosophila melanogaster mutants with altered ethanol tolerance, we identified intolerant (intol), a novel allele of discs large 1 (dlg1). Dlg1 encodes Discs Large 1, a MAGUK (Membrane Associated Guanylate Kinase) family member that is the highly conserved homolog of mammalian PSD-95 and SAP97. The intol mutation disrupted specifically the expression of DlgS97, a SAP97 homolog, and one of two major protein isoforms encoded by dlg1 via alternative splicing. Expression of the major isoform, DlgA, a PSD-95 homolog, appeared unaffected. Ethanol tolerance in the intol mutant could be partially restored by transgenic expression of DlgS97, but not DlgA, in specific neurons of the fly's brain. Based on co-immunoprecipitation, DlgS97 forms a complex with N-methyl-D-aspartate (NMDA) receptors, a known target of ethanol. Consistent with these observations, flies expressing reduced levels of the essential NMDA receptor subunit dNR1 also showed reduced ethanol tolerance, as did mutants in the gene calcium/calmodulin-dependent protein kinase (caki), encoding the fly homolog of mammalian CASK, a known binding partner of DlgS97. Lastly, mice in which SAP97, the mammalian homolog of DlgS97, was conditionally deleted in adults failed to develop rapid tolerance to ethanol's sedative/hypnotic effects. We propose that DlgS97/SAP97 plays an important and conserved role in the development of tolerance to ethanol via NMDA receptor-mediated synaptic plasticity.

Background: The polarized reorganization of the T cell membrane and intracellular signaling molecules in response to T cell receptor (TCR) engagement has been implicated in the modulation of T cell development and effector responses. In siRNA-based studies Dlg1, a MAGUK scaffold protein and member of the Scribble polarity complex, has been shown to play a role in T cell polarity and TCR signal specificity, however the role of Dlg1 in T cell development and function in vivo remains unclear., Methodology/principal Findings: Here we present the combined data from three independently-derived dlg1-knockout mouse models; two germline deficient knockouts and one conditional knockout. While defects were not observed in T cell development, TCR-induced early phospho-signaling, actin-mediated events, or proliferation in any of the models, the acute knockdown of Dlg1 in Jurkat T cells diminished accumulation of actin at the IS. Further, while Th1-type cytokine production appeared unaffected in T cells derived from mice with a dlg1 germline-deficiency, altered production of TCR-dependent Th1 and Th2-type cytokines was observed in T cells derived from mice with a conditional loss of dlg1 expression and T cells with acute Dlg1 suppression, suggesting a differential requirement for Dlg1 activity in signaling events leading to Th1 versus Th2 cytokine induction. The observed inconsistencies between these and other knockout models and siRNA strategies suggest that 1) compensatory upregulation of alternate gene(s) may be masking a role for dlg1 in controlling TCR-mediated events in dlg1 deficient mice and 2) the developmental stage during which dlg1 ablation begins may control the degree to which compensatory events occur., Conclusions/significance: These findings provide a potential explanation for the discrepancies observed in various studies using different dlg1-deficient T cell models and underscore the importance of acute dlg1 ablation to avoid the upregulation of compensatory mechanisms for future functional studies of the Dlg1 protein.

A compact 4 × 25 Gbps optical transceiver has been fabricated for an optical backplane system, which consists of a 4 × 25 Gbps DFB-LD array, a 4 × 25 Gbps PIN-PD array, and a CMOS transceiver chip. These are directly mounted on 9 × 14 mm(2) multi-layer ceramic package with an electromagnetic shield structure to suppress inner-channel crosstalk effectively. The transceiver includes an analog front-end as well as an electrical interface function to interface with the switch LSI or CPU. Power consumption was as low as 20 mW/Gbps, and a transmission experiment was successfully conducted at 25 Gbps., (© 2011 Optical Society of America)

