Publications by authors named "Paul De Koninck"

38 Publications

Zebrafish: a big fish in the study of the gut microbiota.

Curr Opin Biotechnol 2021 Oct 12;73:308-313. Epub 2021 Oct 12.

Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Québec, QC, G1V 0A6, Canada; Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Université Laval, Québec, QC, G1V 0A6, Canada; Félix d'Hérelle Reference Center for Bacterial Viruses, Université Laval, Québec, QC, G1V 0A6, Canada. Electronic address:

The importance of the gut microbiota in host health is now well established, but the underlying mechanisms remain poorly understood. Among the animal models used to investigate microbiota-host interactions, the zebrafish (Danio renio) is gaining attention. Several factors contribute to the recent interest in this model, including its low cost, the ability to assess large cohorts, the possibility to obtain germ-free larvae from non-axenic parents, and the availability of optical methodologies to probe the transparent larvae and adults from various genetic lines. We review recent findings on the zebrafish gut microbiota and its modulation by exogenous microbes, nutrition, and environmental factors. We also highlight the potential of this model for assessing the impact of the gut microbiota on brain development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.copbio.2021.09.007DOI Listing
October 2021

Activity-Dependent Remodeling of Synaptic Protein Organization Revealed by High Throughput Analysis of STED Nanoscopy Images.

Front Neural Circuits 2020 15;14:57. Epub 2020 Oct 15.

CERVO Brain Research Centre, Québec, QC, Canada.

The organization of proteins in the apposed nanodomains of pre- and postsynaptic compartments is thought to play a pivotal role in synaptic strength and plasticity. As such, the alignment between pre- and postsynaptic proteins may regulate, for example, the rate of presynaptic release or the strength of postsynaptic signaling. However, the analysis of these structures has mainly been restricted to subsets of synapses, providing a limited view of the diversity of synaptic protein cluster remodeling during synaptic plasticity. To characterize changes in the organization of synaptic nanodomains during synaptic plasticity over a large population of synapses, we combined STimulated Emission Depletion (STED) nanoscopy with a Python-based statistical object distance analysis (pySODA), in dissociated cultured hippocampal circuits exposed to treatments driving different forms of synaptic plasticity. The nanoscale organization, characterized in terms of coupling properties, of presynaptic (Bassoon, RIM1/2) and postsynaptic (PSD95, Homer1c) scaffold proteins was differently altered in response to plasticity-inducing stimuli. For the Bassoon - PSD95 pair, treatments driving synaptic potentiation caused an increase in their coupling probability, whereas a stimulus driving synaptic depression had an opposite effect. To enrich the characterization of the synaptic cluster remodeling at the population level, we applied unsupervised machine learning approaches to include selected morphological features into a multidimensional analysis. This combined analysis revealed a large diversity of synaptic protein cluster subtypes exhibiting differential activity-dependent remodeling, yet with common features depending on the expected direction of plasticity. The expanded palette of synaptic features revealed by our unbiased approach should provide a basis to further explore the widely diverse molecular mechanisms of synaptic plasticity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fncir.2020.00057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594516PMC
October 2021

The dynamic interplay between ATP/ADP levels and autophagy sustain neuronal migration in vivo.

Elife 2020 09 28;9. Epub 2020 Sep 28.

CERVO Brain Research Center, Quebec City, Canada.

Cell migration is a dynamic process that entails extensive protein synthesis and recycling, structural remodeling, and considerable bioenergetic demand. Autophagy is one of the pathways that maintain cellular homeostasis. Time-lapse imaging of autophagosomes and ATP/ADP levels in migrating cells in the rostral migratory stream of mouse revealed that decreases in ATP levels force cells into the stationary phase and induce autophagy. Pharmacological or genetic impairments of autophagy in neuroblasts using either bafilomycin, inducible conditional mice, or CRISPR/Cas9 gene editing decreased cell migration due to the longer duration of the stationary phase. Autophagy is modulated in response to migration-promoting and inhibiting molecular cues and is required for the recycling of focal adhesions. Our results show that autophagy and energy consumption act in concert in migrating cells to dynamically regulate the pace and periodicity of the migratory and stationary phases to sustain neuronal migration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.56006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556871PMC
September 2020

Neuronal activity remodels the F-actin based submembrane lattice in dendrites but not axons of hippocampal neurons.

Sci Rep 2020 07 20;10(1):11960. Epub 2020 Jul 20.

CERVO Brain Research Center, 2601 de la Canardière, Québec, QC, G1J 2G3, Canada.

