Lu Chen

All Publications

• Retinoic acid receptor RARalpha-dependent synaptic signaling mediates homeostatic synaptic plasticity at the inhibitory synapses of mouse visual cortex. The Journal of neuroscience : the official journal of the Society for Neuroscience Zhong, L., Chen, X., Park, E., Sudhof, T. C., Chen, L. 2018

  Abstract

  Homeostatic synaptic plasticity is a synaptic mechanism through which the nervous system adjusts synaptic excitation and inhibition to maintain network stability. Retinoic acid (RA) and its receptor RARalpha have been established as critical mediators of homeostatic synaptic plasticity. In vitro studies reveal that RA signaling enhances excitatory synaptic strength and decreases inhibitory synaptic strength. However, it is unclear whether RA-mediated homeostatic synaptic plasticity occurs in vivo, and if so, whether it operates at specific types of synapses. Here, we examine the impact of RA/RARalpha signaling in the monocular zone of primary visual cortex (V1m) in mice of either sex. Exogenous RA treatment in acute cortical slices resulted in a reduction in miniature inhibitory post-synaptic currents (mIPSCs) of layer 2/3 pyramidal neurons (PNs), an effect mimicked by visual deprivation induced by binocular enucleation in post-critical period animals. Postnatal deletion of RARalpha blocked RA's effect on mIPSCs. Cell type-specific deletion of RARalpha revealed that RA acted specifically on parvalbumin (PV)-expressing interneurons. RARalpha deletion in PV+ interneurons blocked visual deprivation-induced changes in mIPSCs, demonstrating the critical involvement of RA signaling in PV+ interneurons in vivo Moreover, visual deprivation- or RA-induced downregulation of synaptic inhibition was absent in the visual cortical circuit of constitutive and PV-specific Fmr1 KO mice, strongly suggesting a functional interaction between FMRP and RA signaling pathways. Taken together, our results demonstrate that RA/RARalpha signaling acts as a key component for homeostatic regulation of synaptic transmission at the inhibitory synapses of the visual cortex.SIGNIFICANCE STATEMENT In vitro studies established that retinoic acid (RA) and its receptor RARalpha play key roles in homeostatic synaptic plasticity, a mechanism by which synaptic excitation/inhibition balance and network stability are maintained. However, whether synaptic RA signaling operates in vivo remains undetermined. Here, using a conditional RARalpha knockout mouse and cell type-specific Cre-driver lines, we showed that RARalpha signaling in parvalbumin-expressing interneurons is crucial for visual deprivation-induced homeostatic synaptic plasticity at inhibitory synapses in visual cortical circuits. Importantly, this form of synaptic plasticity is absent when FMRP is selectively deleted in parvalbumin-expressing interneurons, suggesting a functional connection between RARalpha and FMRP signaling pathways in vivo Thus, dysfunction of RA-dependent homeostatic plasticity may contribute to cortical circuit abnormalities in fragile X syndrome.

  View details for DOI 10.1523/JNEUROSCI.1133-18.2018

  View details for PubMedID 30355624

• Homeostatic synaptic plasticity as a metaplasticity mechanism-a molecular and cellular perspective. Current opinion in neurobiology Li, J., Park, E., Zhong, L. R., Chen, L. 2018; 54: 44–53

  Abstract

  The molecular mechanisms underlying various types of synaptic plasticity are historically regarded as separate processes involved in independent cellular events. However, recent progress in our molecular understanding of Hebbian and homeostatic synaptic plasticity supports the observation that these two types of plasticity share common cellular events, and are often altered together in neurological diseases. Here, we discuss the emerging concept of homeostatic synaptic plasticity as a metaplasticity mechanism with a focus on cellular signaling processes that enable a direct interaction between Hebbian and homeostatic plasticity. We also identify distinct and shared molecular players involved in these cellular processes that may be explored experimentally in future studies to test the hypothesis that homeostatic synaptic plasticity serves as a metaplasticity mechanism to integrate changes in neuronal activity and support optimal Hebbian learning.

  View details for DOI 10.1016/j.conb.2018.08.010

  View details for PubMedID 30212714

• The fragile X mutation impairs homeostatic plasticity in human neurons by blocking synaptic retinoic acid signaling. Science translational medicine Zhang, Z., Marro, S. G., Zhang, Y., Arendt, K. L., Patzke, C., Zhou, B., Fair, T., Yang, N., Sudhof, T. C., Wernig, M., Chen, L. 2018; 10 (452)

  Abstract

  Fragile X syndrome (FXS) is an X chromosome-linked disease leading to severe intellectual disabilities. FXS is caused by inactivation of the fragile X mental retardation 1 (FMR1) gene, but how FMR1 inactivation induces FXS remains unclear. Using human neurons generated from control and FXS patient-derived induced pluripotent stem (iPS) cells or from embryonic stem cells carrying conditional FMR1 mutations, we show here that loss of FMR1 function specifically abolished homeostatic synaptic plasticity without affecting basal synaptic transmission. We demonstrated that, in human neurons, homeostatic plasticity induced by synaptic silencing was mediated by retinoic acid, which regulated both excitatory and inhibitory synaptic strength. FMR1 inactivation impaired homeostatic plasticity by blocking retinoic acid-mediated regulation of synaptic strength. Repairing the genetic mutation in the FMR1 gene in an FXS patient cell line restored fragile X mental retardation protein (FMRP) expression and fully rescued synaptic retinoic acid signaling. Thus, our study reveals a robust functional impairment caused by FMR1 mutations that might contribute to neuronal dysfunction in FXS. In addition, our results suggest that FXS patient iPS cell-derived neurons might be useful for studying the mechanisms mediating functional abnormalities in FXS.

  View details for DOI 10.1126/scitranslmed.aar4338

  View details for PubMedID 30068571

• Postnatal ablation of synaptic retinoic acid signaling impairs cortical information processing and sensory discrimination in mice. The Journal of neuroscience : the official journal of the Society for Neuroscience Park, E., Tjia, M., Zuo, Y., Chen, L. 2018

  Abstract

  Retinoic acid (RA) and its receptors (RARs) are well-established essential transcriptional regulators during embryonic development. Recent findings in cultured neurons identified an independent and critical post-transcriptional role of RA and RARalpha in the homeostatic regulation of excitatory and inhibitory synaptic transmission in mature neurons. However, the functional relevance of synaptic RA signaling in vivo has not been established. Here, using somatosensory cortex as a model system and the RARalpha conditional knockout mouse as a tool, we applied multiple genetic manipulations to delete RARalpha postnatally in specific populations of cortical neurons, and asked whether synaptic RA signaling observed in cultured neurons is involved in cortical information processing in vivo Indeed, conditional ablation of RARalpha in mice via a CaMKIIalpha-Cre or a layer 5-Cre driver line or via somatosensory cortex-specific viral expression of Cre-recombinase impaired whisker-dependent texture discrimination, suggesting a critical requirement of RARalpha expression in L5 pyramidal neurons of somatosensory cortex for normal tactile sensory processing. Transcranial two-photon imaging revealed a significant increase in dendritic spine elimination on apical dendrites of somatosensory cortical layer 5 pyramidal neurons in these mice. Interestingly, the enhancement of spine elimination is whisker experience-dependent as whisker trimming rescued the spine elimination phenotype. Additionally, experiencing an enriched environment improved texture discrimination in RARalpha-deficient mice and reduced excessive spine pruning. Thus, RA signaling is essential for normal experience-dependent cortical circuit remodeling and sensory processing.SIGNIFICANCE STATEMENTThe importance of synaptic RA signaling has been demonstrated in in vitro studies. However, whether RA signaling mediated by RARalpha contributes to neural circuit functions in vivo remains largely unknown. In this study, using a RARalpha conditional knockout mouse, we performed multiple regional/cell type-specific manipulation of RARalpha expression in the postnatal brain, and show that RARalpha signaling contributes to normal whisker-dependent texture discrimination as well as regulating spine dynamics of apical dendrites from layer (L5) pyramidal neurons (PNs) in S1. Deletion of RARalpha in excitatory neurons in the forebrain induces elevated spine elimination and impaired sensory discrimination. Our study provides novel insights into the role of RARalpha signaling in cortical processing and experience-dependent spine maturation.

  View details for DOI 10.1523/JNEUROSCI.3028-17.2018

  View details for PubMedID 29760176

• Postsynaptic synaptotagmins mediate AMPA receptor exocytosis during LTP NATURE Wu, D., Bacaj, T., Morishita, W., Goswami, D., Arendt, K. L., Xu, W., Chen, L., Malenka, R. C., Sudhof, T. C. 2017; 544 (7650): 316-?