KIBRA has recently been identified as a gene associated with human memory performance. Despite the elucidation of the role of KIBRA in several diverse processes in nonneuronal cells, the molecular function of KIBRA in neurons is unknown. We found that KIBRA directly binds to the protein interacting with C-kinase 1 (PICK1) and forms a complex with α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs), the major excitatory neurotransmitter receptors in the brain. KIBRA knockdown accelerates the rate of AMPAR recycling following N-methyl-D-aspartate receptor-induced internalization. Genetic deletion of KIBRA in mice impairs both long-term depression and long-term potentiation at hippocampal Schaffer collateral-CA1 synapses. Moreover, KIBRA knockout mice have severe deficits in contextual fear learning and memory. These results indicate that KIBRA regulates higher brain function by regulating AMPAR trafficking and synaptic plasticity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Fast excitatory synaptic transmission in central synapses is mediated primarily by AMPA receptors (AMPARs), which are heteromeric assemblies of four subunits, GluA1-4. Among these subunits, rapidly gating GluA3/4 appears to be the most abundantly expressed to enable neurotransmission with submillisecond precision at fast rates in subsets of central synapses. However, neither definitive identification of the molecular substrate for native AMPARs in these neurons, nor their hypothesized functional roles in vivo has been unequivocally demonstrated, largely due to lack of specific antagonists. Using GluA3 or GluA4 knockout (KO) mice, we investigated these issues at the calyx of Held synapse, which is known as a high-fidelity synapse involved in sound localization. Patch-clamp recordings from postsynaptic neurons showed that deletion of GluA4 significantly slowed the time course of both evoked and miniature AMPAR-mediated excitatory postsynaptic currents (AMPAR-EPSCs), reduced their amplitude, and exacerbated AMPAR desensitization and short-term depression (STD). Surprisingly, presynaptic release probability was also elevated, contributing to severe STD at GluA4-KO synapses. In contrast, only marginal changes in AMPAR-EPSCs were found in GluA3-KO mice. Furthermore, independent of changes in intrinsic excitability of postsynaptic neurons, deletion of GluA4 markedly reduced synaptic drive and increased action potential failures during high-frequency activity, leading to profound deficits in specific components of the auditory brainstem responses associated with synchronized spiking in the calyx of Held synapse and other related neurons in vivo. These observations identify GluA4 as the main determinant for fast synaptic response, indispensable for driving high-fidelity neurotransmission and conveying precise temporal information.

Phosphorylation of the GluA1 subunit of AMPA receptors has been proposed to regulate receptor trafficking and synaptic transmission and plasticity. However, it remains unclear whether GluA1 phosphorylation is permissive or sufficient for enacting these functional changes. Here we investigate the role of GluA1 phosphorylation at S831 and S845 residues in the hippocampus through the analyses of GluA1 S831D/S845D phosphomimetic knock-in mice. S831D/S845D mice showed normal total and surface expression and subcellular localization of GluA1 as well as intact basal synaptic transmission. In addition, theta-burst stimulation, a protocol that was sufficient to induce robust long-term potentiation (LTP) in WT mice, resulted in LTP of similar magnitude in S831D/S845D mice. However, S831D/S845D mice showed LTP induced with 10-Hz stimulation, a protocol that is weaker than theta-burst stimulation and was not sufficient to induce LTP in WT mice. Moreover, S831D/S845D mice exhibited LTP induced with spike-timing-dependent plasticity (STDP) protocol at a long pre-post interval that was subthreshold for WT mice, although a suprathreshold STDP protocol at a short pre-post interval resulted in similarly robust LTP for WT and S831D/S845D mice. These results indicate that phosphorylation of GluA1 at S831 and S845 is sufficient to lower the threshold for LTP induction, increasing the probability of synaptic plasticity.

