Publications by authors named "Richard W Tsien"

77 Publications

Unique dynamics and exocytosis properties of GABAergic synaptic vesicles revealed by three-dimensional single vesicle tracking.

Proc Natl Acad Sci U S A 2021 Mar;118(9)

Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, 999077 Kowloon, Hong Kong;

Maintaining the balance between neuronal excitation and inhibition is essential for proper function of the central nervous system. Inhibitory synaptic transmission plays an important role in maintaining this balance. Although inhibitory transmission has higher kinetic demands compared to excitatory transmission, its properties are poorly understood. In particular, the dynamics and exocytosis of single inhibitory vesicles have not been investigated, due largely to both technical and practical limitations. Using a combination of quantum dots (QDs) conjugated to antibodies against the luminal domain of the vesicular GABA transporter to selectively label GABAergic (i.e., predominantly inhibitory) vesicles together with dual-focus imaging optics, we tracked the real-time three-dimensional position of single GABAergic vesicles up to the moment of exocytosis (i.e., fusion). Using three-dimensional trajectories, we found that GABAergic synaptic vesicles traveled a shorter distance prior to fusion and had a shorter time to fusion compared to synaptotagmin-1 (Syt1)-labeled vesicles, which were mostly from excitatory neurons. Moreover, our analysis revealed that GABAergic synaptic vesicles move more straightly to their release sites than Syt1-labeled vesicles. Finally, we found that GABAergic vesicles have a higher prevalence of kiss-and-run fusion than Syt1-labeled vesicles. These results indicate that inhibitory synaptic vesicles have a unique set of dynamics and exocytosis properties to support rapid synaptic inhibition, thereby maintaining a tightly regulated coordination between excitation and inhibition in the central nervous system.
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http://dx.doi.org/10.1073/pnas.2022133118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936280PMC
March 2021

Synaptic vesicle traffic is supported by transient actin filaments and regulated by PKA and NO.

Nat Commun 2020 10 21;11(1):5318. Epub 2020 Oct 21.

NYU Neuroscience Institute and Department of Neuroscience and Physiology, NYU Langone Medical Center, New York, NY, 10016, USA.

Synaptic vesicles (SVs) can be pooled across multiple synapses, prompting questions about their dynamic allocation for neurotransmission and plasticity. We find that the axonal traffic of recycling vesicles is not supported by ubiquitous microtubule-based motility but relies on actin instead. Vesicles freed from synaptic clusters undergo ~1 µm bouts of active transport, initiated by nearby elongation of actin filaments. Long distance translocation arises when successive bouts of active transport were linked by periods of free diffusion. The availability of SVs for active transport can be promptly increased by protein kinase A, a key player in neuromodulation. Vesicle motion is in turn impeded by shutting off axonal actin polymerization, mediated by nitric oxide-cyclic GMP signaling leading to inhibition of RhoA. These findings provide a potential framework for coordinating post-and pre-synaptic strength, using retrograde regulation of axonal actin dynamics to mobilize and recruit presynaptic SV resources.
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http://dx.doi.org/10.1038/s41467-020-19120-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578807PMC
October 2020

Neuronal Inactivity Co-opts LTP Machinery to Drive Potassium Channel Splicing and Homeostatic Spike Widening.

Cell 2020 06 2;181(7):1547-1565.e15. Epub 2020 Jun 2.

Department of Neuroscience and Physiology, Neuroscience Institute, NYU Grossman Medical Center, New York, NY 10016, USA; Center for Neural Science, New York University, New York, NY 10003, USA. Electronic address:

Homeostasis of neural firing properties is important in stabilizing neuronal circuitry, but how such plasticity might depend on alternative splicing is not known. Here we report that chronic inactivity homeostatically increases action potential duration by changing alternative splicing of BK channels; this requires nuclear export of the splicing factor Nova-2. Inactivity and Nova-2 relocation were connected by a novel synapto-nuclear signaling pathway that surprisingly invoked mechanisms akin to Hebbian plasticity: Ca-permeable AMPA receptor upregulation, L-type Ca channel activation, enhanced spine Ca transients, nuclear translocation of a CaM shuttle, and nuclear CaMKIV activation. These findings not only uncover commonalities between homeostatic and Hebbian plasticity but also connect homeostatic regulation of synaptic transmission and neuronal excitability. The signaling cascade provides a full-loop mechanism for a classic autoregulatory feedback loop proposed ∼25 years ago. Each element of the loop has been implicated previously in neuropsychiatric disease.
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http://dx.doi.org/10.1016/j.cell.2020.05.013DOI Listing
June 2020

Real-time three-dimensional tracking of single synaptic vesicles reveals that synaptic vesicles undergoing kiss-and-run fusion remain close to their original fusion site before reuse.