  Abstract

  Strengthening of synaptic connections by NMDA (N-methyl-d-aspartate) receptor-dependent long-term potentiation (LTP) shapes neural circuits and mediates learning and memory. During the induction of NMDA-receptor-dependent LTP, Ca(2+) influx stimulates recruitment of synaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, thereby strengthening synapses. How Ca(2+) induces the recruitment of AMPA receptors remains unclear. Here we show that, in the pyramidal neurons of the hippocampal CA1 region in mice, blocking postsynaptic expression of both synaptotagmin-1 (Syt1) and synaptotagmin-7 (Syt7), but not of either alone, abolished LTP. LTP was restored by expression of wild-type Syt7 but not of a Ca(2+)-binding-deficient mutant Syt7. Blocking postsynaptic expression of Syt1 and Syt7 did not impair basal synaptic transmission, reduce levels of synaptic or extrasynaptic AMPA receptors, or alter other AMPA receptor trafficking events. Moreover, expression of dominant-negative mutant Syt1 which inhibits Ca(2+)-dependent presynaptic vesicle exocytosis, also blocked Ca(2+)-dependent postsynaptic AMPA receptor exocytosis, thereby abolishing LTP. Our results suggest that postsynaptic Syt1 and Syt7 act as redundant Ca(2+)-sensors for Ca(2+)-dependent exocytosis of AMPA receptors during LTP, and thereby delineate a simple mechanism for the recruitment of AMPA receptors that mediates LTP.

  View details for DOI 10.1038/nature21720

  View details for Web of Science ID 000399524400030

  View details for PubMedID 28355182

• The Retromer Supports AMPA Receptor Trafficking During LTP NEURON Temkin, P., Morishita, W., Goswami, D., Arendt, K., Chen, L., Malenka, R. 2017; 94 (1): 74-?

  Abstract

  Alterations in the function of the retromer, a multisubunit protein complex that plays a specialized role in endosomal sorting, have been linked to Alzheimer's and Parkinson's diseases, yet little is known about the retromer's role in the mature brain. Using in vivo knockdown of the critical retromer component VPS35, we demonstrate a specific role for this endosomal sorting complex in the trafficking of AMPA receptors during NMDA-receptor-dependent LTP at mature hippocampal synapses. The impairment of LTP due to VPS35 knockdown was mechanistically independent of any role of the retromer in the production of Aβ from APP. Finally, we find surprising differences between Alzheimer's- and Parkinson's-disease-linked VPS35 mutations in supporting this pathway. These findings demonstrate a key role for the retromer in LTP and provide insights into how retromer malfunction in the mature brain may contribute to symptoms of common neurodegenerative diseases. VIDEO ABSTRACT.

  View details for DOI 10.1016/j.neuron.2017.03.020

  View details for Web of Science ID 000398262000010

  View details for PubMedID 28384478

• A metaplasticity view of the interaction between homeostatic and Hebbian plasticity PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES Yee, A. X., Hsu, Y., Chen, L. 2017; 372 (1715)

  Abstract

  Hebbian and homeostatic plasticity are two major forms of plasticity in the nervous system: Hebbian plasticity provides a synaptic basis for associative learning, whereas homeostatic plasticity serves to stabilize network activity. While achieving seemingly very different goals, these two types of plasticity interact functionally through overlapping elements in their respective mechanisms. Here, we review studies conducted in the mammalian central nervous system, summarize known circuit and molecular mechanisms of homeostatic plasticity, and compare these mechanisms with those that mediate Hebbian plasticity. We end with a discussion of 'local' homeostatic plasticity and the potential role of local homeostatic plasticity as a form of metaplasticity that modulates a neuron's future capacity for Hebbian plasticity.This article is part of the themed issue 'Integrating Hebbian and homeostatic plasticity'.

  View details for DOI 10.1098/rstb.2016.0155

  View details for Web of Science ID 000393410000005

  View details for PubMedID 28093549

  View details for PubMedCentralID PMC5247587

• Integrating Hebbian and homeostatic plasticity: the current state of the field and future research directions PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES Keck, T., Toyoizumi, T., Chen, L., Doiron, B., Feldman, D. E., Fox, K., Gerstner, W., Haydon, P. G., Huebener, M., Lee, H., Lisman, J. E., Rose, T., Sengpiel, F., Stellwagen, D., Stryker, M. P., Turrigiano, G. G., van Rossum, M. C. 2017; 372 (1715)

  Abstract

  We summarize here the results presented and subsequent discussion from the meeting on Integrating Hebbian and Homeostatic Plasticity at the Royal Society in April 2016. We first outline the major themes and results presented at the meeting. We next provide a synopsis of the outstanding questions that emerged from the discussion at the end of the meeting and finally suggest potential directions of research that we believe are most promising to develop an understanding of how these two forms of plasticity interact to facilitate functional changes in the brain.This article is part of the themed issue 'Integrating Hebbian and homeostatic plasticity'.

  View details for DOI 10.1098/rstb.2016.0158

  View details for Web of Science ID 000393410000016

  View details for PubMedID 28093552

  View details for PubMedCentralID PMC5247590

• Differential regulation of spontaneous and evoked inhibitory synaptic transmission in somatosensory cortex by retinoic acid. Synapse X Yee, A., Chen, L. 2016; 70 (11): 445-452

  Abstract

  Retinoic acid (RA), a developmental morphogen, has emerged in recent studies as a novel synaptic signaling molecule that acts in mature hippocampal neurons to modulate excitatory and inhibitory synaptic transmission in the context of homeostatic synaptic plasticity. However, it is unclear whether RA is capable of modulating neural circuits outside of the hippocampus, and if so, whether the mode of RA's action at synapses is similar to that within the hippocampal network. Here we explore for the first time RA's synaptic function outside the hippocampus and uncover a novel function of all-trans retinoic acid at inhibitory synapses. Acute RA treatment increases spontaneous inhibitory synaptic transmission in L2/3 pyramidal neurons of the somatosensory cortex, and this effect requires expression of RA's receptor RARα both pre- and post-synaptically. Intriguingly, RA does not seem to affect evoked inhibitory transmission assayed with either extracellular stimulation or direct activation of action potentials in presynaptic interneurons at connected pairs of interneurons and pyramidal neurons. Taken together, these results suggest that RA's action at synapses is not monotonous, but is diverse depending on the type of synaptic connection (excitatory versus inhibitory) and circuit (hippocampal versus cortical). Thus, synaptic signaling of RA may mediate multi-faceted regulation of synaptic plasticity. In addition to its classic roles in brain development, retinoic acid (RA) has recently been shown to regulate excitatory and inhibitory transmission in the adult brain. Here, the authors show that in layer 2/3 (L2/3) of the somatosensory cortex (S1), acute RA induces increases in spontaneous but not action-potential evoked transmission, and that this requires retinoic acid receptor (RARα) both in presynaptic PV-positive interneurons and postsynaptic pyramidal (PN) neurons.

  View details for DOI 10.1002/syn.21921

  View details for PubMedID 27348405

• Calcineurin mediates homeostatic synaptic plasticity by regulating retinoic acid synthesis. Proceedings of the National Academy of Sciences of the United States of America Arendt, K. L., Zhang, Z., Ganesan, S., Hintze, M., Shin, M. M., Tang, Y., Cho, A., Graef, I. A., Chen, L. 2015; 112 (42): E5744-52

  Abstract

  Homeostatic synaptic plasticity is a form of non-Hebbian plasticity that maintains stability of the network and fidelity for information processing in response to prolonged perturbation of network and synaptic activity. Prolonged blockade of synaptic activity decreases resting Ca(2+) levels in neurons, thereby inducing retinoic acid (RA) synthesis and RA-dependent homeostatic synaptic plasticity; however, the signal transduction pathway that links reduced Ca(2+)-levels to RA synthesis remains unknown. Here we identify the Ca(2+)-dependent protein phosphatase calcineurin (CaN) as a key regulator for RA synthesis and homeostatic synaptic plasticity. Prolonged inhibition of CaN activity promotes RA synthesis in neurons, and leads to increased excitatory and decreased inhibitory synaptic transmission. These effects of CaN inhibitors on synaptic transmission are blocked by pharmacological inhibitors of RA synthesis or acute genetic deletion of the RA receptor RARα. Thus, CaN, acting upstream of RA, plays a critical role in gating RA signaling pathway in response to synaptic activity. Moreover, activity blockade-induced homeostatic synaptic plasticity is absent in CaN knockout neurons, demonstrating the essential role of CaN in RA-dependent homeostatic synaptic plasticity. Interestingly, in GluA1 S831A and S845A knockin mice, CaN inhibitor- and RA-induced regulation of synaptic transmission is intact, suggesting that phosphorylation of GluA1 C-terminal serine residues S831 and S845 is not required for CaN inhibitor- or RA-induced homeostatic synaptic plasticity. Thus, our study uncovers an unforeseen role of CaN in postsynaptic signaling, and defines CaN as the Ca(2+)-sensing signaling molecule that mediates RA-dependent homeostatic synaptic plasticity.