Long-term depression at parallel fiber-Purkinje cell synapses (PF-PC LTD) has been proposed to be required for cerebellar motor learning. To date, tests of this hypothesis have sought to interfere with receptors (mGluR1) and enzymes (PKC, PKG, or αCamKII) necessary for induction of PF-PC LTD and thereby determine if cerebellar motor learning is impaired. Here, we tested three mutant mice that target the expression of PF-PC LTD by blocking internalization of AMPA receptors. Using three different cerebellar coordination tasks (adaptation of the vestibulo-ocular reflex, eyeblink conditioning, and locomotion learning on the Erasmus Ladder), we show that there is no motor learning impairment in these mutant mice that lack PF-PC LTD. These findings demonstrate that PF-PC LTD is not essential for cerebellar motor learning., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Sensory experience, and the lack thereof, can alter the function of excitatory synapses in the primary sensory cortices. Recent evidence suggests that changes in sensory experience can regulate the synaptic level of Ca(2+)-permeable AMPA receptors (CP-AMPARs). However, the molecular mechanisms underlying such a process have not been determined. We found that binocular visual deprivation, which is a well-established in vivo model to produce multiplicative synaptic scaling in visual cortex of juvenile rodents, is accompanied by an increase in the phosphorylation of AMPAR GluR1 (or GluA1) subunit at the serine 845 (S845) site and the appearance of CP-AMPARs at synapses. To address the role of GluR1-S845 in visual deprivation-induced homeostatic synaptic plasticity, we used mice lacking key phosphorylation sites on the GluR1 subunit. We found that mice specifically lacking the GluR1-S845 site (GluR1-S845A mutants), which is a substrate of cAMP-dependent kinase (PKA), show abnormal basal excitatory synaptic transmission and lack visual deprivation-induced homeostatic synaptic plasticity. We also found evidence that increasing GluR1-S845 phosphorylation alone is not sufficient to produce normal multiplicative synaptic scaling. Our study provides concrete evidence that a GluR1 dependent mechanism, especially S845 phosphorylation, is a necessary pre-requisite step for in vivo homeostatic synaptic plasticity.

Protein interacting with C Kinase 1 (PICK1), a PDZ domain-containing scaffolding protein, interacts with multiple different proteins in the mammalian nervous system and is believed to play important roles in diverse physiological and pathological conditions. In this study, we report that PICK1 is expressed in neurons of the dorsal root ganglion (DRG) and spinal cord dorsal horn, two major pain-related regions. PICK1 was present in approximately 29.7% of DRG neurons, most of which were small-less than 750 μm(2) in cross-sectional area. Some of these PICK1-positive cells co-labeled with isolectin B4 or calcitonin-gene-related peptide. In the dorsal horn, PICK1 immunoreactivity was concentrated in the superficial dorsal horn, where it was prominent in the postsynaptic density, axons, and dendrites. Targeted disruption of PICK1 gene did not affect basal paw withdrawal responses to acute noxious thermal and mechanical stimuli or locomotor reflex activity, but it completely blocked the induction of peripheral nerve injury-induced mechanical and thermal pain hypersensitivities. PICK1 appears to be required for peripheral nerve injury-induced neuropathic pain development and to be a potential biochemical target for treating this disorder.

AMPA-type glutamate receptors (AMPARs) mediate the majority of fast excitatory neurotransmission in the mammalian central nervous system. Modulation of AMPAR trafficking supports several forms of synaptic plasticity thought to underlie learning and memory. Protein interacting with C kinase 1 (PICK1) is an AMPAR-binding protein shown to regulate both AMPAR trafficking and synaptic plasticity at many distinct synapses. However, studies examining the requirement for PICK1 in maintaining basal synaptic transmission and regulating synaptic plasticity at hippocampal Schaffer collateral-cornu ammonis 1 (SC-CA1) synapses have produced conflicting results. In addition, the effect of PICK1 manipulation on learning and memory has not been investigated. In the present study we analyzed the effect of genetic deletion of PICK1 on basal synaptic transmission and synaptic plasticity at hippocampal Schaffer collateral-CA1 synapses in adult and juvenile mice. Surprisingly, we find that loss of PICK1 has no significant effect on synaptic plasticity in juvenile mice but impairs some forms of long-term potentiation and multiple distinct forms of long-term depression in adult mice. Moreover, inhibitory avoidance learning is impaired only in adult KO mice. These results suggest that PICK1 is selectively required for hippocampal synaptic plasticity and learning in adult rodents.