The nanoscale organization of the F-actin cytoskeleton in neurons comprises membrane-associated periodical rings, bundles, and longitudinal fibers. The F-actin rings have been observed predominantly in axons but only sporadically in dendrites, where fluorescence nanoscopy reveals various patterns of F-actin arranged in mixed patches. These complex dendritic F-actin patterns pose a challenge for investigating quantitatively their regulatory mechanisms. We developed here a weakly supervised deep learning segmentation approach of fluorescence nanoscopy images of F-actin in cultured hippocampal neurons. This approach enabled the quantitative assessment of F-actin remodeling, revealing the disappearance of the rings during neuronal activity in dendrites, but not in axons. The dendritic F-actin cytoskeleton of activated neurons remodeled into longitudinal fibers. We show that this activity-dependent remodeling involves [Formula: see text] and NMDA receptor-dependent mechanisms. This highly dynamic restructuring of dendritic F-actin based submembrane lattice into longitudinal fibers may serve to support activity-dependent membrane remodeling, protein trafficking and neuronal plasticity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-68180-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371643PMC
July 2020

Opposite Control of Excitatory and Inhibitory Synapse Formation by Slitrk2 and Slitrk5 on Dopamine Neurons Modulates Hyperactivity Behavior.

Cell Rep 2020 02;30(7):2374-2386.e5

CERVO Brain Research Centre, 2601 de la Canardière, Québec, QC G1J 2G3, Canada; Department of Psychiatry and Neuroscience, Faculty of Medicine, Université Laval, Québec, QC G1V 0A6, Canada. Electronic address:

The neurodevelopmental origin of hyperactivity disorder has been suggested to involve the dopaminergic system, but the underlying mechanisms are still unknown. Here, transcription factors Lmx1a and Lmx1b are shown to be essential for midbrain dopaminergic (mDA) neuron excitatory synaptic inputs and dendritic development. Strikingly, conditional knockout (cKO) of Lmx1a/b in postmitotic mDA neurons results in marked hyperactivity. In seeking Lmx1a/b target genes, we identify positively regulated Slitrk2 and negatively regulated Slitrk5. These two synaptic adhesion proteins promote excitatory and inhibitory synapses on mDA neurons, respectively. Knocking down Slitrk2 reproduces some of the Lmx1a/b cKO cellular and behavioral phenotypes, whereas Slitrk5 knockdown has opposite effects. The hyperactivity caused by this imbalance in excitatory/inhibitory synaptic inputs on dopamine neurons is reproduced by chronically inhibiting the ventral tegmental area during development using pharmacogenetics. Our study shows that alterations in developing dopaminergic circuits strongly impact locomotor activity, shedding light on mechanisms causing hyperactivity behaviors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.01.084DOI Listing
February 2020

Spatial dynamics of the insulin receptor in living neurons.

J Neurochem 2021 01 22;156(1):88-105. Epub 2020 Jan 22.

CERVO Brain Research Center, Québec, QC, Canada.

Insulin signaling through the insulin receptor has long been studied in classic target organs, such as adipose tissue and skeletal muscle, where one of its effects is to increase glucose uptake. Insulin and insulin receptor are present in many areas of the brain, but the functions of brain insulin signaling outside feeding circuits are not well defined. It has been proposed that hippocampal insulin signaling is important for memory, that brain insulin signaling is deficient in Alzheimer's disease, and that intranasal insulin treatment improves cognition, but the mechanisms remain unclear and do not seem to involve increased glucose uptake by neurons. The molecular behavior of the insulin receptor itself is not well known in living neurons; therefore, we investigated the spatial dynamics of the insulin receptor on somatodendritic membranes of live rat hippocampal neurons in culture. Using single-molecule tracking of quantum dot-tagged insulin receptors and single-particle tracking photoactivation localization microscopy, we show that the insulin receptor is distributed over both dendritic shafts and spines. Using colocalization with synaptic markers, we also show that in contrast to the glutamate receptor subunit glutamate receptor subunit A1, the dynamics of the insulin receptor are not affected by association with excitatory synapses; however, the insulin receptor is immobilized by components of inhibitory synapses. The mobility of the insulin receptor is reduced both by low concentrations of the pro-inflammatory cytokine tumor necrosis factor α and by cholesterol depletion, suggesting an association with sphingolipid-rich membrane domains. On the other hand, the insulin receptor dynamics in hippocampal neurons are not affected by increased excitatory signaling. Finally, using real-time single-event quantification, we find evidence of strong insulin receptor exocytosis on dendritic shafts. Our results suggest an association of the neuronal insulin receptor with specific elements of the dendritic shaft, rather than excitatory synapses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jnc.14950DOI Listing
January 2021

Fluorescence lifetime imaging nanoscopy for measuring Förster resonance energy transfer in cellular nanodomains.

Neurophotonics 2019 Jan 31;6(1):015002. Epub 2019 Jan 31.

CERVO Brain Research Center, Québec (QC), Canada.

Microscopy methods used to measure Förster resonance energy transfer (FRET) between fluorescently labeled proteins can provide information on protein interactions in cells. However, these methods are diffraction-limited, thus do not enable the resolution of the nanodomains in which such interactions occur in cells. To overcome this limitation, we assess FRET with an imaging system combining fluorescence lifetime imaging microscopy with stimulated emission depletion, termed fluorescence lifetime imaging nanoscopy (FLIN). The resulting FRET-FLIN approach utilizes immunolabeling of proteins in fixed cultured neurons. We demonstrate the capacity to discriminate nanoclusters of synaptic proteins exhibiting variable degrees of interactions with labeled binding partners inside dendritic spines of hippocampal neurons. This method enables the investigation of FRET within nanodomains of cells, approaching the scale of molecular signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1117/1.NPh.6.1.015002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6354015PMC
January 2019

A machine learning approach for online automated optimization of super-resolution optical microscopy.