  View details for DOI 10.1073/pnas.1510239112

  View details for PubMedID 26443861

• Aldehyde dehydrogenase 1a1 mediates a GABA synthesis pathway in midbrain dopaminergic neurons. Science Kim, J., Ganesan, S., Luo, S. X., Wu, Y., Park, E., Huang, E. J., Chen, L., Ding, J. B. 2015; 350 (6256): 102-106

  Abstract

  Midbrain dopamine neurons are an essential component of the basal ganglia circuitry, playing key roles in the control of fine movement and reward. Recently, it has been demonstrated that γ-aminobutyric acid (GABA), the chief inhibitory neurotransmitter, is co-released by dopamine neurons. Here, we show that GABA co-release in dopamine neurons does not use the conventional GABA-synthesizing enzymes, glutamate decarboxylases GAD65 and GAD67. Our experiments reveal an evolutionarily conserved GABA synthesis pathway mediated by aldehyde dehydrogenase 1a1 (ALDH1a1). Moreover, GABA co-release is modulated by ethanol (EtOH) at concentrations seen in blood alcohol after binge drinking, and diminished ALDH1a1 leads to enhanced alcohol consumption and preference. These findings provide insights into the functional role of GABA co-release in midbrain dopamine neurons, which may be essential for reward-based behavior and addiction.

  View details for DOI 10.1126/science.aac4690

  View details for PubMedID 26430123

• beta-Neurexins Control Neural Circuits by Regulating Synaptic Endocannabinoid Signaling CELL Anderson, G. R., Aoto, J., Tabuchi, K., Foeldy, C., Covy, J., Yee, A. X., Wu, D., Lee, S., Chen, L., Malenka, R. C., Suedhof, T. C. 2015; 162 (3): 593-606

  Abstract

  α- and β-neurexins are presynaptic cell-adhesion molecules implicated in autism and schizophrenia. We find that, although β-neurexins are expressed at much lower levels than α-neurexins, conditional knockout of β-neurexins with continued expression of α-neurexins dramatically decreased neurotransmitter release at excitatory synapses in cultured cortical neurons. The β-neurexin knockout phenotype was attenuated by CB1-receptor inhibition, which blocks presynaptic endocannabinoid signaling, or by 2-arachidonoylglycerol synthesis inhibition, which impairs postsynaptic endocannabinoid release. In synapses formed by CA1-region pyramidal neurons onto burst-firing subiculum neurons, presynaptic in vivo knockout of β-neurexins aggravated endocannabinoid-mediated inhibition of synaptic transmission and blocked LTP; presynaptic CB1-receptor antagonists or postsynaptic 2-arachidonoylglycerol synthesis inhibition again reversed this block. Moreover, conditional knockout of β-neurexins in CA1-region neurons impaired contextual fear memories. Thus, our data suggest that presynaptic β-neurexins control synaptic strength in excitatory synapses by regulating postsynaptic 2-arachidonoylglycerol synthesis, revealing an unexpected role for β-neurexins in the endocannabinoid-dependent regulation of neural circuits.

  View details for DOI 10.1016/j.cell.2015.06.056

  View details for Web of Science ID 000358801800018

• Retinoic Acid and LTP Recruit Postsynaptic AMPA Receptors Using Distinct SNARE-Dependent Mechanisms NEURON Arendt, K. L., Zhang, Y., Jurado, S., Malenka, R. C., Suedhof, T. C., Chen, L. 2015; 86 (2): 442-456

  Abstract

  Retinoic acid (RA)-dependent homeostatic plasticity and NMDA receptor-dependent long-term potentiation (LTP), a form of Hebbian plasticity, both enhance synaptic strength by increasing the abundance of postsynaptic AMPA receptors (AMPARs). However, it is unclear whether the molecular mechanisms mediating AMPAR trafficking during homeostatic and Hebbian plasticity differ, and it is unknown how RA signaling impacts Hebbian plasticity. Here, we show that RA increases postsynaptic AMPAR abundance using an activity-dependent mechanism that requires a unique SNARE (soluble NSF-attachment protein receptor)-dependent fusion machinery different from that mediating LTP. Specifically, RA-induced AMPAR trafficking did not involve complexin, which activates SNARE complexes containing syntaxin-1 or -3, but not complexes containing syntaxin-4, whereas LTP required complexin. Moreover, RA-induced AMPAR trafficking utilized the Q-SNARE syntaxin-4, whereas LTP utilized syntaxin-3; both additionally required the Q-SNARE SNAP-47 and the R-SNARE synatobrevin-2. Finally, acute RA treatment blocked subsequent LTP expression, probably by increasing AMPAR trafficking. Thus, RA-induced homeostatic plasticity involves a novel, activity-dependent postsynaptic AMPAR-trafficking pathway mediated by a unique SNARE-dependent fusion machinery.

  View details for DOI 10.1016/j.neuron.2015.03.009

  View details for Web of Science ID 000353410000013

  View details for PubMedID 25843403

• Synaptic retinoic acid signaling and homeostatic synaptic plasticity. Neuropharmacology Chen, L., Lau, A. G., Sarti, F. 2014; 78: 3-12

  Abstract

  One of the defining features of the nervous system is its ability to modify synaptic strength in an experience-dependent manner. Chronic elevation or reduction of network activity activates compensatory mechanisms that modulate synaptic strength in the opposite direction (i.e. reduced network activity leads to increased synaptic strength), a process called homeostatic synaptic plasticity. Among the many mechanisms that mediate homeostatic synaptic plasticity, retinoic acid (RA) has emerged as a novel signaling molecule that is critically involved in homeostatic synaptic plasticity induced by blockade of synaptic activity. In neurons, silencing of synaptic transmission triggers RA synthesis. RA then acts at synapses by a non-genomic mechanism that is independent of its well-known function as a transcriptional regulator, but operates through direct activation of protein translation in neuronal dendrites. Protein synthesis is activated by RA-binding to its receptor RARα, which functions locally in dendrites in a non-canonical manner as an RNA-binding protein that mediate RA's effect on translation. The present review will discuss recent progress in our understanding of the novel role of RA, which led to the identification of RA as a critical synaptic signaling molecule that mediates activity-dependent regulation of protein synthesis in neuronal dendrites. This article is part of a Special Issue entitled 'Homeostatic Plasticity'.

  View details for DOI 10.1016/j.neuropharm.2012.12.004

  View details for PubMedID 23270606

• Accelerated Experience-Dependent Pruning of Cortical Synapses in Ephrin-A2 Knockout Mice NEURON Yu, X., Wang, G., Gilmore, A., Yee, A. X., Li, X., Xu, T., Smith, S. J., Chen, L., Zuo, Y. 2013; 80 (1): 64-71

  Abstract

  Refinement of mammalian neural circuits involves substantial experience-dependent synapse elimination. Using in vivo two-photon imaging, we found that experience-dependent elimination of postsynaptic dendritic spines in the cortex was accelerated in ephrin-A2 knockout (KO) mice, resulting in fewer adolescent spines integrated into adult circuits. Such increased spine removal in ephrin-A2 KOs depended on activation of glutamate receptors, as blockade of the N-methyl-D-aspartate (NMDA) receptors eliminated the difference in spine loss between wild-type and KO mice. We also showed that ephrin-A2 in the cortex colocalized with glial glutamate transporters, which were significantly downregulated in ephrin-A2 KOs. Consistently, glial glutamate transport was reduced in ephrin-A2 KOs, resulting in an accumulation of synaptic glutamate. Finally, inhibition of glial glutamate uptake promoted spine elimination in wild-type mice, resembling the phenotype of ephrin-A2 KOs. Together, our results suggest that ephrin-A2 regulates experience-dependent, NMDA receptor-mediated synaptic pruning through glial glutamate transport during maturation of the mouse cortex.

  View details for DOI 10.1016/j.neuron.2013.07.014

  View details for Web of Science ID 000326305300008

  View details for PubMedID 24094103

  View details for PubMedCentralID PMC3792401

• Rapid Suppression of Inhibitory Synaptic Transmission by Retinoic Acid JOURNAL OF NEUROSCIENCE Sarti, F., Zhang, Z., Schroeder, J., Chen, L. 2013; 33 (28): 11440-11450

  Abstract

  In brain, properly balanced synaptic excitation and inhibition is critically important for network stability and efficient information processing. Here, we show that retinoic acid (RA), a synaptic signaling molecule whose synthesis is activated by reduced neural activity, induces rapid internalization of synaptic GABAA receptors in mouse hippocampal neurons, leading to significant reduction of inhibitory synaptic transmission. Similar to its action at excitatory synapses, action of RA at inhibitory synapses requires protein translation and is mediated by a nontranscriptional function of the RA-receptor RARα. Different from RA action at excitatory synapses, however, RA at inhibitory synapses causes a loss instead of the gain of a synaptic protein (i.e., GABAARs). Moreover, the removal of GABAARs from the synapses and the reduction of synaptic inhibition do not require the execution of RA's action at excitatory synapses (i.e., downscaling of synaptic inhibition is intact when upscaling of synaptic excitation is blocked). Thus, the action of RA at inhibitory and excitatory synapses diverges significantly after the step of RARα-mediated protein synthesis, and the regulations of GABAAR and AMPAR trafficking are independent processes. When both excitatory and inhibitory synapses are examined together in the same neuron, the synaptic excitation/inhibition ratio is significantly enhanced by RA. Importantly, RA-mediated downscaling of synaptic inhibition is completely absent in Fmr1 knock-out neurons. Thus, RA acts as a central organizer for coordinated homeostatic plasticity in both excitatory and inhibitory synapses, and impairment of this overall process alters the excitatory/inhibitory balance of a circuit and likely represents a major feature of fragile X-syndrome.