Biochem Biophys Res Commun 2019 06 12;514(3):1004-1008. Epub 2019 May 12.

Department of Physics, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. Electronic address:

The release of neurotransmitters via the fusion between synaptic vesicles and the presynaptic membrane is an essential step in synaptic transmission. Synaptic vesicles generally undergo two distinct modes of exocytosis called full-collapse fusion and kiss-and-run fusion. In kiss-and-run fusion, the fusion pore of the synaptic vesicle opens transiently without the vesicle collapsing fully into the plasma membrane; thus, each synaptic vesicle can be used multiple times to release neurotransmitters. Despite considerable research, the detailed mechanisms that underlie kiss-and-run fusion remain elusive, particularly the location of synaptic vesicles after kiss-and-run events. To address this question, we performed real-time three-dimensional tracking of single synaptic vesicles labeled with a single quantum dot in the presynaptic terminal of cultured hippocampal neurons and analyzed the three-dimensional trajectories of these vesicles undergoing kiss-and-run fusion. We found that the majority of these synaptic vesicles underwent another exocytosis event within 120 nm of their original fusion site and underwent a second exocytosis event within 10 s of the first fusion event. These results indicate that after kiss-and-run fusion, synaptic vesicles remain relatively close to their original fusion site and can release repeatedly at brief intervals, allowing neurons to maintain neurotransmitter release during bursting activity.
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http://dx.doi.org/10.1016/j.bbrc.2019.05.043DOI Listing
June 2019

Oxytocin Transforms Firing Mode of CA2 Hippocampal Neurons.

Neuron 2018 11 4;100(3):593-608.e3. Epub 2018 Oct 4.

NYU Neuroscience Institute, New York University School of Medicine, New York, NY 10016, USA. Electronic address:

Oxytocin is an important neuromodulator in the mammalian brain that increases information salience and circuit plasticity, but its signaling mechanisms and circuit effect are not fully understood. Here we report robust oxytocinergic modulation of intrinsic properties and circuit operations in hippocampal area CA2, a region of emerging importance for hippocampal function and social behavior. Upon oxytocin receptor activation, CA2 pyramidal cells depolarize and fire bursts of action potentials, a consequence of phospholipase C signaling to modify two separate voltage-dependent ionic processes. A reduction of potassium current carried by KCNQ-based M channels depolarizes the cell; protein kinase C activity attenuates spike rate of rise and overshoot, dampening after-hyperpolarizations. These actions, in concert with activation of fast-spiking interneurons, promote repetitive firing and CA2 bursting; bursting then governs short-term plasticity of CA2 synaptic transmission onto CA1 and, thus, efficacy of information transfer in the hippocampal network.
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http://dx.doi.org/10.1016/j.neuron.2018.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6516476PMC
November 2018

Calmodulin shuttling mediates cytonuclear signaling to trigger experience-dependent transcription and memory.

Nat Commun 2018 06 22;9(1):2451. Epub 2018 Jun 22.

Institute of Neuroscience, and Department of Neurology of Second Affiliated Hospital, Mental Health Center, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, 310058, China.

Learning and memory depend on neuronal plasticity originating at the synapse and requiring nuclear gene expression to persist. However, how synapse-to-nucleus communication supports long-term plasticity and behavior has remained elusive. Among cytonuclear signaling proteins, γCaMKII stands out in its ability to rapidly shuttle Ca/CaM to the nucleus and thus activate CREB-dependent transcription. Here we show that elimination of γCaMKII prevents activity-dependent expression of key genes (BDNF, c-Fos, Arc), inhibits persistent synaptic strengthening, and impairs spatial memory in vivo. Deletion of γCaMKII in adult excitatory neurons exerts similar effects. A point mutation in γCaMKII, previously uncovered in a case of intellectual disability, selectively disrupts CaM sequestration and CaM shuttling. Remarkably, this mutation is sufficient to disrupt gene expression and spatial learning in vivo. Thus, this specific form of cytonuclear signaling plays a key role in learning and memory and contributes to neuropsychiatric disease.
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http://dx.doi.org/10.1038/s41467-018-04705-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015085PMC
June 2018

Direct Visualization of Wide Fusion-Fission Pores and Their Highly Varied Dynamics.