Nat Commun 2018 12 7;9(1):5247. Epub 2018 Dec 7.

CERVO Brain Research Center, 2601 de la Canardière, Québec, QC, G1J 2G3, Canada.

Traditional approaches for finding well-performing parameterizations of complex imaging systems, such as super-resolution microscopes rely on an extensive exploration phase over the illumination and acquisition settings, prior to the imaging task. This strategy suffers from several issues: it requires a large amount of parameter configurations to be evaluated, it leads to discrepancies between well-performing parameters in the exploration phase and imaging task, and it results in a waste of time and resources given that optimization and final imaging tasks are conducted separately. Here we show that a fully automated, machine learning-based system can conduct imaging parameter optimization toward a trade-off between several objectives, simultaneously to the imaging task. Its potential is highlighted on various imaging tasks, such as live-cell and multicolor imaging and multimodal optimization. This online optimization routine can be integrated to various imaging systems to increase accessibility, optimize performance and improve overall imaging quality.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-07668-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286316PMC
December 2018

Activity-Dependent Netrin-1 Secretion Drives Synaptic Insertion of GluA1-Containing AMPA Receptors in the Hippocampus.

Cell Rep 2018 10;25(1):168-182.e6

Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC H3A 2B4, Canada; McGill Program in Neuroengineering, McGill University, Montreal, QC H3A 2B4, Canada. Electronic address:

Dynamic trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPARs) to synapses is critical for activity-dependent synaptic plasticity underlying learning and memory, but the identity of key molecular effectors remains elusive. Here, we demonstrate that membrane depolarization and N-methyl-D-aspartate receptor (NMDAR) activation triggers secretion of the chemotropic guidance cue netrin-1 from dendrites. Using selective genetic deletion, we show that netrin-1 expression by excitatory neurons is required for NMDAR-dependent long-term potentiation (LTP) in the adult hippocampus. Furthermore, we demonstrate that application of exogenous netrin-1 is sufficient to trigger the potentiation of excitatory glutamatergic transmission at hippocampal Schaffer collateral synapses via Ca-dependent recruitment of GluA1-containing AMPARs, promoting the maturation of immature or nascent synapses. These findings identify a central role for activity-dependent release of netrin-1 as a critical effector of synaptic plasticity in the adult hippocampus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.09.028DOI Listing
October 2018

CaMKIIα Expression Defines Two Functionally Distinct Populations of Granule Cells Involved in Different Types of Odor Behavior.

Curr Biol 2017 Nov 26;27(21):3315-3329.e6. Epub 2017 Oct 26.

CERVO Brain Research Center, Quebec City, QC G1J 2G3, Canada; Department of Psychiatry and Neuroscience, Université Laval, Quebec City, QC G1V 0A6, Canada. Electronic address:

Granule cells (GCs) in the olfactory bulb (OB) play an important role in odor information processing. Although they have been classified into various neurochemical subtypes, the functional roles of these subtypes remain unknown. We used in vivo two-photon Ca imaging combined with cell-type-specific identification of GCs in the mouse OB to examine whether functionally distinct GC subtypes exist in the bulbar network. We showed that half of GCs express Ca/calmodulin-dependent protein kinase IIα (CaMKIIα) and that these neurons are preferentially activated by olfactory stimulation. The higher activity of CaMKIIα neurons is due to the weaker inhibitory input that they receive compared to their CaMKIIα-immunonegative (CaMKIIα) counterparts. In line with these functional data, immunohistochemical analyses showed that 75%-90% of GCs expressing the immediate early gene cFos are CaMKIIα in naive animals and in mice that have been exposed to a novel odor and go/no-go operant conditioning, or that have been subjected to long-term associative memory and spontaneous habituation/dishabituation odor discrimination tasks. On the other hand, a perceptual learning task resulted in increased activation of CaMKIIα cells. Pharmacogenetic inhibition of CaMKIIα GCs revealed that this subtype is involved in habituation/dishabituation and go/no-go odor discrimination, but not in perceptual learning. In contrast, pharmacogenetic inhibition of GCs in a subtype-independent manner affected perceptual learning. Our results indicate that functionally distinct populations of GCs exist in the OB and that they play distinct roles during different odor tasks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2017.09.058DOI Listing
November 2017

Tracking the Fragile X Mental Retardation Protein in a Highly Ordered Neuronal RiboNucleoParticles Population: A Link between Stalled Polyribosomes and RNA Granules.

PLoS Genet 2016 07 27;12(7):e1006192. Epub 2016 Jul 27.

Institut universitaire en santé mentale de Québec, Quebec, Canada.