PSD-95/SAP90/DLG/ZO-1 (PDZ) domain-mediated protein-protein interactions play important roles in regulating AMPA receptor trafficking and neuronal plasticity. GRIP1 and GRIP2 are homologous multi-PDZ domain-containing proteins that bind to the C-termini of AMPA-R GluA2 and GluA3 subunits. Previous attempts to determine the cellular roles of GRIP1 and GRIP2 in neurons have been complicated by nonspecific reagents, and by the embryonic lethality of conventional GRIP1 KO mice. To circumvent these issues we developed a conditional targeted deletion strategy to knock out GRIP1 in postnatal neurons derived from GRIP2 KO mice. Loss of GRIP1 and 2 did not affect normal AMPA-R steady-state trafficking and endocytosis, but strikingly impaired activity-dependent AMPA-R recycling. This previously uncharacterized role for GRIP1 appears to be mediated by novel interactions with the cellular trafficking machinery via the exocyst protein complex. Indeed, disruption of GRIP1-exocyst binding caused a strikingly similar deficit in AMPA-R recycling. Together these findings reveal a previously unidentified role for AMPA-R-GRIP1-exocyst protein complexes in activity-dependent AMPA-R trafficking.

Activity-dependent changes in excitatory synaptic transmission in the CNS have been shown to depend on the regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). In particular, several lines of evidence suggest that reversible phosphorylation of AMPAR subunit glutamate receptor 1 (GluR1, also referred to as GluA1 or GluR-A) plays a role in long-term potentiation (LTP) and long-term depression (LTD). We previously reported that regulation of serines (S) 831 and 845 on the GluR1 subunit may play a critical role in bidirectional synaptic plasticity in the Schaffer collateral inputs to CA1. Specifically, gene knockin mice lacking both S831 and S845 phosphorylation sites ("double phosphomutants"), where both serine residues were replaced by alanines (A), showed a faster decaying LTP and a deficit in LTD. To determine which of the two phosphorylation sites was responsible for the phenotype, we have now generated two lines of gene knockin mice: one that specifically lacks S831 (S831A mutants) and another that lacks only S845 (S845A mutants). We found that S831A mutants display normal LTP and LTD, whereas S845A mutants show a specific deficit in LTD. Taken together with our previous results from the "double phosphomutants," our data suggest that either S831 or S845 alone may support LTP, whereas the S845 site is critical for LTD expression.

Spinal cord GluR2-lacking AMPA receptors (AMPARs) contribute to nociceptive hypersensitivity in persistent pain, but the molecular mechanisms underlying this event are not completely understood. We report that complete Freund's adjuvant (CFA)-induced peripheral inflammation induces synaptic GluR2 internalization in dorsal horn neurons during the maintenance of CFA-evoked nociceptive hypersensitivity. This internalization is initiated by GluR2 phosphorylation at Ser(880) and subsequent disruption of GluR2 binding to its synaptic anchoring protein (GRIP), resulting in a switch of GluR2-containing AMPARs to GluR2-lacking AMPARs and an increase of AMPAR Ca(2+) permeability at the synapses in dorsal horn neurons. Spinal cord NMDA receptor-mediated triggering of protein kinase C (PKC) activation is required for the induction and maintenance of CFA-induced dorsal horn GluR2 internalization. Moreover, preventing CFA-induced spinal GluR2 internalization through targeted mutation of the GluR2 PKC phosphorylation site impairs CFA-evoked nociceptive hypersensitivity during the maintenance period. These results suggest that dorsal horn GluR2 internalization might participate in the maintenance of NMDA receptor/PKC-dependent nociceptive hypersensitivity in persistent inflammatory pain.

Activity-dependent dendrite elaboration influences the pattern of interneuronal connectivity and network function. In the present study, we examined the mechanism by which the GluR1 subunit of AMPA receptors controls dendrite morphogenesis. GluR1 binds to SAP97, a scaffolding protein that is a component of the postsynaptic density, via its C-terminal 7 aa. We find that elimination of this interaction in vitro or in vivo (by deleting the C-terminal 7 aa of GluR1, GluR1Delta7) does not influence trafficking, processing, or cell surface GluR1 expression but does prevent translocation of SAP97 from the cytosol to membranes. GluR1 and SAP97 together at the plasma membrane promotes dendrite branching in an activity-dependent manner, although this does not require physical association. Our findings suggest that the C-terminal 7 aa of GluR1 are essential for bringing SAP97 to the plasma membrane, where it acts to translate the activity of AMPA receptors into dendrite growth.