Cell 2018 05;173(4):819-821

Department of Neuroscience and Physiology, Neuroscience Institute, New York University, New York, NY 10016, USA. Electronic address:

In this issue of Cell, Shin et al. report the first live-cell imaging of a fusion pore. Directly visualized pores in neuroendocrine cells can be much larger than expected yet not require vesicular full-collapse. These fusion-fission pores have diverse fates arising from opposing dynamin-driven pore constriction and F-actin-mediated pore expansion.
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http://dx.doi.org/10.1016/j.cell.2018.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475904PMC
May 2018

Discovery of peptide ligands through docking and virtual screening at nicotinic acetylcholine receptor homology models.

Proc Natl Acad Sci U S A 2017 09 5;114(38):E8100-E8109. Epub 2017 Sep 5.

Department of Chemistry, Belfer Research Center-Hunter College, New York, NY 10021;

Venom peptide toxins such as conotoxins play a critical role in the characterization of nicotinic acetylcholine receptor (nAChR) structure and function and have potential as nervous system therapeutics as well. However, the lack of solved structures of conotoxins bound to nAChRs and the large size of these peptides are barriers to their computational docking and design. We addressed these challenges in the context of the α4β2 nAChR, a widespread ligand-gated ion channel in the brain and a target for nicotine addiction therapy, and the 19-residue conotoxin α-GID that antagonizes it. We developed a docking algorithm, ToxDock, which used ensemble-docking and extensive conformational sampling to dock α-GID and its analogs to an α4β2 nAChR homology model. Experimental testing demonstrated that a virtual screen with ToxDock correctly identified three bioactive α-GID mutants (α-GID[A10V], α-GID[V13I], and α-GID[V13Y]) and one inactive variant (α-GID[A10Q]). Two mutants, α-GID[A10V] and α-GID[V13Y], had substantially reduced potency at the human α7 nAChR relative to α-GID, a desirable feature for α-GID analogs. The general usefulness of the docking algorithm was highlighted by redocking of peptide toxins to two ion channels and a binding protein in which the peptide toxins successfully reverted back to near-native crystallographic poses after being perturbed. Our results demonstrate that ToxDock can overcome two fundamental challenges of docking large toxin peptides to ion channel homology models, as exemplified by the α-GID:α4β2 nAChR complex, and is extendable to other toxin peptides and ion channels. ToxDock is freely available at rosie.rosettacommons.org/tox_dock.
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http://dx.doi.org/10.1073/pnas.1703952114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617267PMC
September 2017

Roger Yonchien Tsien (1952-2016).

Nature 2016 10;538(7624):172

New York University, New York, USA.

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http://dx.doi.org/10.1038/538172aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960232PMC
October 2016

What is memory? The present state of the engram.

BMC Biol 2016 05 19;14:40. Epub 2016 May 19.

Salk Institute for Biological Studies, Laboratory of Genetics, 10010 N. Torrey Pines Road, La Jolla, CA, 92037, USA.

The mechanism of memory remains one of the great unsolved problems of biology. Grappling with the question more than a hundred years ago, the German zoologist Richard Semon formulated the concept of the engram, lasting connections in the brain that result from simultaneous "excitations", whose precise physical nature and consequences were out of reach of the biology of his day. Neuroscientists now have the knowledge and tools to tackle this question, however, and this Forum brings together leading contemporary views on the mechanisms of memory and what the engram means today.
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http://dx.doi.org/10.1186/s12915-016-0261-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4874022PMC
May 2016

Excitation-Transcription Coupling in Parvalbumin-Positive Interneurons Employs a Novel CaM Kinase-Dependent Pathway Distinct from Excitatory Neurons.

Neuron 2016 04 31;90(2):292-307. Epub 2016 Mar 31.

Department of Neuroscience and Physiology and NYU Neuroscience Institute, NYU Langone Medical Center, New York, NY 10016, USA; Center for Neural Science, New York University, New York, NY 10003, USA. Electronic address:

Properly functional CNS circuits depend on inhibitory interneurons that in turn rely upon activity-dependent gene expression for morphological development, connectivity, and excitatory-inhibitory coordination. Despite its importance, excitation-transcription coupling in inhibitory interneurons is poorly understood. We report that PV+ interneurons employ a novel CaMK-dependent pathway to trigger CREB phosphorylation and gene expression. As in excitatory neurons, voltage-gated Ca(2+) influx through CaV1 channels triggers CaM nuclear translocation via local Ca(2+) signaling. However, PV+ interneurons are distinct in that nuclear signaling is mediated by γCaMKI, not γCaMKII. CREB phosphorylation also proceeds with slow, sigmoid kinetics, rate-limited by paucity of CaMKIV, protecting against saturation of phospho-CREB in the face of higher firing rates and bigger Ca(2+) transients. Our findings support the generality of CaM shuttling to drive nuclear CaMK activity, and they are relevant to disease pathophysiology, insofar as dysfunction of PV+ interneurons and molecules underpinning their excitation-transcription coupling both relate to neuropsychiatric disease.
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http://dx.doi.org/10.1016/j.neuron.2016.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4866871PMC
April 2016

Unifying Views of Autism Spectrum Disorders: A Consideration of Autoregulatory Feedback Loops.