Local translation at the synapse plays key roles in neuron development and activity-dependent synaptic plasticity. mRNAs are translocated from the neuronal soma to the distant synapses as compacted ribonucleoparticles referred to as RNA granules. These contain many RNA-binding proteins, including the Fragile X Mental Retardation Protein (FMRP), the absence of which results in Fragile X Syndrome, the most common inherited form of intellectual disability and the leading genetic cause of autism. Using FMRP as a tracer, we purified a specific population of RNA granules from mouse brain homogenates. Protein composition analyses revealed a strong relationship between polyribosomes and RNA granules. However, the latter have distinct architectural and structural properties, since they are detected as close compact structures as observed by electron microscopy, and converging evidence point to the possibility that these structures emerge from stalled polyribosomes. Time-lapse video microscopy indicated that single granules merge to form cargoes that are transported from the soma to distal locations. Transcriptomic analyses showed that a subset of mRNAs involved in cytoskeleton remodelling and neural development is selectively enriched in RNA granules. One third of the putative mRNA targets described for FMRP appear to be transported in granules and FMRP is more abundant in granules than in polyribosomes. This observation supports a primary role for FMRP in granules biology. Our findings open new avenues for the study of RNA granule dysfunctions in animal models of nervous system disorders, such as Fragile X syndrome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1006192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4963131PMC
July 2016

An E3-ligase-based method for ablating inhibitory synapses.

Nat Methods 2016 08 6;13(8):673-8. Epub 2016 Jun 6.

Department of Biology, Section of Molecular and Computational Biology, University of Southern California, Los Angeles, Los Angeles, California, USA.

Although neuronal activity can be modulated using a variety of techniques, there are currently few methods for controlling neuronal connectivity. We introduce a tool (GFE3) that mediates the fast, specific and reversible elimination of inhibitory synaptic inputs onto genetically determined neurons. GFE3 is a fusion between an E3 ligase, which mediates the ubiquitination and rapid degradation of proteins, and a recombinant, antibody-like protein (FingR) that binds to gephyrin. Expression of GFE3 leads to a strong and specific reduction of gephyrin in culture or in vivo and to a substantial decrease in phasic inhibition onto cells that express GFE3. By temporarily expressing GFE3 we showed that inhibitory synapses regrow following ablation. Thus, we have created a simple, reversible method for modulating inhibitory synaptic input onto genetically determined cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.3894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5312699PMC
August 2016

Hyperspectral multiplex single-particle tracking of different receptor subtypes labeled with quantum dots in live neurons.

J Biomed Opt 2016 Apr;21(4):46008

Institut Universitaire en santé mentale de Québec, 2601 Chemin de la Canardière, Québec G1J 2G3, CanadacUniversité Laval, Department of Biochemistry, Microbiology, and Bio-informatics, Québec G1K 7P4, Canada.

The efficacy of existing therapies and the discovery of innovative treatments for central nervous system (CNS) diseases have been limited by the lack of appropriate methods to investigate complex molecular processes at the synaptic level. To improve our capability to investigate complex mechanisms of synaptic signaling and remodeling, we designed a fluorescence hyperspectral imaging platform to simultaneously track different subtypes of individual neurotransmitter receptors trafficking in and out of synapses. This imaging platform allows simultaneous image acquisition of at least five fluorescent markers in living neurons with a high-spatial resolution. We used quantum dots emitting at different wavelengths and functionalized to specifically bind to single receptors on the membrane of living neurons. The hyperspectral imaging platform enabled the simultaneous optical tracking of five different synaptic proteins, including subtypes of glutamate receptors (mGluR and AMPAR) and postsynaptic signaling proteins. It also permitted the quantification of their mobility after treatments with various pharmacological agents. This technique provides an efficient method to monitor several synaptic proteins at the same time, which could accelerate the screening of effective compounds for treatment of CNS disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1117/1.JBO.21.4.046008DOI Listing
April 2016

New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein.

PLoS One 2016 25;11(2):e0150000. Epub 2016 Feb 25.

Institute of Cellular Neurosciences, Medical Faculty, University of Bonn, Bonn, Germany.

Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3'UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150000PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767366PMC
July 2016

Gold nanoparticle-assisted all optical localized stimulation and monitoring of Ca²⁺ signaling in neurons.

Sci Rep 2016 Feb 9;6:20619. Epub 2016 Feb 9.

Institut Universitaire en Santé Mentale de Québec, 2601 de la Canardière, Québec, QC, G1J 2G3, Canada.

Light-assisted manipulation of cells to control membrane activity or intracellular signaling has become a major avenue in life sciences. However, the ability to perform subcellular light stimulation to investigate localized signaling has been limited. Here, we introduce an all optical method for the stimulation and the monitoring of localized Ca(2+) signaling in neurons that takes advantage of plasmonic excitation of gold nanoparticles (AuNPs). We show with confocal microscopy that 800 nm laser pulse application onto a neuron decorated with a few AuNPs triggers a transient increase in free Ca(2+), measured optically with GCaMP6s. We show that action potentials, measured electrophysiologically, can be induced with this approach. We demonstrate activation of local Ca(2+) transients and Ca(2+) signaling via CaMKII in dendritic domains, by illuminating a single or few functionalized AuNPs specifically targeting genetically-modified neurons. This NP-Assisted Localized Optical Stimulation (NALOS) provides a new complement to light-dependent methods for controlling neuronal activity and cell signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep20619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4746645PMC
February 2016

FRET-FLIM investigation of PSD95-NMDA receptor interaction in dendritic spines; control by calpain, CaMKII and Src family kinase.