  View details for DOI 10.1523/JNEUROSCI.1710-13.2013

  View details for Web of Science ID 000321622600012

  View details for PubMedID 23843516

• Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron Zhang, Y., Pak, C., Han, Y., Ahlenius, H., Zhang, Z., Chanda, S., Marro, S., Patzke, C., Acuna, C., Covy, J., Xu, W., Yang, N., Danko, T., Chen, L., Wernig, M., Südhof, T. C. 2013; 78 (5): 785-798

  Abstract

  Available methods for differentiating human embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) into neurons are often cumbersome, slow, and variable. Alternatively, human fibroblasts can be directly converted into induced neuronal (iN) cells. However, with present techniques conversion is inefficient, synapse formation is limited, and only small amounts of neurons can be generated. Here, we show that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2 weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin, form mature pre- and postsynaptic specializations, and integrate into existing synaptic networks when transplanted into mouse brain. As illustrated by selected examples, our approach enables large-scale studies of human neurons for questions such as analyses of human diseases, examination of human-specific genes, and drug screening.

  View details for DOI 10.1016/j.neuron.2013.05.029

  View details for PubMedID 23764284

  View details for PubMedCentralID PMC3751803

• AMPA receptor/TARP stoichiometry visualized by single-molecule subunit counting. Proceedings of the National Academy of Sciences of the United States of America Hastie, P., Ulbrich, M. H., Wang, H., Arant, R. J., Lau, A. G., Zhang, Z., Isacoff, E. Y., Chen, L. 2013; 110 (13): 5163-5168

  Abstract

  Members of the transmembrane AMPA receptor-regulatory protein (TARP) family modulate AMPA receptor (AMPA-R) trafficking and function. AMPA-Rs consist of four pore-forming subunits. Previous studies show that TARPs are an integral part of the AMPA-R complex, acting as accessory subunits for mature receptors in vivo. The TARP/AMPA-R stoichiometry was previously measured indirectly and found to be variable and dependent on TARP expression level, with at most four TARPs associated with each AMPA-R complex. Here, we use a single-molecule technique in live cells that selectively images proteins located in the plasma membrane to directly count the number of TARPs associated with each AMPA-R complex. Although individual GFP-tagged TARP subunits are observed as freely diffusing fluorescent spots on the surface of Xenopus laevis oocytes when expressed alone, coexpression with AMPA-R-mCherry immobilizes the stargazin-GFP spots at sites of AMPA-R-mCherry, consistent with complex formation. We determined the number of TARP molecules associated with each AMPA-R by counting bleaching steps for three different TARP family members: γ-2, γ-3, and γ-4. We confirm that the TARP/AMPA-R stoichiometry depends on TARP expression level and discover that the maximum number of TARPs per AMPA-R complex falls into two categories: up to four γ-2 or γ-3 subunits, but rarely above two for γ-4 subunit. This unexpected AMPA-R/TARP stoichiometry difference has important implications for the assembly and function of TARP/AMPA-R complexes.

  View details for DOI 10.1073/pnas.1218765110

  View details for PubMedID 23479622

  View details for PubMedCentralID PMC3612642

• Chronic Inactivation of a Neural Circuit Enhances LTP by Inducing Silent Synapse Formation JOURNAL OF NEUROSCIENCE Arendt, K. L., Sarti, F., Chen, L. 2013; 33 (5): 2087-2096

  Abstract

  Chronic inactivation of a neural network is known to induce homeostatic upregulation of synaptic strength, a form of synaptic plasticity that differs from Hebbian-type synaptic plasticity in that it is not input-specific, but involves all synapses of an individual neuron. However, it is unclear how homeostatic and Hebbian synaptic plasticity interact in the same neuron. Here we show that long-term potentiation (LTP) at Schaffer collateral-CA1 synapses is greatly enhanced in cultured mouse hippocampal slices after chronic (60 h) network-activity blockade with tetrodotoxin (TTX). This increase in LTP is not due to an altered synaptic NMDA receptor composition or presynaptic function. Instead, we found that silencing neural network activity not only increases the abundance of both AMPA and NMDA receptors at existing synapses as previously described, but also promotes the presence of new glutamatergic synapses that contain only NMDA receptors-a class of synapses that are functionally silent due to the absence of AMPA receptors. Induction of LTP in TTX-treated neurons leads to insertion of AMPA receptors into the silent synapses, thereby "switching on" these silent synapses, which produces the observed enhancement of LTP magnitude. Our findings suggest that homeostatic synaptic plasticity manifests not only in the adjustment of the strength of existing synapses, but also in the modulation of new synapse formation/maintenance. Moreover, presence of new but functionally silent synapses enables more robust LTP to occur through rapid conversion of silent synapses to active synapses, resulting in a stronger input-specific modulation of synapses following prolonged network silencing.

  View details for DOI 10.1523/JNEUROSCI.3880-12.2013

  View details for Web of Science ID 000314351300032

  View details for PubMedID 23365245

• Conditional RARa knockout mice reveal acute requirement for retinoic acid and RARa in homeostatic plasticity. Frontiers in molecular neuroscience Sarti, F., Schroeder, J., Aoto, J., Chen, L. 2012; 5: 16-?

  Abstract

  All-trans retinoic acid (RA) plays important roles in brain development through regulating gene transcription. Recently, a novel post-developmental role of RA in mature brain was proposed. Specifically, RA rapidly enhanced excitatory synaptic transmission independent of transcriptional regulation. RA synthesis was induced when excitatory synaptic transmission was chronically blocked, and RA then activated dendritic protein synthesis and synaptic insertion of homomeric GluA1 AMPA receptors, thereby compensating for the loss of neuronal activity in a homeostatic fashion. This action of RA was suggested to be mediated by its canonical receptor RARα but no genetic evidence was available. Thus, we here tested the fundamental requirement of RARα in homeostatic plasticity using conditional RARα knockout (KO) mice, and additionally performed a structure-function analysis of RARα. We show that acutely deleting RARα in neurons eliminated RA's effect on excitatory synaptic transmission, and inhibited activity blockade-induced homeostatic synaptic plasticity. By expressing various RARα rescue constructs in RARα KO neurons, we found that the DNA-binding domain of RARα was dispensable for its role in regulating synaptic strength, further supporting the notion that RA and RARα act in a non-transcriptional manner in this context. By contrast, the ligand-binding domain (LBD) and the mRNA-binding domain (F-domain) are both necessary and sufficient for the function of RARα in homeostatic plasticity. Furthermore, we found that homeostatic regulation performed by the LBD/F-domains leads to insertion of calcium-permeable AMPA receptors. Our results confirm with unequivocal genetic approaches that RA and RARα perform essential non-transcriptional functions in regulating synaptic strength, and establish a functional link between the various domains of RARα and their involvement in regulating protein synthesis and excitatory synaptic transmission during homeostatic plasticity.

  View details for DOI 10.3389/fnmol.2012.00016

  View details for PubMedID 22419906

• Conditional RAR alpha knockout mice reveal acute requirement for retinoic acid and RAR alpha in homeostatic plasticity FRONTIERS IN MOLECULAR NEUROSCIENCE Sarti, F., Schroeder, J., Aoto, J., Chen, L. 2012; 5

  View details for DOI 10.3389/fnmol.2012.00016

  View details for Web of Science ID 000209202600016

• Decrease in Calcium Concentration Triggers Neuronal Retinoic Acid Synthesis during Homeostatic Synaptic Plasticity JOURNAL OF NEUROSCIENCE Wang, H., Zhang, Z., Hintze, M., Chen, L. 2011; 31 (49): 17764-17771

  Abstract

  Blockade of synaptic activity induces homeostatic plasticity, in part by stimulating synthesis of all-trans retinoic acid (RA), which in turn increases AMPA receptor synthesis. However, the synaptic signal that triggers RA synthesis remained unknown. Using multiple activity-blockade protocols that induce homeostatic synaptic plasticity, here we show that RA synthesis is activated whenever postsynaptic Ca(2+) entry is significantly decreased and that RA is required for upregulation of synaptic strength under these homeostatic plasticity conditions, suggesting that Ca(2+) plays an inhibitory role in RA synthesis. Consistent with this notion, we demonstrate that both transient Ca(2+) depletion by membrane-permeable Ca(2+) chelators and chronic blockage of L-type Ca(2+)-channels induces RA synthesis. Moreover, the source of dendritic Ca(2+) entry that regulates RA synthesis is not specific because mild depolarization with KCl is sufficient to reverse synaptic scaling induced by L-type Ca(2+)-channel blocker. By expression of a dihydropyridine-insensitive L-type Ca(2+) channel, we further show that RA acts cell autonomously to modulate synaptic transmission. Our findings suggest that, in synaptically active neurons, modest "basal" levels of postsynaptic Ca(2+) physiologically suppress RA synthesis, whereas in synaptically inactive neurons, decreases in the resting Ca(2+) levels induce homeostatic plasticity by stimulating synthesis of RA that then acts in a cell-autonomous manner to increase AMPA receptor function.