Neuron 2016 Mar;89(6):1131-1156

Department of Neuroscience and Physiology, Neuroscience Institute, New York University Langone Medical Center, New York, NY 10016, USA. Electronic address:

Understanding the mechanisms underlying autism spectrum disorders (ASDs) is a challenging goal. Here we review recent progress on several fronts, including genetics, proteomics, biochemistry, and electrophysiology, that raise motivation for forming a viable pathophysiological hypothesis. In place of a traditionally unidirectional progression, we put forward a framework that extends homeostatic hypotheses by explicitly emphasizing autoregulatory feedback loops and known synaptic biology. The regulated biological feature can be neuronal electrical activity, the collective strength of synapses onto a dendritic branch, the local concentration of a signaling molecule, or the relative strengths of synaptic excitation and inhibition. The sensor of the biological variable (which we have termed the homeostat) engages mechanisms that operate as negative feedback elements to keep the biological variable tightly confined. We categorize known ASD-associated gene products according to their roles in such feedback loops and provide detailed commentary for exemplar genes within each module.
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http://dx.doi.org/10.1016/j.neuron.2016.02.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5757244PMC
March 2016

Sequential ionic and conformational signaling by calcium channels drives neuronal gene expression.

Science 2016 Feb;351(6275):863-7

Department of Neuroscience and Physiology and New York University Neuroscience Institute, New York, NY 10016, USA. Department of Molecular and Cellular Physiology, Beckman Center, School of Medicine, Stanford University, Stanford, CA 94305, USA.

Voltage-gated CaV1.2 channels (L-type calcium channel α1C subunits) are critical mediators of transcription-dependent neural plasticity. Whether these channels signal via the influx of calcium ion (Ca(2+)), voltage-dependent conformational change (VΔC), or a combination of the two has thus far been equivocal. We fused CaV1.2 to a ligand-gated Ca(2+)-permeable channel, enabling independent control of localized Ca(2+) and VΔC signals. This revealed an unexpected dual requirement: Ca(2+) must first mobilize actin-bound Ca(2+)/calmodulin-dependent protein kinase II, freeing it for subsequent VΔC-mediated accumulation. Neither signal alone sufficed to activate transcription. Signal order was crucial: Efficiency peaked when Ca(2+) preceded VΔC by 10 to 20 seconds. CaV1.2 VΔC synergistically augmented signaling by N-methyl-d-aspartate receptors. Furthermore, VΔC mistuning correlated with autistic symptoms in Timothy syndrome. Thus, nonionic VΔC signaling is vital to the function of CaV1.2 in synaptic and neuropsychiatric processes.
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http://dx.doi.org/10.1126/science.aad3647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5467645PMC
February 2016

Cannabinoids and Epilepsy.

Neurotherapeutics 2015 Oct;12(4):747-68

Department of Neurology, Comprehensive Epilepsy Center, New York University School of Medicine, New York, NY, 10016, UK.

Cannabis has been used for centuries to treat seizures. Recent anecdotal reports, accumulating animal model data, and mechanistic insights have raised interest in cannabis-based antiepileptic therapies. In this study, we review current understanding of the endocannabinoid system, characterize the pro- and anticonvulsive effects of cannabinoids [e.g., Δ9-tetrahydrocannabinol and cannabidiol (CBD)], and highlight scientific evidence from pre-clinical and clinical trials of cannabinoids in epilepsy. These studies suggest that CBD avoids the psychoactive effects of the endocannabinoid system to provide a well-tolerated, promising therapeutic for the treatment of seizures, while whole-plant cannabis can both contribute to and reduce seizures. Finally, we discuss results from a new multicenter, open-label study using CBD in a population with treatment-resistant epilepsy. In all, we seek to evaluate our current understanding of cannabinoids in epilepsy and guide future basic science and clinical studies.
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http://dx.doi.org/10.1007/s13311-015-0375-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604191PMC
October 2015

Evolutionary and functional perspectives on signaling from neuronal surface to nucleus.