PLoS One 2014 13;9(11):e112170. Epub 2014 Nov 13.

Institut Universitaire en Santé Mentale de Québec, Université Laval, Québec, QC, Canada; Département de Biochimie, Microbiologie et Bio-informatique, Université Laval, Québec, QC, Canada.

Little is known about the changes in protein interactions inside synapses during synaptic remodeling, as their live monitoring in spines has been limited. We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR) and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction. NMDAR stimulation caused a transient decrease in FRET between the NMDAR and PSD95 in spines of young and mature neurons. The activity of both CaMKII and calpain were essential for this effect in both developmental stages. Meanwhile, inhibition of Src family kinase (SFK) had opposing impacts on this decrease in FRET in young versus mature neurons. Our data suggest concerted roles for CaMKII, SFK and calpain activity in regulating activity-dependent separation of PSD95 from GluN2A or GluN2B. Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0112170PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4230936PMC
January 2016

Fast gene transfer into the adult zebrafish brain by herpes simplex virus 1 (HSV-1) and electroporation: methods and optogenetic applications.

Front Neural Circuits 2014 6;8:41. Epub 2014 May 6.

Friedrich Miescher Institute for Biomedical Research Basel, Switzerland ; University of Basel Basel, Switzerland.

The zebrafish has various advantages as a model organism to analyze the structure and function of neural circuits but efficient viruses or other tools for fast gene transfer are lacking. We show that transgenes can be introduced directly into the adult zebrafish brain by herpes simplex type I viruses (HSV-1) or electroporation. We developed a new procedure to target electroporation to defined brain areas and identified promoters that produced strong long-term expression. The fast workflow of electroporation was exploited to express multiple channelrhodopsin-2 variants and genetically encoded calcium indicators in telencephalic neurons for measurements of neuronal activity and synaptic connectivity. The results demonstrate that HSV-1 and targeted electroporation are efficient tools for gene delivery into the zebrafish brain, similar to adeno-associated viruses and lentiviruses in other species. These methods fill an important gap in the spectrum of molecular tools for zebrafish and are likely to have a wide range of applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fncir.2014.00041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4018551PMC
September 2014

Nuclear Fragile X Mental Retardation Protein is localized to Cajal bodies.

PLoS Genet 2013 Oct 31;9(10):e1003890. Epub 2013 Oct 31.

Centre de recherche, Institut en santé mentale de Québec, Québec, Québec, Canada ; Département de psychiatrie et des neurosciences, Faculté de médecine, Université Laval, Québec, Québec, Canada.

Fragile X syndrome is caused by loss of function of a single gene encoding the Fragile X Mental Retardation Protein (FMRP). This RNA-binding protein, widely expressed in mammalian tissues, is particularly abundant in neurons and is a component of messenger ribonucleoprotein (mRNP) complexes present within the translational apparatus. The absence of FMRP in neurons is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. A prevalent model posits that FMRP is a nucleocytoplasmic shuttling protein that transports its mRNA targets from the nucleus to the translation machinery. However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process. Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7) do not localize to the nucleus. Instead, specific FMRP isoforms 6 and 12 (ISO6 and 12), containing a novel C-terminal domain, were the only isoforms that localized to the nuclei in cultured human cells. These isoforms localized to specific p80-coilin and SMN positive structures that were identified as Cajal bodies. The Cajal body localization signal was confined to a 17 amino acid stretch in the C-terminus of human ISO6 and is lacking in a mouse Iso6 variant. As FMRP is an RNA-binding protein, its presence in Cajal bodies suggests additional functions in nuclear post-transcriptional RNA metabolism. Supporting this hypothesis, a missense mutation (I304N), known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies. These findings open unexplored avenues in search for new insights into the pathophysiology of Fragile X Syndrome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1003890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814324PMC
October 2013

Translocation of CaMKII to dendritic microtubules supports the plasticity of local synapses.

J Cell Biol 2012 Sep 10;198(6):1055-73. Epub 2012 Sep 10.

Institut universitaire en santé mentale de Québec, Québec G1J 2G3, Canada.

The processing of excitatory synaptic inputs involves compartmentalized dendritic Ca(2+) oscillations. The downstream signaling evoked by these local Ca(2+) transients and their impact on local synaptic development and remodeling are unknown. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an important decoder of Ca(2+) signals and mediator of synaptic plasticity. In addition to its known accumulation at spines, we observed with live imaging the dynamic recruitment of CaMKII to dendritic subdomains adjacent to activated synapses in cultured hippocampal neurons. This localized and transient enrichment of CaMKII to dendritic sites coincided spatially and temporally with dendritic Ca(2+) transients. We show that it involved an interaction with microtubular elements, required activation of the kinase, and led to localized dendritic CaMKII autophosphorylation. This process was accompanied by the adjacent remodeling of spines and synaptic AMPA receptor insertion. Replacement of endogenous CaMKII with a mutant that cannot translocate within dendrites lessened this activity-dependent synaptic plasticity. Thus, CaMKII could decode compartmental dendritic Ca(2+) transients to support remodeling of local synapses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201202058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3444784PMC
September 2012

Interaction between αCaMKII and GluN2B controls ERK-dependent plasticity.