  View details for DOI 10.1523/JNEUROSCI.3964-11.2011

  View details for Web of Science ID 000298035400003

  View details for PubMedID 22159093

  View details for PubMedCentralID PMC3457695

• Acute knockdown of AMPA receptors reveals a trans-synaptic signal for presynaptic maturation EMBO JOURNAL Tracy, T. E., Yan, J. J., Chen, L. 2011; 30 (8): 1577-1592

  Abstract

  Newly formed glutamatergic synapses often lack postsynaptic AMPA-type glutamate receptors (AMPARs). Aside from 'unsilencing' the postsynaptic site, however, the significance of postsynaptic AMPAR insertion during synapse maturation remains unclear. To investigate the role of AMPAR in synapse maturation, we used RNA interference (RNAi) to knockdown AMPARs in cultured hippocampal neurons. Surprisingly, loss of postsynaptic AMPARs increased the occurrence of presynaptically inactive synapses without changing the release probability of the remaining active synapses. Additionally, heterologous synapses formed between axons and AMPAR-expressing HEK cells develop significantly fewer inactive presynaptic terminals. The extracellular domain of the AMPAR subunit GluA2 was sufficient to reproduce this effect at heterologous synapses. Indeed, the retrograde signalling by AMPARs is independent of their channel function as RNAi-resistant AMPARs restore synaptic transmission in neurons lacking AMPARs despite chronic receptor antagonist treatment. Our findings suggest that postsynaptic AMPARs perform an organizational function at synapses that exceeds their standard role as ionotropic receptors by conveying a retrograde trans-synaptic signal that increases the transmission efficacy at a synapse.

  View details for DOI 10.1038/emboj.2011.59

  View details for Web of Science ID 000290306300017

  View details for PubMedID 21378752

• Fragile X Protein FMRP Is Required for Homeostatic Plasticity and Regulation of Synaptic Strength by Retinoic Acid JOURNAL OF NEUROSCIENCE Soden, M. E., Chen, L. 2010; 30 (50): 16910-16921

  Abstract

  Homeostatic synaptic plasticity adjusts the strength of synapses during global changes in neural activity, thereby stabilizing the overall activity of neural networks. Suppression of synaptic activity increases synaptic strength by inducing synthesis of retinoic acid (RA), which activates postsynaptic synthesis of AMPA-type glutamate receptors (AMPARs) in dendrites and promotes synaptic insertion of newly synthesized AMPARs. Here, we show that fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates dendritic protein synthesis, is essential for increases in synaptic strength induced by RA or by blockade of neural activity in the mouse hippocampus. Although activity-dependent RA synthesis is maintained in Fmr1 knock-out neurons, RA-dependent dendritic translation of GluR1-type AMPA receptors is impaired. Intriguingly, FMRP is only required for the form of homeostatic plasticity that is dependent on both RA signaling and local protein synthesis. Postsynaptic expression of wild-type or mutant FMRP(I304N) in knock-out neurons reduced the total, surface, and synaptic levels of AMPARs, implying a role for FMRP in regulating AMPAR abundance. Expression of FMRP lacking the RGG box RNA-binding domain had no effect on AMPAR levels. Importantly, postsynaptic expression of wild-type FMRP, but not FMRP(I304N) or FMRPΔRGG, restored synaptic scaling when expressed in knock-out neurons. Together, these findings identify an unanticipated role for FMRP in regulating homeostatic synaptic plasticity downstream of RA. Our results raise the possibility that at least some of the symptoms of fragile X syndrome reflect impaired homeostatic plasticity and impaired RA signaling.

  View details for DOI 10.1523/JNEUROSCI.3660-10.2010

  View details for Web of Science ID 000285342300017

  View details for PubMedID 21159962

• Retinoic acid-gated sequence-specific translational control by RAR alpha PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Poon, M. M., Chen, L. 2008; 105 (51): 20303-20308

  Abstract

  Retinoic acid (RA) plays important roles in development by modulating gene transcription through nuclear receptor activation. Increasing evidence supports a role for RA and RA receptors (RARs) in synaptic plasticity in the brain. We have recently reported that RA mediates a type of homeostatic synaptic plasticity through activation of dendritic protein synthesis, a process that requires dendritically localized RARalpha and is independent of transcriptional regulation. The molecular basis of this translational regulation by RA/RARalpha signaling, however, is unknown. Here we show that RARalpha is actively exported from the nucleus. Cytoplasmic RARalpha acts as an RNA-binding protein that associates with a subset of mRNAs, including dendritically localized glutamate receptor 1 (GluR1) mRNA. This binding is mediated by the RARalpha carboxyl terminal F-domain and specific sequence motifs in the 5'UTR of the GluR1 mRNA. Moreover, RARalpha association with the GluR1 mRNA directly underlies the translational control of GluR1 by RA: RARalpha represses GluR1 translation, while RA binding to RARalpha reduces its association with the GluR1 mRNA and relieves translational repression. Taken together, our results demonstrate a ligand-gated translational regulation mechanism mediated by a non-genomic function of RA/RARalpha signaling.

  View details for DOI 10.1073/pnas.0807740105

  View details for Web of Science ID 000261995600053

  View details for PubMedID 19073915

• Synaptic Signaling by All-Trans Retinoic Acid in Homeostatic Synaptic Plasticity NEURON Aoto, J., Nam, C. I., Poon, M. M., Ting, P., Chen, L. 2008; 60 (2): 308-320

  Abstract

  Normal brain function requires that the overall synaptic activity in neural circuits be kept constant. Long-term alterations of neural activity lead to homeostatic regulation of synaptic strength by a process known as synaptic scaling. The molecular mechanisms underlying synaptic scaling are largely unknown. Here, we report that all-trans retinoic acid (RA), a well-known developmental morphogen, unexpectedly mediates synaptic scaling in response to activity blockade. We show that activity blockade increases RA synthesis in neurons and that acute RA treatment enhances synaptic transmission. The RA-induced increase in synaptic strength is occluded by activity blockade-induced synaptic scaling. Suppression of RA synthesis prevents synaptic scaling. This form of RA signaling operates via a translation-dependent but transcription-independent mechanism, causes an upregulation of postsynaptic glutamate receptor levels, and requires RARalpha receptors. Together, our data suggest that RA functions in homeostatic plasticity as a signaling molecule that increases synaptic strength by a protein synthesis-dependent mechanism.

  View details for DOI 10.1016/j.neuron.2008.08.012

  View details for Web of Science ID 000260549300013

  View details for PubMedID 18957222

• Retinoic acid regulates RAR alpha-mediated control of translation in dendritic RNA granules during homeostatic synaptic plasticity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Maghsoodi, B., Poon, M. M., Nam, C. I., Aoto, J., Ting, P., Chen, L. 2008; 105 (41): 16015-16020

  Abstract

  Homeostatic plasticity is thought to play an important role in maintaining the stability of neuronal circuits. During one form of homeostatic plasticity, referred to as synaptic scaling, activity blockade leads to a compensatory increase in synaptic transmission by stimulating in dendrites the local translation and synaptic insertion of the AMPA receptor subunit GluR1. We have previously shown that all-trans retinoic acid (RA) mediates activity blockade-induced synaptic scaling by activating dendritic GluR1 synthesis and that this process requires RARalpha, a member of the nuclear RA receptor family. This result raised the question of where RARalpha is localized in dendrites and whether its localization is regulated by RA and/or activity blockade. Here, we show that activity blockade or RA treatment in neurons enhances the concentration of RARalpha in the dendritic RNA granules and activates local GluR1 synthesis in these RNA granules. Importantly, the same RNA granules that contain RARalpha also exhibit an accumulation of GluR1 protein but with a much slower time course than that of RARalpha, suggesting that the former regulates the latter. Taken together, our results provide a direct link between dendritically localized RARalpha and local GluR1 synthesis in RNA granules during RA-mediated synaptic signaling in homeostatic synaptic plasticity.

  View details for DOI 10.1073/pnas.0804801105

  View details for Web of Science ID 000260240900067

  View details for PubMedID 18840692

• Bidirectional ephrin/Eph signaling in synaptic functions BRAIN RESEARCH Aoto, J., Chen, L. 2007; 1184: 72-80

  Abstract

  Eph receptors, the largest family of receptor tyrosine kinases, and their membrane bound ligands, the ephrins, are involved in multiple developmental and adult processes within and outside of the nervous system. Bi-directional signaling from both the receptor and the ligand is initiated by ephrin-Eph binding upon cell-cell contact, and involves interactions with distinct subsets of downstream signaling molecules related to specific functions. In the CNS, Ephs and ephrins act as attractive/repulsive, migratory and cell adhesive cues during development and participate in synaptic functions in adult animals. In this review, we will focus on recent findings highlighting the functions of ephrin/Eph signaling in dendritic spine morphogenesis, synapse formation and synaptic plasticity.