Biochem Biophys Res Commun 2015 Apr;460(1):88-99

NYU Neuroscience Institute and Department of Neuroscience and Physiology, NYU Langone Medical Center, New York, NY 10016, USA. Electronic address:

Reliance on Ca(2+) signaling has been well-preserved through the course of evolution. While the complexity of Ca(2+) signaling pathways has increased, activation of transcription factors including CREB by Ca(2+)/CaM-dependent kinases (CaMKs) has remained critical for long-term plasticity. In C. elegans, the CaMK family is made up of only three members, and CREB phosphorylation is mediated by CMK-1, the homologue of CaMKI. CMK-1 nuclear translocation directly regulates adaptation of thermotaxis behavior in response to changes in the environment. In mammals, the CaMK family has been expanded from three to ten members, enabling specialization of individual elements of a signal transduction pathway and increased reliance on the CaMKII subfamily. This increased complexity enables private line communication between Ca(2+) sources at the cell surface and specific cellular targets. Using both new and previously published data, we review the mechanism of a γCaMKII-CaM nuclear translocation. This intricate pathway depends on a specific role for multiple Ca(2+)/CaM-dependent kinases and phosphatases: α/βCaMKII phosphorylates γCaMKII to trap CaM; CaN dephosphorylates γCaMKII to dispatch it to the nucleus; and PP2A induces CaM release from γCaMKII so that CaMKK and CaMKIV can trigger CREB phosphorylation. Thus, while certain basic elements have been conserved from C. elegans, evolutionary modifications offer opportunities for targeted communication, regulation of key nodes and checkpoints, and greater specificity and flexibility in signaling.
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http://dx.doi.org/10.1016/j.bbrc.2015.02.146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701207PMC
April 2015

Protein mutated in paroxysmal dyskinesia interacts with the active zone protein RIM and suppresses synaptic vesicle exocytosis.

Proc Natl Acad Sci U S A 2015 Mar 17;112(10):2935-41. Epub 2015 Feb 17.

Department of Neurology, Howard Hughes Medical Institute, University of California, San Francisco, CA 94158;

Paroxysmal nonkinesigenic dyskinesia (PNKD) is an autosomal dominant episodic movement disorder precipitated by coffee, alcohol, and stress. We previously identified the causative gene but the function of the encoded protein remains unknown. We also generated a PNKD mouse model that revealed dysregulated dopamine signaling in vivo. Here, we show that PNKD interacts with synaptic active zone proteins Rab3-interacting molecule (RIM)1 and RIM2, localizes to synapses, and modulates neurotransmitter release. Overexpressed PNKD protein suppresses release, and mutant PNKD protein is less effective than wild-type at inhibiting exocytosis. In PNKD KO mice, RIM1/2 protein levels are reduced and synaptic strength is impaired. Thus, PNKD is a novel synaptic protein with a regulatory role in neurotransmitter release.
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http://dx.doi.org/10.1073/pnas.1501364112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4364199PMC
March 2015

Distinct roles of multiple isoforms of CaMKII in signaling to the nucleus.

Biochim Biophys Acta 2015 Sep 17;1853(9):1953-7. Epub 2015 Feb 17.

NYU Neuroscience Institute, NYU Langone Medical Center, New York, NY 10016, USA. Electronic address:

Long-lasting synaptic changes following information acquisition are critical steps for memory. In this process, long-term potentiation (LTP) is widely considered as one of the major cellular mechanisms modifying synaptic strength. It can be classified into early phase LTP (E-LTP) and late phase LTP (L-LTP) based on its duration. Using genetically modified mice, investigators have recognized the critical role of CaMKII in E-LTP and memory. However, its function in L-LTP, which is strongly dependent on gene transcription and protein synthesis, is still unclear. In this review, we discuss how different isoforms of CaMKII are coordinated to regulate gene expression in an activity-dependent manner, and thus contribute to L-LTP and memory. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
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http://dx.doi.org/10.1016/j.bbamcr.2015.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522395PMC
September 2015

The impact of NMDA receptor hypofunction on GABAergic neurons in the pathophysiology of schizophrenia.

Schizophr Res 2015 Sep 9;167(1-3):98-107. Epub 2015 Jan 9.

NYU Neuroscience Institute, Department of Neuroscience and Physiology, NYU Langone Medical Center, New York, NY 10016, USA; Department of Psychiatry, NYU Langone Medical Center, 550 First Avenue, New York, NY 10016, USA. Electronic address:

While the dopamine hypothesis has dominated schizophrenia research for several decades, more recent studies have highlighted the role of fast synaptic transmitters and their receptors in schizophrenia etiology. Here we review evidence that schizophrenia is associated with a reduction in N-methyl-d-aspartate receptor (NMDAR) function. By highlighting postmortem, neuroimaging and electrophysiological studies, we provide evidence for preferential disruption of GABAergic circuits in the context of NMDAR hypo-activity states. The functional relationship between NMDARs and GABAergic neurons is realized at the molecular, cellular, microcircuit and systems levels. A synthesis of findings across these levels explains how NMDA-mediated inhibitory dysfunction may lead to aberrant interactions among brain regions, accounting for key clinical features of schizophrenia. This synthesis of schizophrenia unifies observations from diverse fields and may help chart pathways for developing novel diagnostics and therapeutics.
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http://dx.doi.org/10.1016/j.schres.2014.12.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4724170PMC
September 2015

γCaMKII shuttles Ca²⁺/CaM to the nucleus to trigger CREB phosphorylation and gene expression.