J Neurosci 2012 Aug;32(31):10767-79

Université de Caen-Basse Normandie, CNRS UMR 6232, CINAPS-PSY, 14 000 Caen, France.

Understanding how brief synaptic events can lead to sustained changes in synaptic structure and strength is a necessary step in solving the rules governing learning and memory. Activation of ERK1/2 (extracellular signal regulated protein kinase 1/2) plays a key role in the control of functional and structural synaptic plasticity. One of the triggering events that activates ERK1/2 cascade is an NMDA receptor (NMDAR)-dependent rise in free intracellular Ca(2+) concentration. However the mechanism by which a short-lasting rise in Ca(2+) concentration is transduced into long-lasting ERK1/2-dependent plasticity remains unknown. Here we demonstrate that although synaptic activation in mouse cultured cortical neurons induces intracellular Ca(2+) elevation via both GluN2A and GluN2B-containing NMDARs, only GluN2B-containing NMDAR activation leads to a long-lasting ERK1/2 phosphorylation. We show that αCaMKII, but not βCaMKII, is critically involved in this GluN2B-dependent activation of ERK1/2 signaling, through a direct interaction between GluN2B and αCaMKII. We then show that interfering with GluN2B/αCaMKII interaction prevents synaptic activity from inducing ERK-dependent increases in synaptic AMPA receptors and spine volume. Thus, in a developing circuit model, the brief activity of synaptic GluN2B-containing receptors and the interaction between GluN2B and αCaMKII have a role in long-term plasticity via the control of ERK1/2 signaling. Our findings suggest that the roles that these major molecular elements have in learning and memory may operate through a common pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.5622-11.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6621385PMC
August 2012

Activity-dependent subcellular cotrafficking of the small GTPase Rem2 and Ca2+/CaM-dependent protein kinase IIα.

PLoS One 2012 18;7(7):e41185. Epub 2012 Jul 18.

Department of Physiology and Pharmacology, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada.

Background: Rem2 is a small monomeric GTP-binding protein of the RGK family, whose known functions are modulation of calcium channel currents and alterations of cytoskeletal architecture. Rem2 is the only RGK protein found predominantly in the brain, where it has been linked to synaptic development. We wished to determine the effect of neuronal activity on the subcellular distribution of Rem2 and its interacting partners.

Results: We show that Rem2 undergoes activity-and N-Methyl-D-Aspartate Receptor (NMDAR)-dependent translocation in rat hippocampal neurons. This redistribution of Rem2, from a diffuse pattern to one that is highly punctate, is dependent on Ca(2+) influx, on binding to calmodulin (CaM), and also involves an auto-inhibitory domain within the Rem2 distal C-terminus region. We found that Rem2 can bind to Ca(2+)/CaM-dependent protein kinase IIα (CaMKII) a in Ca(2+)/CaM-dependent manner. Furthermore, our data reveal a spatial and temporal correlation between NMDAR-dependent clustering of Rem2 and CaMKII in neurons, indicating co-assembly and co-trafficking in neurons. Finally, we show that inhibiting CaMKII aggregation in neurons and HEK cells reduces Rem2 clustering, and that Rem2 affects the baseline distribution of CaMKII in HEK cells.

Conclusions: Our data suggest a novel function for Rem2 in co-trafficking with CaMKII, and thus potentially expose a role in neuronal plasticity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041185PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3399833PMC
January 2013

Fragile X mental retardation protein interacts with the RNA-binding protein Caprin1 in neuronal RiboNucleoProtein complexes [corrected].

PLoS One 2012 21;7(6):e39338. Epub 2012 Jun 21.

Centre de Recherche, Institut Universitaire en Santé Mentale, Québec, PQ, Canada.

Fragile X syndrome is caused by the absence of the Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein. FMRP is associated with messenger RiboNucleoParticles (mRNPs) present in polyribosomes and its absence in neurons leads to alteration in synaptic plasticity as a result of translation regulation defects. The molecular mechanisms by which FMRP plays a role in translation regulation remain elusive. Using immunoprecipitation approaches with monoclonal Ab7G1-1 and a new generation of chicken antibodies, we identified Caprin1 as a novel FMRP-cellular partner. In vivo and in vitro evidence show that Caprin1 interacts with FMRP at the level of the translation machinery as well as in trafficking neuronal granules. As an RNA-binding protein, Caprin1 has in common with FMRP at least two RNA targets that have been identified as CaMKIIα and Map1b mRNAs. In view of the new concept that FMRP species bind to RNA regardless of known structural motifs, we propose that protein interactors might modulate FMRP functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0039338PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3380850PMC
December 2012

Optophysiological approach to resolve neuronal action potentials with high spatial and temporal resolution in cultured neurons.

Front Cell Neurosci 2011 10;5:20. Epub 2011 Oct 10.

Centre de Recherche Université Laval Robert-Giffard, Université Laval Québec, QC, Canada.