Glutamate receptor-interacting protein 1 (GRIP1) and GRIP2 are closely related proteins that bind GluR2-containing AMPA receptors and couple them to structural and signaling complexes in neurons. Cerebellar long-term synaptic depression (LTD) is a model system of synaptic plasticity that is expressed by persistent internalization of GluR2-containing AMPA receptors. Here, we show that genetic deletion of both GRIP1 and GRIP2 blocks LTD expression in primary cultures of mouse cerebellar neurons but that single deletion of either isoform allows LTD to occur. In GRIP1/2 double knock-out Purkinje cells, LTD can be fully rescued by a plasmid-driving expression of GRIP1 and partially rescued by a GRIP2 plasmid. These results indicate that the GRIP family comprises an essential molecular component for cerebellar LTD.

Constitutive activation of FMS-like tyrosine kinase 3 (FLT3) by internal tandem duplication (ITD) mutations is one of the most common molecular alterations known in acute myeloid leukemia (AML). To investigate the role FLT3/ITD mutations play in the development of leukemia, we generated a FLT3/ITD knock-in mouse model by inserting an ITD mutation into the juxtamembrane domain of murine Flt3. FLT3wt/ITD mice developed myeloproliferative disease, characterized by splenomegaly, leukocytosis, and myeloid hypercellularity, which progressed to mortality by 6 to 20 months. Bone marrow (BM) and spleen from FLT3wt/ITD mice had an increased fraction of granulocytes/monocytes and dendritic cells, and a decreased fraction of B-lymphocytes. No sign of acute leukemia was observed over the lifetime of these mice. BM from FLT3wt/ITD mice showed enhanced potential to generate myeloid colonies in vitro. BM from FLT3wt/ITD mice also produced more spleen colonies in the in vivo colony-forming unit (CFU)-spleen assay. In the long-term competitive repopulation assay, BM cells from FLT3wt/ITD mice outgrew the wild-type competitor cells and showed increased myeloid and reduced lymphoid expansion activity. In summary, our data indicate that expression of FLT3/ITD mutations alone is capable of conferring normal hematopoietic stem/progenitor cells (HSPCs) with enhanced myeloid expansion. It also appears to suppress B lymphoid maturation. Additional cooperative events appear to be required to progress to acute leukemia.

Neuronal activity regulated pentraxin (Narp) is a secreted neuronal product which clusters AMPA receptors and regulates excitatory synaptogenesis. Although Narp is selectively enriched in brain, its role in behavior is not known. As Narp is expressed prominently in limbic regions, we examined whether Narp deletion affects performance on tasks used to assess motivational consequences of food-rewarded learning. Narp knock-out (KO) mice were unimpaired in learning simple pavlovian discriminations, instrumental lever pressing, and in acquisition of at least two aspects of pavlovian incentive learning, conditioned reinforcement and pavlovian-instrumental transfer. In contrast, Narp deletion resulted in a substantial deficit in the ability to use specific outcome expectancies to modulate instrumental performance in a devaluation task. In this task, mice were trained to respond on two levers for two different rewards. After training, mice were prefed with one of the two rewards, devaluing it. Responding on both levers was then assessed in extinction. Whereas control mice showed a significant preference in responding on the lever associated with the nondevalued reward, Narp KO mice responded equally on both levers, failing to suppress responding on the lever associated with the devalued reward. Both groups consumed more of the nondevalued reward in a subsequent choice test, indicating Narp KO mice could distinguish between the rewards themselves. These data suggest Narp has a selective role in processing sensory-specific information necessary for appropriate devaluation performance, but not in general motivational effects of reward-predictive cues on performance.