Cell 2014 Oct;159(2):281-94

Department of Neuroscience and Physiology, Neuroscience Institute, NYU Langone Medical Center, New York, NY 10016, USA. Electronic address:

Activity-dependent CREB phosphorylation and gene expression are critical for long-term neuronal plasticity. Local signaling at CaV1 channels triggers these events, but how information is relayed onward to the nucleus remains unclear. Here, we report a mechanism that mediates long-distance communication within cells: a shuttle that transports Ca(2+)/calmodulin from the surface membrane to the nucleus. We show that the shuttle protein is γCaMKII, its phosphorylation at Thr287 by βCaMKII protects the Ca(2+)/CaM signal, and CaN triggers its nuclear translocation. Both βCaMKII and CaN act in close proximity to CaV1 channels, supporting their dominance, whereas γCaMKII operates as a carrier, not as a kinase. Upon arrival within the nucleus, Ca(2+)/CaM activates CaMKK and its substrate CaMKIV, the CREB kinase. This mechanism resolves long-standing puzzles about CaM/CaMK-dependent signaling to the nucleus. The significance of the mechanism is emphasized by dysregulation of CaV1, γCaMKII, βCaMKII, and CaN in multiple neuropsychiatric disorders.
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http://dx.doi.org/10.1016/j.cell.2014.09.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4201038PMC
October 2014

Virtual finger boosts three-dimensional imaging and microsurgery as well as terabyte volume image visualization and analysis.

Nat Commun 2014 Jul 11;5:4342. Epub 2014 Jul 11.

1] Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA [2] Allen Institute for Brain Science, 551 North 34th Street, Suite 200, Seattle, Washington 98103, USA [3] BioImage, L.L.C., Bellevue, Washington 98005, USA.

Three-dimensional (3D) bioimaging, visualization and data analysis are in strong need of powerful 3D exploration techniques. We develop virtual finger (VF) to generate 3D curves, points and regions-of-interest in the 3D space of a volumetric image with a single finger operation, such as a computer mouse stroke, or click or zoom from the 2D-projection plane of an image as visualized with a computer. VF provides efficient methods for acquisition, visualization and analysis of 3D images for roundworm, fruitfly, dragonfly, mouse, rat and human. Specifically, VF enables instant 3D optical zoom-in imaging, 3D free-form optical microsurgery, and 3D visualization and annotation of terabytes of whole-brain image volumes. VF also leads to orders of magnitude better efficiency of automated 3D reconstruction of neurons and similar biostructures over our previous systems. We use VF to generate from images of 1,107 Drosophila GAL4 lines a projectome of a Drosophila brain.
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http://dx.doi.org/10.1038/ncomms5342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104457PMC
July 2014

CaV1.2 calcium channels: just cut out to be regulated?

Neuron 2014 Jun;82(5):939-40

NYU Neuroscience Institute and Department of Neuroscience and Physiology, NYU Langone Medical Center, New York, NY 10016, USA. Electronic address:

Tight regulation of calcium entry through the L-type calcium channel CaV1.2 ensures optimal excitation-response coupling. In this issue of Neuron, Michailidis et al. (2014) demonstrate that CaV1.2 activity triggers negative feedback regulation through proteolytic cleavage of the channel within the core of the pore-forming subunit.
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http://dx.doi.org/10.1016/j.neuron.2014.05.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960233PMC
June 2014

Ca2+ channel nanodomains boost local Ca2+ amplitude.

Proc Natl Acad Sci U S A 2013 Sep 9;110(39):15794-9. Epub 2013 Sep 9.

Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147.