Cell to cell communication in the central nervous system is encoded into transient and local membrane potential changes (ΔVm). Deciphering the rules that govern synaptic transmission and plasticity entails to be able to perform V(m) recordings throughout the entire neuronal arborization. Classical electrophysiology is, in most cases, not able to do so within small and fragile neuronal subcompartments. Thus, optical techniques based on the use of fluorescent voltage-sensitive dyes (VSDs) have been developed. However, reporting spontaneous or small ΔV(m) from neuronal ramifications has been challenging, in part due to the limited sensitivity and phototoxicity of VSD-based optical measurements. Here we demonstrate the use of water soluble VSD, ANNINE-6plus, with laser-scanning microscopy to optically record ΔV(m) in cultured neurons. We show that the sensitivity (>10% of fluorescence change for 100 mV depolarization) and time response (sub millisecond) of the dye allows the robust detection of action potentials (APs) even without averaging, allowing the measurement of spontaneous neuronal firing patterns. In addition, we show that back-propagating APs can be recorded, along distinct dendritic sites and within dendritic spines. Importantly, our approach does not induce any detectable phototoxic effect on cultured neurons. This optophysiological approach provides a simple, minimally invasive, and versatile optical method to measure electrical activity in cultured neurons with high temporal (ms) resolution and high spatial (μm) resolution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fncel.2011.00020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3191737PMC
November 2011

Adaptive movement compensation for in vivo imaging of fast cellular dynamics within a moving tissue.

PLoS One 2011 24;6(5):e19928. Epub 2011 May 24.

Centre de Recherche Université Laval Robert-Giffard, Université Laval, Québec, Canada.

In vivo non-linear optical microscopy has been essential to advance our knowledge of how intact biological systems work. It has been particularly enabling to decipher fast spatiotemporal cellular dynamics in neural networks. The power of the technique stems from its optical sectioning capability that in turn also limits its application to essentially immobile tissue. Only tissue not affected by movement or in which movement can be physically constrained can be imaged fast enough to conduct functional studies at high temporal resolution. Here, we show dynamic two-photon Ca(2+) imaging in the spinal cord of a living rat at millisecond time scale, free of motion artifacts using an optical stabilization system. We describe a fast, non-contact adaptive movement compensation approach, applicable to rough and weakly reflective surfaces, allowing real-time functional imaging from intrinsically moving tissue in live animals. The strategy involves enslaving the position of the microscope objective to that of the tissue surface in real-time through optical monitoring and a closed feedback loop. The performance of the system allows for efficient image locking even in conditions of random or irregular movements.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019928PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3101223PMC
September 2011

CaMKII triggers the diffusional trapping of surface AMPARs through phosphorylation of stargazin.

Neuron 2010 Jul;67(2):239-52

CNRS UMR 5091, Cellular Physiology of the Synapse, Bordeaux, France.

The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is critically required for the synaptic recruitment of AMPA-type glutamate receptors (AMPARs) during both development and plasticity. However, the underlying mechanism is unknown. Using single-particle tracking of AMPARs, we show that CaMKII activation and postsynaptic translocation induce the synaptic trapping of AMPARs diffusing in the membrane. AMPAR immobilization requires both phosphorylation of the auxiliary subunit Stargazin and its binding to PDZ domain scaffolds. It does not depend on the PDZ binding domain of GluA1 AMPAR subunit nor its phosphorylation at Ser831. Finally, CaMKII-dependent AMPAR immobilization regulates short-term plasticity. Thus, NMDA-dependent Ca(2+) influx in the post-synapse triggers a CaMKII- and Stargazin-dependent decrease in AMPAR diffusional exchange at synapses that controls synaptic function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2010.06.007DOI Listing
July 2010

CaMKII control of spine size and synaptic strength: role of phosphorylation states and nonenzymatic action.

Proc Natl Acad Sci U S A 2010 Aug 26;107(32):14437-42. Epub 2010 Jul 26.

Quantitative Biology Program, Physics Department, Brandeis University, Waltham, MA 02454, USA.

CaMKII is an abundant synaptic protein strongly implicated in plasticity. Overexpression of autonomous (T286D) CaMKII in CA1 hippocampal cells enhances synaptic strength if T305/T306 sites are not phosphorylated, but decreases synaptic strength if they are phosphorylated. It has generally been thought that spine size and synaptic strength covary; however, the ability of CaMKII and its various phosphorylation states to control spine size has not been previously examined. Using a unique method that allows the effects of overexpressed protein to be monitored over time, we found that all autonomous forms of CaMKII increase spine size. Thus, for instance, the T286D/T305D/T306D form increases spine size but decreases synaptic strength. Further evidence for such dissociation is provided by experiments with the T286D form that has been made catalytically dead. This form fails to enhance synaptic strength but increases spine size, presumably by a structural process. Thus very different mechanisms govern how CaMKII affects spine structure and synaptic function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1009268107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2922610PMC
August 2010

Autonomous CaMKII can promote either long-term potentiation or long-term depression, depending on the state of T305/T306 phosphorylation.

J Neurosci 2010 Jun;30(26):8704-9

Physics Department, Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02454, USA.