  View details for DOI 10.1016/j.brainres.2006.11.033

  View details for Web of Science ID 000252096600009

  View details for PubMedID 17166489

• Postsynaptic EphrinB3 promotes shaft glutamatergic synapse formation JOURNAL OF NEUROSCIENCE Aoto, J., Ting, P., Maghsoodi, B., Xu, N., Henkemeyer, M., Chen, L. 2007; 27 (28): 7508-7519

  Abstract

  Excitatory synapses in the CNS are formed on both dendritic spines and shafts. Recent studies show that the density of shaft synapses may be independently regulated by behavioral learning and the induction of synaptic plasticity, suggesting that distinct mechanisms are involved in regulating these two types of synapses. Although the molecular mechanisms underlying spinogenesis and spine synapse formation are being delineated, those regulating shaft synapses are still unknown. Here, we show that postsynaptic ephrinB3 expression promotes the formation of glutamatergic synapses specifically on the shafts, not on spines. Reducing or increasing postsynaptic ephrinB3 expression selectively decreases or increases shaft synapse density, respectively. In the ephrinB3 knock-out mouse, although spine synapses are normal, shaft synapse formation is reduced in the hippocampus. Overexpression of glutamate receptor-interacting protein 1 (GRIP1) rescues ephrinB3 knockdown phenotype by restoring shaft synapse density. GRIP1 knockdown prevents the increase in shaft synapse density induced by ephrinB3 overexpression. Together, our results reveal a novel mechanism for independent modulation of shaft synapses through ephrinB3 reverse signaling.

  View details for DOI 10.1523/JNEUROSCI.0705-07.2007

  View details for Web of Science ID 000248147900016

  View details for PubMedID 17626212

• Dynamics of postsynaptic glutamate receptor targeting CURRENT OPINION IN NEUROBIOLOGY Chen, L., Tracy, T., Nam, C. I. 2007; 17 (1): 53-58

  Abstract

  Targeting of glutamate receptors to synapses is an important event in both developing and mature neurons. Glutamate receptors are delivered to nascent synapses during synaptogenesis and to existing synapses during activity-dependent synaptic strengthening. Increasing evidence suggests that glutamate receptors are inserted into the plasma membrane before they accumulate at the synapse. Lateral diffusion of receptors occurs at both synaptic and non-synaptic membranes, and glutamate receptors can exchange rapidly between synaptic and extrasynaptic sites. In addition, recent studies show that postsynaptic scaffold molecules can be highly mobile. The dynamic nature of the synapse suggests that many mechanisms might be involved in regulating synapse formation and synaptic plasticity.

  View details for DOI 10.1016/j.conb.2006.11.001

  View details for Web of Science ID 000244771100008

  View details for PubMedID 17161597

• Postsynaptic assembly induced by neurexin-neuroligin interaction and neurotransmitter PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nam, C. I., Chen, L. 2005; 102 (17): 6137-6142

  Abstract

  Presynaptic and postsynaptic differentiation occurs at axodendritic contacts between CNS neurons. Synaptic adhesion mediated by synaptic cell adhesion molecule (SynCAM) and beta-neurexins/neuroligins triggers presynaptic differentiation. The signals that trigger postsynaptic differentiation are, however, unknown. Here we report that beta-neurexin expressed in nonneuronal cells induced postsynaptic density (PSD)-95 clustering in contacting dendrites of hippocampal neurons. The effect is specific to beta-neurexin and was not observed with other synaptic cell adhesion molecules such as N-cadherin or SynCAM. NMDA receptors, but not alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptors (AMPARs), were recruited to this beta-neurexin-induced PSD-95 scaffold. Remarkably, AMPARs were inserted into this scaffold upon glutamate application or expression of a constitutively active form of calmodulin kinase II in neurons. Expression of a dominant-negative neuroligin-1 in cultured neurons markedly reduced the sizes and densities of PSD-95 puncta and AMPAR clusters. In addition, excitatory, but not inhibitory, synaptic functions were impaired in these neurons, confirming that PSD-95/neuroligin-1 interaction is involved in postsynaptic assembly at glutamatergic synapses. These results demonstrate that postsynaptic assembly of the glutamatergic synapse may be initiated by presynaptic beta-neurexin and that glutamate release also is required for maturation of synapses.

  View details for DOI 10.1073/pnas.0502038102

  View details for Web of Science ID 000228738800045

  View details for PubMedID 15837930

• Stargazin differentially controls the trafficking of alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate and kainate receptors MOLECULAR PHARMACOLOGY Chen, L., El-Husseini, A., Tomita, S., Bredt, D. S., Nicoll, R. A. 2003; 64 (3): 703-706

  Abstract

  Synaptic plasticity at excitatory synapses in the brain is largely achieved by rapid changes in the number of synaptic alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptors. Stargazin, a membrane protein that interacts with AMPA receptors, is believed to play a pivotal role in trafficking AMPA receptors to the plasma membrane and targeting them to the synapse. However, it is unclear whether the trafficking of kainate receptors, which are structurally very similar to AMPA receptors, is also dependent on stargazin. Here we show that in both cerebellar granule cells and in Xenopus laevis oocytes expression system, surface delivery of kainate receptor is independent of stargazin. These results suggest that stargazin action is highly selective for AMPA receptors.

  View details for Web of Science ID 000184764300020

  View details for PubMedID 12920207

• Functional studies and distribution define a family of transmembrane AMPA receptor regulatory proteins JOURNAL OF CELL BIOLOGY Tomita, S., Chen, L., Kawasaki, Y., Petralia, R. S., Wenthold, R. J., Nicoll, R. A., Bredt, D. S. 2003; 161 (4): 805-816

  Abstract

  Functional expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in cerebellar granule cells requires stargazin, a member of a large family of four-pass transmembrane proteins. Here, we define a family of transmembrane AMPA receptor regulatory proteins (TARPs), which comprise stargazin, gamma-3, gamma-4, and gamma-8, but not related proteins, that mediate surface expression of AMPA receptors. TARPs exhibit discrete and complementary patterns of expression in both neurons and glia in the developing and mature central nervous system. In brain regions that express multiple isoforms, such as cerebral cortex, TARP-AMPA receptor complexes are strictly segregated, suggesting distinct roles for TARP isoforms. TARPs interact with AMPA receptors at the postsynaptic density, and surface expression of mature AMPA receptors requires a TARP. These studies indicate a general role for TARPs in controlling synaptic AMPA receptors throughout the central nervous system.

  View details for DOI 10.1083/jcb.200212116

  View details for Web of Science ID 000183286700016

  View details for PubMedID 12771129

• Direct interactions between PSD-95 and stargazin control synaptic AMPA receptor number PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schnell, E., Sizemore, M., Karimzadegan, S., Chen, L., Bredt, D. S., Nicoll, R. A. 2002; 99 (21): 13902-13907

  Abstract

  Excitatory synapses in the brain exhibit a remarkable degree of functional plasticity, which largely reflects changes in the number of synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). However, mechanisms involved in recruiting AMPARs to synapses are unknown. Here we use hippocampal slice cultures and biolistic gene transfections to study the targeting of AMPARs to synapses. We show that AMPARs are localized to synapses through direct binding of the first two PDZ domains of synaptic PSD-95 (postsynaptic density protein of 95 kDa) to the AMPAR-associated protein, stargazin. Increasing the level of synaptic PSD-95 recruits new AMPARs to synapses without changing the number of surface AMPARs. At the same time, we show that stargazin overexpression drastically increases the number of extra-synaptic AMPARs, but fails to alter synaptic currents if synaptic PSD-95 levels are kept constant. Finally, we make compensatory mutations to both PSD-95 and stargazin to demonstrate the central role of direct interactions between them in determining the number of synaptic AMPARs.

  View details for DOI 10.1073/pnas.192511199

  View details for Web of Science ID 000178635700100

  View details for PubMedID 12359873

• Phosphorylation of the postsynaptic density-95 (PSD-95)/discs large/zona occludens-1 binding site of stargazin regulates binding to PSD-95 and synaptic targeting of AMPA receptors JOURNAL OF NEUROSCIENCE Chetkovich, D. M., Chen, L., Stocker, T. J., Nicoll, R. A., Bredt, D. S. 2002; 22 (14): 5791-5796

  Abstract

  Dynamic regulation of AMPA-type receptors at the synapse is proposed to play a critical role in alterations of the synaptic strength seen in cellular models of learning and memory such as long-term potentiation in the hippocampus. Stargazin, previously identified as an AMPA receptor (AMPAR)-interacting protein, is critical for surface expression and synaptic targeting of AMPARs. Stargazin interacts with postsynaptic density-95/discs large/zona occludens-1 (PDZ) proteins via a C-terminal PDZ binding motif. Interestingly, the C terminal of stargazin also predicts phosphorylation at a threonine residue critical for PDZ protein binding. Because protein phosphorylation regulates synaptic plasticity, we characterized this site and the effects of stargazin phosphorylation on AMPAR function. In vitro peptide phosphorylation assays and Western blot analysis with phospho-stargazin-specific antibodies indicate that the critical threonine within the stargazin PDZ binding site is phosphorylated by protein kinase A. This phosphorylation disrupts stargazin interaction and clustering with postsynaptic density-95 (PSD-95) in transfected COS-7 cells. Furthermore, a stargazin construct with a Thr-to-Glu mutation that mimics phosphorylation fails to cluster at synaptic spines and downregulates synaptic AMPAR function in cultured hippocampal neurons. These data suggest that phosphorylation of the stargazin PDZ ligand can disrupt stargazin interaction with PSD-95 and thereby regulate synaptic AMPAR function.