Many central excitatory synapses undergo developmental alterations in the molecular and biophysical characteristics of postsynaptic ionotropic glutamate receptors via changes in subunit composition. Concerning AMPA receptors (AMPARs), glutamate receptor 2 subunit (GluR2)-containing, Ca2+-impermeable AMPARs (CI-AMPARs) prevail at synapses between mature principal neurons; however, accumulating evidence indicates that GluR2-lacking, Ca2+-permeable AMPARs (CP-AMPARs) contribute at these synapses early in development. Here, we used a combination of imaging and electrophysiological recording techniques to investigate potential roles for CP-AMPARs at developing hippocampal mossy fiber-CA3 pyramidal cell (MF-PYR) synapses. We found that transmission at nascent MF-PYR synapses is mediated by a mixed population of CP- and CI-AMPARs as evidenced by polyamine-dependent inwardly rectifying current-voltage (I-V) relationships, and partial philanthotoxin sensitivity of synaptic events. CP-AMPAR expression at MF-PYR synapses is transient, being limited to the first 3 postnatal weeks. Moreover, the expression of CP-AMPARs is regulated by the PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain-containing protein interacting with C kinase 1 (PICK1), because MF-PYR synapses in young PICK1 knock-out mice are philanthotoxin insensitive with linear I-V relationships. Strikingly, MF-PYR transmission via CP-AMPARs is selectively depressed during depolarization-induced long-term depression (DiLTD), a postsynaptic form of MF-PYR plasticity observed only at young MF-PYR synapses. The selective depression of CP-AMPARs during DiLTD was evident as a loss of postsynaptic CP-AMPAR-mediated Ca2+ transients in PYR spines and reduced rectification of MF-PYR synaptic currents. Preferential targeting of CP-AMPARs during DiLTD is further supported by a lack of DiLTD in young PICK1 knock-out mice. Together, these findings indicate that the transient participation of CP-AMPARs at young MF-PYR synapses dictates the developmental window to observe DiLTD.

Emotion enhances our ability to form vivid memories of even trivial events. Norepinephrine (NE), a neuromodulator released during emotional arousal, plays a central role in the emotional regulation of memory. However, the underlying molecular mechanism remains elusive. Toward this aim, we have examined the role of NE in contextual memory formation and in the synaptic delivery of GluR1-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors during long-term potentiation (LTP), a candidate synaptic mechanism for learning. We found that NE, as well as emotional stress, induces phosphorylation of GluR1 at sites critical for its synaptic delivery. Phosphorylation at these sites is necessary and sufficient to lower the threshold for GluR1 synaptic incorporation during LTP. In behavioral experiments, NE can lower the threshold for memory formation in wild-type mice but not in mice carrying mutations in the GluR1 phosphorylation sites. Our results indicate that NE-driven phosphorylation of GluR1 facilitates the synaptic delivery of GluR1-containing AMPARs, lowering the threshold for LTP, thereby providing a molecular mechanism for how emotion enhances learning and memory.

Near coincidental pre- and postsynaptic action potentials induce associative long-term potentiation (LTP) or long-term depression (LTD), depending on the order of their timing. Here, we show that in visual cortex the rules of this spike-timing-dependent plasticity are not rigid, but shaped by neuromodulator receptors coupled to adenylyl cyclase (AC) and phospholipase C (PLC) signaling cascades. Activation of the AC and PLC cascades results in phosphorylation of postsynaptic glutamate receptors at sites that serve as specific "tags" for LTP and LTD. As a consequence, the outcome (i.e., whether LTP or LTD) of a given pattern of pre- and postsynaptic firing depends not only on the order of the timing, but also on the relative activation of neuromodulator receptors coupled to AC and PLC. These findings indicate that cholinergic and adrenergic neuromodulation associated with the behavioral state of the animal can control the gating and the polarity of cortical plasticity.

PSD-95 is a major protein found in virtually all mature excitatory glutamatergic synapses in the brain. Here, we have addressed the role of PSD-95 in controlling glutamatergic synapse function by generating and characterizing a PSD-95 KO mouse. We found that the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)subtype of glutamate receptor (AMPAR)-mediated synaptic transmission was reduced in these mice. Two-photon (2P) uncaging of MNI-glutamate onto individual spines suggested that the decrease in AMPAR function in the PSD-95 KO mouse stems from an increase in the proportion of "silent" synapses i.e., synapses containing N-methyl-d-aspartate (NMDA) receptors (NMDARs) but no AMPARs. Unexpectedly, the silent synapses in the KO mouse were located onto morphologically mature spines. We also observed that a significant population of synapses appeared unaffected by PSD-95 gene deletion, suggesting that the functional role of PSD-95 displays synapse-specificity. In addition, we report that the decay of NMDAR-mediated current was slower in KO mice: The contribution of NR2B subunit containing receptors to the NMDAR-mediated synaptic current was greater in KO mice. The greater occurrence of silent synapses might be related to the greater magnitude of potentiation after long-term potentiation induction observed in these mice. Together, these results suggest a synapse-specific role for PSD-95 in controlling synaptic function that is independent of spine morphology.