Ca(2+)/calmodulin-dependent kinase II (CaMKII) is a key mediator of long-term potentiation (LTP). Whereas acute intracellular injection of catalytically active CaMKII fragments saturates LTP (Lledo et al., 1995), an autonomously active form (T286D) of CaMKII holoenzyme expressed in transgenic mice did not saturate potentiation (Mayford et al., 1995). To better understand the role of the holoenzyme in the control of synaptic strength, we transfected hippocampal neurons with constructs encoding forms of CaMKII mimicking different phosphorylation states. Surprisingly, T286D not only failed to potentiate synaptic strength, but produced synaptic depression through an long-term depression (LTD)-like process. T305/T306 phosphorylation was critical for this depression because overexpression of the pseudophosphorylated form (T286D/T305D/T306D) caused depression that occluded LTD, and overexpression of an autonomous form in which T305/T306 could not be phosphorylated (T286D/T305A/T306A) prevented LTD (instead producing potentiation). Therefore, autonomous CaMKII can lead to either LTP or LTD, depending on the phosphorylation state of the control point, T305/T306.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.0133-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2903435PMC
June 2010

Beyond photobleaching, laser illumination unbinds fluorescent proteins.

J Phys Chem B 2009 Apr;113(15):5225-33

Department of Physics and Department of Chemistry, McGill University, Montreal, Quebec, Canada.

Confocal and two-photon fluorescence microscopy techniques using genetically encoded fluorescent probes are widely used in cell biology. Beyond the common problems of photobleaching and phototoxicity, we present evidence that photounbinding also has the potential to compromise such methods, especially in quantitative studies. We show that laser intensities within excitation regimes typical for imaging approaches such as as fluorescence recovery after photobleaching (FRAP), photolysis, or fluorescence correlation spectroscopy (FCS) experiments can cause the dissociation of antibodies from their ligands. Indeed, both one- and two-photon excitation of a fluorescent anti-GFP antibody caused its dissociation from immobilized GFP in vitro. Importantly, with two-photon excitation, the laser intensity threshold for photobleaching was the same as for photounbinding. By contrast, with single-photon excitation, we found a range of laser intensities where photobleaching can be separated from photounbinding. This photounbinding effect was visualized and measured by rebinding a second fluorescent anti-GFP (Green Fluorescent Protein) antibody, indicating that the GFP remained functional for reassociation following the photoinduced dissociation. Finally, we show that this unbinding effect occurs only when at least one binding partner carries a fluorescent label. Our results show that this photounbinding effect can readily remain masked or be misinterpreted as photobleaching, which can compromise the quantitative interpretation of binding studies made using fluorescence microscopy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jp8060152DOI Listing
April 2009

Probing synaptic signaling with quantum dots.

HFSP J 2007 May 2;1(1):5-10. Epub 2007 May 2.

Synapses are the principal substrates of neuronal communication in the brain. Neuroscientists are trying to understand how the remodeling of synapses at the molecular level leads to changes in learning and memory. The lateral movement of neurotransmitter receptors is emerging as an important mechanism in the control of synaptic transmission. However, our understanding of the spatial dynamics of membrane receptors at synapses has been limited largely because of a lack of appropriate tools to resolve single receptors in living cells. Fluorescent quantum dots represent promising probes to monitor individual synaptic receptors in living neural circuits. Bats and colleagues (Bats, Groc, and Choquet, Neuron 53, 719, 2007) used quantum dots to track the ins and outs of glutamate receptors at synapses, showing that the receptors bring company as they diffuse in the synapse to become trapped via their partner's local connections. Their study sheds new light on the mechanisms used by synapses to change their efficacy, which may impact on our understanding of the cellular and molecular basis of learning and memory.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2976/1.2735016/10.2976/1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2645557PMC
May 2007

Dendritic spine viscoelasticity and soft-glassy nature: balancing dynamic remodeling with structural stability.

Biophys J 2007 Feb 17;92(4):1419-30. Epub 2006 Nov 17.

Department of Physics, McGill University, Montreal, QC, Canada H3A 2T8.

Neuronal dendritic spines are a key component of brain circuitry, implicated in many mechanisms for plasticity and long-term stability of synaptic communication. They can undergo rapid actin-based activity-dependent shape fluctuations, an intriguing biophysical property that is believed to alter synaptic transmission. Yet, because of their small size (approximately 1 microm or less) and metastable behavior, spines are inaccessible to most physical measurement techniques. Here we employ atomic force microscopy elasticity mapping and novel dynamic indentation methods to probe the biomechanics of dendritic spines in living neurons. We find that spines exhibit 1), a wide range of rigidities, correlated with morphological characteristics, axonal association, and glutamatergic stimulation, 2), a uniquely large viscosity, four to five times that of other cell types, consistent with a high density of solubilized proteins, and 3), weak power-law rheology, described by the soft-glassy model for cellular mechanics. Our findings provide a new perspective on spine functionality and identify key mechanical properties that govern the ability of spines to rapidly remodel and regulate internal protein trafficking but also maintain structural stability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1529/biophysj.106.092361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1783894PMC
February 2007
-->