  View details for Web of Science ID 000176840500001

  View details for PubMedID 12122038

• Cerebellar cortical inhibition and classical eyeblink conditioning PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bao, S. W., Chen, L., Kim, J. J., Thompson, R. F. 2002; 99 (3): 1592-1597

  Abstract

  The cerebellum is considered a brain structure in which memories for learned motor responses (e.g., conditioned eyeblink responses) are stored. Within the cerebellum, however, the relative importance of the cortex and the deep nuclei in motor learning/memory is not entirely clear. In this study, we show that the cerebellar cortex exerts both basal and stimulus-activated inhibition to the deep nuclei. Sequential application of a gamma-aminobutyric acid type A receptor (GABA(A)R) agonist and a noncompetitive GABA(A)R antagonist allows selective blockade of stimulus-activated inhibition. By using the same sequential agonist and antagonist methods in behaving animals, we demonstrate that the conditioned response (CR) expression and timing are completely dissociable and involve different inhibitory inputs; although the basal inhibition modulates CR expression, the conditioned stimulus-activated inhibition is required for the proper timing of the CR. In addition, complete blockade of cerebellar deep nuclear GABA(A)Rs prevents CR acquisition. Together, these results suggest that different aspects of the memories for eyeblink CRs are encoded in the cerebellar cortex and the cerebellar deep nuclei.

  View details for Web of Science ID 000173752500089

  View details for PubMedID 11805298

• Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms NATURE Chen, L., Chetkovich, D. M., Petralia, R. S., Sweeney, N. T., Kawasaki, Y., Wenthold, R. J., Bredt, D. S., Nicoll, R. A. 2000; 408 (6815): 936-943

  Abstract

  Stargazer, an ataxic and epileptic mutant mouse, lacks functional AMPA (alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate) receptors on cerebellar granule cells. Stargazin, the mutated protein, interacts with both AMPA receptor subunits and synaptic PDZ proteins, such as PSD-95. The interaction of stargazin with AMPA receptor subunits is essential for delivering functional receptors to the surface membrane of granule cells, whereas its binding with PSD-95 and related PDZ proteins through a carboxy-terminal PDZ-binding domain is required for targeting the AMPA receptor to synapses. Expression of a mutant stargazin lacking the PDZ-binding domain in hippocampal pyramidal cells disrupts synaptic AMPA receptors, indicating that stargazin-like mechanisms for targeting AMPA receptors may be widespread in the central nervous system.

  View details for Web of Science ID 000165951100039

  View details for PubMedID 11140673

• Learning- and cerebellum-dependent neuronal activity in the lateral pontine nucleus BEHAVIORAL NEUROSCIENCE Bao, S. W., Chen, L., Thompson, R. F. 2000; 114 (2): 254-261

  Abstract

  The effects of inactivation of cerebellar deep nuclei and the lateral pontine nucleus on classical eyeblink conditioning with tone or lateral reticular nucleus (LRN) stimulation as conditioned stimuli (CSs) were examined. Inactivation of cerebellar deep nuclei abolished eyeblink conditioned responses (CRs) when the CS was either a tone or LRN stimulation. Inactivation of the lateral pontine nucleus prevented only the acquisition and retention of tone-evoked eyeblink CRs. Multiple-unit recording demonstrated that when LRN stimulation was used as the CS, inactivation of the interpositus nucleus abolished learning-related neuronal activity in the lateral pontine nucleus, whereas inactivation of pontine nucleus had little effect on similar activity in the interpositus nucleus. Thus, the learning-induced neuronal activity in the lateral pontine nucleus was most likely driven by the cerebellar interpositus nucleus.

  View details for DOI 10.1037//0735-7044.114.2.254

  View details for Web of Science ID 000087492200004

  View details for PubMedID 10832787

• Impaired cerebellar synapse maturation in waggler, a mutant mouse with a disrupted neuronal calcium channel gamma subunit PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, L., Bao, S. W., Qiao, X. X., Thompson, R. F. 1999; 96 (21): 12132-12137

  Abstract

  The waggler, a neurological mutant mouse with a disrupted putative neuronal Ca(2+) channel gamma subunit, exhibits a cerebellar granule cell-specific brain-derived neurotrophic factor deficit, severe ataxia, and impaired eyeblink conditioning. Here, we show that multiple synapses of waggler cerebellar granule cells are arrested at an immature stage during development. Synaptic transmission is reduced at parallel fiber-Purkinje cell synapses. The Golgi cell-granule cell synaptic currents show immature kinetics associated with reduced gamma-aminobutyric acid type A receptor alpha6 subunit expression in granule cells. In addition, the mossy fiber-granule cell synapses exhibit N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs), but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated EPSCs. Our results suggest that voltage-dependent Ca(2+) channels are involved in synapse maturation. This deficient synaptic transmission in the waggler cerebellum may account for their behavioral deficits.

  View details for Web of Science ID 000083166800081

  View details for PubMedID 10518588

• Transgenic brain-derived neurotrophic factor modulates a developing cerebellar inhibitory synapse LEARNING & MEMORY Bao, S. W., Chen, L., Qiao, X. X., Thompson, R. F. 1999; 6 (3): 276-283

  Abstract

  Brain-derived neurotrophic factor (BDNF) has been shown to promote synapse formation and maturation in neurons of many brain regions, including inhibitory synapses. In the cerebellum, the Golgi cell-granule cell GABAergic synaptic responses undergo developmental transition from slow-decaying to fast-decaying kinetics, which parallels a developmental increase of GABA(A) receptor alpha6 subunit expression in the cerebellar granule cells. In culture, BDNF accelerates the expression of GABA(A) receptor alpha6 subunit expression in granule cells. Here we examined synaptic GABA(A) response kinetics in BDNF transgenic mice. The mutant mouse, which carries a BDNF transgene driven by a beta-actin promoter, overexpresses BDNF (two- to fivefold increase compared with wild types) in all brain regions. Recordings of the spontaneous GABA(A) responses indicate that the decay time constant of the GABAergic responses decreases during early postnatal development; this transition is accelerated in the BDNF transgenic mouse. The amplitude of the spontaneous GABA(A) responses was also larger in the transgenic mouse than in the wild-type mouse. However, the frequency of the spontaneous GABA(A) responses were not different between the two groups. Our results suggest that BDNF may modulate GABAergic synapse maturation in the cerebellum.

  View details for Web of Science ID 000082396600007

  View details for PubMedID 10492009

• Bilateral lesions of the interpositus nucleus completely prevent eyeblink conditioning in Purkinje cell-degeneration mutant mice BEHAVIORAL NEUROSCIENCE Chen, L., Bao, S. W., Thompson, R. F. 1999; 113 (1): 204-210

  Abstract

  The authors have previously demonstrated that Purkinje cell-degeneration (pcd) mutant mice are impaired in eyeblink conditioning (L. Chen et al., 1996a). The present study addresses the following 3 questions: (a) whether pcd mice perceive the conditioned and unconditioned stimuli as well as the wild-type mice, (b) whether pcd mice have a normal sensitization level, and (c) whether the residual learning in pcd mice is cerebellum-dependent. Results indicated that the pcd mice exhibited normal tone-induced responses in the cochlear nucleus and normal sensitivity to heat-induced pain. They showed a similar level of sensitization as the wild-type mice and were completely unable to learn conditioned eyeblinks after bilateral lesions aimed at the anterior interpositus nucleus. Thus, pcd mice are partially impaired in eyeblink conditioning because of a deficiency in learning mechanisms, and the residual learning in the pcd mice is mediated by the cerebellar nuclei.

  View details for Web of Science ID 000079191100020

  View details for PubMedID 10197920

• Impaired eye-blink conditioning in waggler, a mutant mouse with cerebellar BDNF deficiency LEARNING & MEMORY Bao, S. W., Chen, L., Qiao, X. X., Knusel, B., Thompson, R. F. 1998; 5 (4-5): 355-364

  Abstract

  In addition to their trophic functions, neurotrophins are also implicated in synaptic modulation and learning and memory. Although gene knockout techniques have been used widely in studying the roles of neurotrophins at molecular and cellular levels, behavioral studies using neurotrophin knockouts are limited by the early-onset lethality and various sensory deficits associated with the gene knockout mice. In the present study, we found that in a spontaneous mutant mouse, waggler, the expression of brain-derived neurotrophic factor (BDNF) was selectively absent in the cerebellar granule cells. The cytoarchitecture of the waggler cerebellum appeared to be normal at the light microscope level. The mutant mice exhibited no sensory deficits to auditory stimuli or heat-induced pain. However, they were massively impaired in classic eye-blink conditioning. These results suggest that BDNF may have a role in normal cerebellar neuronal function, which, in turn, is essential for classic eye-blink conditioning.