Local Ca(2+) signals through voltage-gated Ca(2+) channels (CaVs) drive synaptic transmission, neural plasticity, and cardiac contraction. Despite the importance of these events, the fundamental relationship between flux through a single CaV channel and the Ca(2+) signaling concentration within nanometers of its pore has resisted empirical determination, owing to limitations in the spatial resolution and specificity of fluorescence-based Ca(2+) measurements. Here, we exploited Ca(2+)-dependent inactivation of CaV channels as a nanometer-range Ca(2+) indicator specific to active channels. We observed an unexpected and dramatic boost in nanodomain Ca(2+) amplitude, ten-fold higher than predicted on theoretical grounds. Our results uncover a striking feature of CaV nanodomains, as diffusion-restricted environments that amplify small Ca(2+) fluxes into enormous local Ca(2+) concentrations. This Ca(2+) tuning by the physical composition of the nanodomain may represent an energy-efficient means of local amplification that maximizes information signaling capacity, while minimizing global Ca(2+) load.
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http://dx.doi.org/10.1073/pnas.1313898110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3785779PMC
September 2013

GABAergic projection neurons route selective olfactory inputs to specific higher-order neurons.

Neuron 2013 Sep;79(5):917-31

Department of Biology and Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA; Department of Applied Physics, Stanford University, Stanford, CA 94305, USA.

We characterize an inhibitory circuit motif in the Drosophila olfactory system, parallel inhibition, which differs from feedforward or feedback inhibition. Excitatory and GABAergic inhibitory projection neurons (ePNs and iPNs) each receive input from antennal lobe glomeruli and send parallel output to the lateral horn, a higher center implicated in regulating innate olfactory behavior. Ca(2+) imaging of specific lateral horn neurons as an olfactory readout revealed that iPNs selectively suppressed food-related odor responses, but spared signal transmission from pheromone channels. Coapplying food odorant did not affect pheromone signal transmission, suggesting that the differential effects likely result from connection specificity of iPNs, rather than a generalized inhibitory tone. Ca(2+) responses in the ePN axon terminals show no detectable suppression by iPNs, arguing against presynaptic inhibition as a primary mechanism. The parallel inhibition motif may provide specificity in inhibition to funnel specific olfactory information, such as food and pheromone, into distinct downstream circuits.
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http://dx.doi.org/10.1016/j.neuron.2013.06.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3838762PMC
September 2013

GluA1 phosphorylation at serine 831 in the lateral amygdala is required for fear renewal.

Nat Neurosci 2013 Oct 25;16(10):1436-44. Epub 2013 Aug 25.

1] School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Korea. [2].

Fear renewal, a widely pursued model of post-traumatic stress disorder and phobias, refers to the context-specific relapse of conditioned fear after extinction. However, its molecular mechanisms are largely unknown. We found that renewal-inducing stimuli, generally believed to be insufficient to induce synaptic plasticity, enhanced excitatory synaptic strength, activity of synaptic GluA2-lacking AMPA receptors and Ser831 phosphorylation of synaptic surface GluA1 in the lateral nucleus of the amygdala (LAn) of fear-extinguished rats. Consistently, the induction threshold for LAn synaptic potentiation was considerably lowered after extinction, and renewal occluded this low-threshold potentiation. The low-threshold potentiation (a potential cellular substrate for renewal), but not long-term potentiation, was attenuated by dialysis into LAn neurons of a GluA1-derived peptide that competes with Ser831-phosphorylated GluA1. Microinjections of the same peptide into the LAn attenuated fear renewal, but not fear learning. Our findings suggest that GluA1 phosphorylation constitutes a promising target for clinical treatment of aberrant fear-related disorders.
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http://dx.doi.org/10.1038/nn.3491DOI Listing
October 2013

Oxytocin enhances hippocampal spike transmission by modulating fast-spiking interneurons.

Nature 2013 Aug 4;500(7463):458-62. Epub 2013 Aug 4.

Department of Molecular and Cellular Physiology, 279 Campus Drive, Stanford University School of Medicine, Stanford, California 94305, USA.

Neuromodulatory control by oxytocin is essential to a wide range of social, parental and stress-related behaviours. Autism spectrum disorders (ASD) are associated with deficiencies in oxytocin levels and with genetic alterations of the oxytocin receptor (OXTR). Thirty years ago, Mühlethaler et al. found that oxytocin increases the firing of inhibitory hippocampal neurons, but it remains unclear how elevated inhibition could account for the ability of oxytocin to improve information processing in the brain. Here we describe in mammalian hippocampus a simple yet powerful mechanism by which oxytocin enhances cortical information transfer while simultaneously lowering background activity, thus greatly improving the signal-to-noise ratio. Increased fast-spiking interneuron activity not only suppresses spontaneous pyramidal cell firing, but also enhances the fidelity of spike transmission and sharpens spike timing. Use-dependent depression at the fast-spiking interneuron-pyramidal cell synapse is both necessary and sufficient for the enhanced spike throughput. We show the generality of this novel circuit mechanism by activation of fast-spiking interneurons with cholecystokinin or channelrhodopsin-2. This provides insight into how a diffusely delivered neuromodulator can improve the performance of neural circuitry that requires synapse specificity and millisecond precision.
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http://dx.doi.org/10.1038/nature12330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283693PMC
August 2013

AMPA receptor exchange underlies transient memory destabilization on retrieval.