In vertebrates, the receptor families deleted in colorectal cancer (DCC) and UNC5 mediate responses to the bifunctional guidance cue netrin-1. DCC mediates attraction, whereas a complex of DCC and UNC5 mediates repulsion. Thus, a primary determinant of the responsiveness of an axon to netrin-1 is the presence or absence of UNC5 family members on the cell surface. Currently, little is known about the role of receptor trafficking in regulating neuronal responses to netrin-1. We show that protein interacting with C-kinase 1 (PICK1) recruits activated protein kinase Calpha (PKCalpha) to MycUNC5A at the plasma membrane, stimulating its endocytosis. We identify two PKCalpha phosphorylation sites at serines 408 and 587, as well as dileucine internalization motifs, which are required for this endocytosis. We find that PKCalpha-stimulated internalization of UNC5A alters the functional response of developing hippocampal axons to netrin-1, preventing UNC5A-mediated growth cone collapse and converting netrin-1-stimulated chemorepulsion to attraction. To address whether this conversion in axonal response occurs in neurons expressing endogenous levels of UNC5, we show that mouse cerebellar granule axons exhibit chemorepulsion in a netrin-1 gradient and that this chemorepulsion is converted to chemoattraction after PKCalpha activation. We demonstrate that this repulsion depends on UNC5A because Unc5a-/- axons are not repelled and show this conversion depends on PICK1 because PICK1-/- axons are not converted to chemoattraction after PKCalpha activation. Together, these data provide a potential mechanism to explain how developing neurons alter their responsiveness to netrin-1 at intermediate choice points as they navigate to their targets.

Cerebellar long-term depression (LTD) is a major form of synaptic plasticity that is thought to be critical for certain types of motor learning. Phosphorylation of the AMPA receptor subunit GluR2 on serine-880 as well as interaction of GluR2 with PICK1 have been suggested to contribute to the endocytic removal of postsynaptic AMPA receptors during LTD. Here, we show that targeted mutation of PICK1, the GluR2 C-terminal PDZ ligand, or the GluR2 PKC phosphorylation site eliminates cerebellar LTD in mice. LTD can be rescued in cerebellar cultures from mice lacking PICK1 by transfection of wild-type PICK1 but not by a PDZ mutant or a BAR domain mutant deficient in lipid binding, indicating the importance of these domains in PICK1 function. These results demonstrate that PICK1-GluR2 PDZ-based interactions and GluR2 phosphorylation are required for LTD expression in the cerebellum.

A recently described form of synaptic plasticity results in dynamic changes in the calcium permeability of synaptic AMPA receptors. Since the AMPA receptor GluR2 subunit confers calcium permeability, this plasticity is thought to occur through the dynamic exchange of synaptic GluR2-lacking and GluR2-containing receptors. To investigate the molecular mechanisms underlying this calcium-permeable AMPA receptor plasticity (CARP), we examined whether AMPA receptor exchange was mediated by subunit-specific protein-protein interactions. We found that two GluR2-interacting proteins, the PDZ domain-containing Protein interacting with C kinase (PICK1) and N-ethylmaleimide sensitive fusion protein (NSF), are specifically required for CARP. Furthermore, PICK1, but not NSF, regulates the formation of extrasynaptic plasma membrane pools of GluR2-containing receptors that may be laterally mobilized into synapses during CARP. These results demonstrate that PICK1 and NSF dynamically regulate the synaptic delivery of GluR2-containing receptors during CARP and thus regulate the calcium permeability of AMPA receptors at excitatory synapses.