  View details for Web of Science ID 000076812700009

  View details for PubMedID 10454360

• Cerebellar brain-derived neurotrophic factor-TrkB defect associated with impairment of eyeblink conditioning in stargazer mutant mice JOURNAL OF NEUROSCIENCE Qiao, X. X., Gao, H., Bao, S. W., HEFTI, F., Thompson, R. F., Knusel, B. 1998; 18 (17): 6990-6999

  Abstract

  In the spontaneous ataxic mutant mouse stargazer, there is a selective reduction of brain-derived neurotrophic factor (BDNF) mRNA expression in the cerebellum. BDNF protein levels in the cerebellum are reduced by 70%. Despite normal levels of full-length and truncated TrkB receptor, constitutive and neurotrophin-4/5-induced tyrosine phosphorylation was significantly reduced in several signal transduction molecules, including phospholipase-Cgamma1, erk1, and erk2. Morphological examination revealed an increased number of external granule cells at postnatal day 15 and the presence of abnormal neurons resembling immature granule cells in the adult. These abnormalities are associated with a severe impairment in the acquisition of classical eyeblink conditioning, indicating cerebellar malfunction. Our data suggest that normal BDNF expression and TrkB signal transduction in the cerebellum are necessary for learning and plasticity in this model.

  View details for Web of Science ID 000075501200035

  View details for PubMedID 9712667

• Classical eyeblink conditioning in two strains of mice: Conditioned responses, sensitization, and spontaneous eyeblinks BEHAVIORAL NEUROSCIENCE Bao, S. W., Chen, L., Thompson, R. F. 1998; 112 (3): 714-718

  Abstract

  Conditioned eyeblink responses (CRs), sensitization, and spontaneous eyeblinks were studied in C57BL/6J and BALB/c mice. Both strains of mice acquired CRs during 10 days of classical delay eyeblink conditioning. The BALB/c mice reached a higher asymptotic CR level than the C57BL/6J mice. The CRs were extinguished and recovered in both strains following conditioned stimulus-alone and paired conditioned stimulus-unconditioned stimulus training. During 10 days of explicitly unpaired training, the control groups showed no signs of sensitization and low incidence of spontaneous eyeblinks. When switched to paired training, the unpaired groups exhibited significant conditioned inhibition. These results suggest that strain differences must be considered in experimental design and data interpretation for these basic aspects of associative learning and memory.

  View details for Web of Science ID 000074529700022

  View details for PubMedID 9676986

• Selective enhancement of emotional, but not motor, learning in monoamine oxidase A-deficient mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, J. J., Shih, J. C., Chen, K., Chen, L., Bao, S. W., Maren, S., Anagnostaras, S. G., Fanselow, M. S., DEMAEYER, E., Seif, I., Thompson, R. F. 1997; 94 (11): 5929-5933

  Abstract

  Mice deficient in monoamine oxidase A (MAOA), an enzyme that metabolizes monoamines such as norepinephrine and serotonin, have elevated norepinephrine and serotonin levels in the frontal cortex, hippocampus, and cerebellum, compared with normal wild-type mice. Since monoamines in these areas are critically involved in a variety of behaviors, we examined learning and memory (using emotional and motor tasks) in MAOA mutant mice. The MAOA-deficient mice exhibited significantly enhanced classical fear conditioning (freezing to both tone and contextual stimuli) and step-down inhibitory avoidance learning. In contrast, eyeblink conditioning was normal in these mutant mice. The female MAOA-deficient mice also displayed normal species-typical maternal behaviors (nesting, nursing, and pup retrieval). These results suggest that chronic elevations of monoamines, due to a deletion of the gene encoding MAOA, lead to selective alterations in emotional behavior.

  View details for Web of Science ID A1997XB71100082

  View details for PubMedID 9159177

• Associative learning CEREBELLUM AND COGNITION Thompson, R. F., Bao, S. W., Chen, L., Cipriano, B. D., Grethe, J. S., Kim, J. J., Thompson, J. K., Tracy, J. A., Weninger, M. S., Krupa, D. J. 1997; 41: 151-189

  Abstract

  This chapter reviews evidence demonstrating the essential role of the cerebellum and its associated circuitry in the learning and memory of classical conditioning of discrete behavioral responses (e.g., eyeblink, limb flexion, head turn). It now seems conclusive that the memory traces for this basic category of associative learning are formed and stored in the cerebellum. Lesion, neuronal recording, electrical microstimulation, and anatomical procedures have been used to identify the essential conditioned stimulus (CS) circuit, including the pontine mossy fiber projections to the cerebellum; the essential unconditioned stimulus (US) reinforcing or teaching circuit, including neurons in the inferior olive (dorsal accessory olive) projecting to the cerebellum as climbing fibers; and the essential conditioned response (CR) circuit, including the interpositus nucleus, its projection via the superior cerebellar peduncle to the magnocellular red nucleus, and rubral projections to premotor and motor nuclei. Each major component of the eyeblink CR circuit was reversibly inactivated both in trained animals and over the course of training. In all cases in trained animals, inactivation abolished the CR (and the UR as well when motor nuclei were inactivated). When animals were trained during inactivation (and not exhibiting CRs) and then tested without inactivation, animals with inactivation of the motor nuclei, red nucleus, and superior peduncle had fully learned, whereas animals with inactivation of a very localized region of the cerebellum (anterior interpositus and overlying cortex) had not learned at all. Consequently, the memory traces are formed and stored in the cerebellum. Several alternative possibilities are considered and ruled out. Both the cerebellar cortex and the interpositus nucleus are involved in the memory storage process, suggesting that a phenomenon-like long-term depression (LTD) is involved in the cerebellar cortex and long-term potentiation (LTP) is involved in the interpositus. The experimental findings reviewed in this chapter provide perhaps the first conclusive evidence for the localization of a basic form of memory storage to a particular brain region, namely the cerebellum, and indicate that the cerebellum is indeed a cognitive machine.

  View details for Web of Science ID A1997BJ66A00007

  View details for PubMedID 9378587

• Deficient cerebellar long-term depression, impaired eyeblink conditioning, and normal motor coordination in GFAP mutant mice NEURON Shibuki, K., Gomi, H., Chen, L., Bao, S. W., Kim, J. S., Wakatsuki, H., Fujisaki, T., Fujimoto, J., Katoh, A., Ikeda, T., Chen, C., Thompson, R. F., Itohara, S. 1996; 16 (3): 587-599

  Abstract

  Mice devoid of glial fibrillary acidic protein (GFAP), an intermediate filament protein specifically expressed in astrocytes, develop normally and do not show any detectable abnormalities in the anatomy of the brain. In the cerebellum, excitatory synaptic transmission from parallel fibers (PFs) or climbing fibers (CFs) to Purkinje cells is unaltered, and these synapses display normal short-term synaptic plasticity to paired stimuli in GFAP mutant mice. In contrast, long-term depression (LTD) at PF-Purkinje cell synapses is clearly deficient. Furthermore, GFAP mutant mice exhibited a significant impairment of eyeblink conditioning without any detectable deficits in motor coordination tasks. These results suggest that GFAP is required for communications between Bergmann glia and Purkinje cells during LTD induction and maintenance. The data support the notion that cerebellar LTD is a cellular mechanism closely associated with eyeblink conditioning, but is not essential for motor coordination tasks tested.

  View details for Web of Science ID A1996UC16800015

  View details for PubMedID 8785056

• Impaired motor coordination correlates with persistent multiple climbing fiber innervation in PKC gamma mutant mice CELL Chen, C., Kano, M., Abeliovich, A., Chen, L., Bao, S. W., Kim, J. J., HASHIMOTO, K., Thompson, R. F., Tonegawa, S. 1995; 83 (7): 1233-1242

  Abstract

  It is generally believed that a smooth execution of a compound movement, or motor coordination, requires learning of component movements as well as experience-based refinement of the motor program as a whole. PKC gamma mutant mice display impaired motor coordination but intact eyeblink conditioning, a form of component movement learning. Cerebellar long-term depression, a putative cellular mechanism for component motor learning, is also unimpaired. Thus, PKC gamma mutant mice are defective in refinement of the motor program. In the accompanying paper, we demonstrate that innervation of multiple climbing fibers onto Purkinje cells persists in adulthood in these mutant mice. We propose that this defective elimination of surplus climbing fibers underlies motor discoordination.

  View details for Web of Science ID A1995TM76200018

  View details for PubMedID 8548809