Proc Natl Acad Sci U S A 2013 May 29;110(20):8218-23. Epub 2013 Apr 29.

School of Biological Sciences, Seoul National University, Seoul 151-747, Korea.

A consolidated memory can be transiently destabilized by memory retrieval, after which memories are reconsolidated within a few hours; however, the molecular substrates underlying this destabilization process remain essentially unknown. Here we show that at lateral amygdala synapses, fear memory consolidation correlates with increased surface expression of calcium-impermeable AMPA receptors (CI-AMPARs), which are known to be more stable at the synapse, whereas memory retrieval induces an abrupt exchange of CI-AMPARs to calcium-permeable AMPARs (CP-AMPARs), which are known to be less stable at the synapse. We found that blockade of either CI-AMPAR endocytosis or NMDA receptor activity during memory retrieval, both of which blocked the exchange to CP-AMPARs, prevented memory destabilization, indicating that this transient exchange of AMPARs may underlie the transformation of a stable memory into an unstable memory. These newly inserted CP-AMPARs gradually exchanged back to CI-AMPARs within hours, which coincided with the course of reconsolidation. Furthermore, blocking the activity of these newly inserted CP-AMPARs after retrieval impaired reconsolidation, suggesting that they serve as synaptic "tags" that support synapse-specific reconsolidation. Taken together, our results reveal unexpected physiological roles of CI-AMPARs and CP-AMPARs in transforming a consolidated memory into an unstable memory and subsequently guiding reconsolidation.
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http://dx.doi.org/10.1073/pnas.1305235110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3657785PMC
May 2013

Perspectives on kiss-and-run: role in exocytosis, endocytosis, and neurotransmission.

Annu Rev Physiol 2013 ;75:393-422

Department of Molecular and Cellular Physiology, Stanford Institute for Neuro-Innovation and Translational Neurosciences, Stanford Medical School, Stanford, California 94305, USA.

Regulated exocytosis and endocytosis are critical to the function of many intercellular networks, particularly the complex neural circuits underlying mammalian behavior. Kiss-and-run (KR) is an unconventional fusion between secretory vesicles and a target membrane that releases intravesicular content through a transient, nanometer-sized fusion pore. The fusing vesicle retains its gross shape, precluding full integration into the planar membrane, and enough molecular components for rapid retrieval, reacidification, and reuse. KR makes judicious use of finite presynaptic resources, and mounting evidence suggests that it influences synaptic information transfer. Here we detail emerging perspectives on KR and its role in neurotransmission. We additionally formulate a restraining force hypothesis as a plausible mechanistic basis for KR and its physiological modulation in small nerve terminals. Clarification of the mechanism and function of KR has bearing on understanding the kinetic transitions underlying SNARE-mediated fusion, interactions between vesicles and their local environment, and the influence of release dynamics on neural information processing.
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http://dx.doi.org/10.1146/annurev-physiol-020911-153305DOI Listing
August 2013

Exploring the dominant role of Cav1 channels in signalling to the nucleus.

Biosci Rep 2012 Dec 20;33(1):97-101. Epub 2012 Dec 20.

NYU Neuroscience Institute, NYU Langone Medical Center, New York, NY 10016, USA.

Calcium is important in controlling nuclear gene expression through the activation of multiple signal-transduction pathways in neurons. Compared with other voltage-gated calcium channels, Ca(V)1 channels demonstrate a considerable advantage in signalling to the nucleus. In this review, we summarize the recent progress in elucidating the mechanisms involved. Ca(V)1 channels, already advantaged in their responsiveness to depolarization, trigger communication with the nucleus by attracting colocalized clusters of activated CaMKII (Ca(2+)/calmodulin-dependent protein kinase II). Ca(V)2 channels lack this ability, but must work at a distance of >1 μm from the Ca(V)1-CaMKII co-clusters, which hampers their relative efficiency for a given rise in bulk [Ca(2+)](i) (intracellular [Ca(2+)]). Moreover, Ca(2+) influx from Ca(V)2 channels is preferentially buffered by the ER (endoplasmic reticulum) and mitochondria, further attenuating their effectiveness in signalling to the nucleus.
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http://dx.doi.org/10.1042/BSR20120099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3546354PMC
December 2012