Mouse organic anion transporter 1 (mOAT1) belongs to a family of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, anti-tumour drugs, antibiotics, anti-hypertensives and anti-inflammatories. mOAT1-mediated transport of organic anion PAH ( p -aminohippurate) in HeLa cells was inhibited by the cysteine-modifying reagent PCMBS (p-chloromercuribenzenesulphonate). Therefore the role of cysteine residues in the function of mOAT1 was examined by site-directed mutagenesis. All 13 cysteine residues in mOAT1 were replaced by alanine, singly or in combination. Single replacement of these residues had no significant effect on mOAT1-mediated PAH transport, indicating that no individual cysteine residue is necessary for function. Multiple replacements at a C-terminal region (C335/379/427/434A; Cys(335/379/427/434)-->Ala) resulted in a substantial decrease in transport activity. A simultaneous replacement of all 13 cysteine residues (C-less) led to a complete loss of transport function. The decreased or lack of transport activity of the mutants C335/379/427/434A and C-less was due to the impaired trafficking of the mutant transporters to the cell surface. These results suggest that although cysteine residues are not required for function in mOAT1, their presence appears to be important for the targeting of the transporter to the plasma membrane. We also showed that, although all cysteine mutants of mOAT1 were sensitive to the inhibition by PCMBS, C49A was less sensitive than the wild-type mOAT1, suggesting that the modification of Cys49 may play a role in the inhibition of mOAT1 by PCMBS.

Plasticity of the nervous system is dependent on mechanisms that regulate the strength of synaptic transmission. Excitatory synapses in the brain undergo long-term potentiation (LTP) and long-term depression (LTD), cellular models of learning and memory. Protein phosphorylation is required for the induction of many forms of synaptic plasticity, including LTP and LTD. However, the critical kinase substrates that mediate plasticity have not been identified. We previously reported that phosphorylation of the GluR1 subunit of AMPA receptors, which mediate rapid excitatory transmission in the brain, is modulated during LTP and LTD. To test if GluR1 phosphorylation is necessary for plasticity and learning and memory, we generated mice with knockin mutations in the GluR1 phosphorylation sites. The phosphomutant mice show deficits in LTD and LTP and have memory defects in spatial learning tasks. These results demonstrate that phosphorylation of GluR1 is critical for LTD and LTP expression and the retention of memories.

Synaptic GTPase-activating protein (SynGAP) is a neuronal RasGAP (Ras GTPase-activating protein) that is selectively expressed in brain and highly enriched at excitatory synapses, where it negatively regulates Ras activity and its downstream signaling pathways. To investigate the physiological role of SynGAP in the brain, we have generated mutant mice lacking the SynGAP protein. These mice exhibit postnatal lethality, indicating that SynGAP plays a critical role during neuronal development. In addition, cell biological experiments show that neuronal cultures from mutant mice have more synaptic AMPA receptor clusters, suggesting that SynGAP regulates glutamate receptor synaptic targeting. Moreover, electrophysiological studies demonstrated that heterozygous mutant mice have a specific defect in hippocampal long-term potentiation (LTP). These studies show that the regulation of synaptic Ras signaling by SynGAP is important for proper neuronal development and glutamate receptor trafficking and is critical for the induction of LTP.

A 51-year-old female presented with an extremely unusual cerebellar porencephalic lesion manifesting as progressive cerebellar signs and cranial nerve pareses. T1-weighted magnetic resonance imaging demonstrated a low-signal-intensity area in the left cerebellar hemisphere suggesting a cavity communicating with the fourth ventricle. Histological study showed the wall of the cavity contained nonspecific gliosis. A diagnosis of porencephaly was made.

Sequential and regional changes in ischemic edema following various durations of focal cerebral ischemia were studied by magnetic resonance (MR) imaging in a rat unilateral intraluminal middle cerebral artery occlusion model. Occlusion was performed from 5 minutes to 5 hours. T2-weighted images were obtained chronologically 6 hours after onset of ischemia, on day 1 and day 7. An immunohistochemical study using antibodies to calcineurin and glial fibrillary acidic protein was performed to observe histological changes in the ischemic brain. The T2 high-signal-intensity areas representing ischemic edema were observed in the lateral striatum and/or the cerebral cortex by day 1 in all rats with 1- to 5-hour ischemia, and the areas were larger and detected earlier with longer durations of ischemia. In three of six rats with 15-minute ischemia and five of six rats with 30-minute ischemia, the T2 high-signal-intensity areas appeared transiently on day 1 in the dorsolateral striatum where loss of neurons expressing calcineurin immunoreactivity and associated gliosis were found. MR imaging in animal models of reversible focal ischemia can achieve sequential and noninvasive evaluation of dynamic regional changes in ischemic edema.