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Antoine Triller

Publications and source records attributed to Antoine Triller.

31 records · Page 2Linked to original sources
PubMed2004

Glycine receptors and GABA receptor alpha 1 and gamma 2 subunits during the development of mouse hypoglossal nucleus.

In the hypoglossal nucleus, GABA and glycine mediate inhibition at separate or mixed synapses containing glycine receptors (GlyRs) and/or GABA(A) receptors (GABA(A)Rs). The functional development of mixed inhibitory synapses depends on the brain area studied, but their relative proportion to total synapses generally decreases with time. We have determined the sequential process of inhibitory synapse maturation in the hypoglossal nucleus in vivo. Immunocytochemistry and confocal microscopy were used for codetection of VIAAT, the common presynaptic vesicular transporter of glycine and GABA, GlyRs, GABA(A)R alpha1 and gamma2 subunits, and gephyrin, the scaffold protein implicated in the synaptic localization of inhibitory receptors. In E17 embryos, GlyRs were already clustered while GABA(A)R alpha1 and gamma2 subunit immunoreactivity (IR) displayed both diffuse and clustered patterns. Quantitative analysis at this stage revealed that the majority of GlyR clusters were apposed to VIAAT-IR accumulation and that 30% of them colocalized with gamma2GABA(A)R clusters. This proportion increased with age to 50% at P30. GlyR clusters that did not colocalize with gamma2GABA(A)R clusters were associated with GABA(A)R gamma2 diffuse IR. Interestingly, the percentage of GlyR clusters surrounded by GABA(A)R gamma2 diffuse IR decreased with age, while GlyR clusters colocalized with gamma2GABA(A)R clusters increased. The developmental coclustered pattern of gephyrin and GABA(A)R alpha1 and gamma2 subunits paralleled the coclustered pattern of GlyRs and GABA(A)R alpha1 and gamma2 subunits. Our results indicate that the proportion of GlyR-GABA(A)R coclusters increases until adulthood. A developmental sequence of the postsynaptic events is proposed in which diffuse extrasynaptic GABA(A)Rs accumulate at inhibitory synapses to form postsynaptic clusters, most of them being colocalized with GlyR clusters in the adult.

Animals↗
PubMed2004

Preservation of immunoreactivity and fine structure of adult C. elegans tissues using high-pressure freezing.

The location of a protein labeled by immunogold techniques can be resolved under an electron beam to within nanometers of its epitope, a resolution that makes immunoelectron microscopy a valuable tool for studies of cell biology. However, tissues in the nematode Caenorhabditis elegans are difficult to preserve for immunoelectron microscopic studies. The animal's cuticle slows the diffusion of solutions into the animal and thus makes it difficult to preserve both immunoreactivity and cell morphology. Here we describe a protocol that circumvents these problems. Specifically, we instantly immobilized tissue in vitreous ice by freezing living adult animals under high pressure. Frozen specimens were then chemically fixed, dehydrated, and embedded at low temperatures. As a result, chemical diffusion across the cuticle could occur over an extended period without morphological deterioration. We show that this method is capable of preserving both cell morphology, including fine structures, and immunoreactivity. Therefore, it provides a means to characterize the localization of endogenous proteins and exogenous proteins, such as the green fluorescent protein (GFP), with respect to subcellular compartments in C. elegans tissues by using postembedding immunogold labeling.

Actins↗
PubMed2003

Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking.

Semiconductor quantum dots (QDs) are nanometer-sized fluorescent probes suitable for advanced biological imaging. We used QDs to track individual glycine receptors (GlyRs) and analyze their lateral dynamics in the neuronal membrane of living cells for periods ranging from milliseconds to minutes. We characterized multiple diffusion domains in relation to the synaptic, perisynaptic, or extrasynaptic GlyR localization. The entry of GlyRs into the synapse by diffusion was observed and further confirmed by electron microscopy imaging of QD-tagged receptors.

Animals↗
PubMed2003

Lysosomal amino acid transporter LYAAT-1 in the rat central nervous system: an in situ hybridization and immunohistochemical study.

A first mammalian lysosomal transporter (LYAAT-1) was recently identified and functionally characterized. Preliminary immunocytochemical data revealed that LYAAT-1 localizes to lysosomes in some neurons. In order to determine whether it is expressed in specific neuron populations and other cell types, and to confirm whether it is localized at the membrane of lysosomes, we used in situ hybridization and immunohistochemistry methods in adult rat central nervous system (CNS). We found that LYAAT-1 is expressed in most areas of the CNS, specifically in neurons, but also in choroid plexus and ependymal epithelium cells. LYAAT-1-IR (immunoreactivity) levels varied among different neuroanatomical structures but were present in neurons independently of the neurotransmitter used (glutamate, GABA, acetylcholine, noradrenaline, serotonin, or glycine). Light and confocal microscopy demonstrated that LYAAT-1 and the lysosomal marker cathepsin D colocalized throughout the brain and electron microscopy showed that LYAAT-1-IR was associated with lysosomal membranes. In addition, LYAAT-1-IR was also found associated with other membranes belonging to the Golgi apparatus and lateral saccules and less frequently with multivesicular bodies, endoplasmic reticulum, and occasionally with the plasma membrane. The localization of LYAAT-1 at the lysosomal membrane is consistent with the view that it mediates amino acid efflux from lysosomes. Furthermore, its cell expression pattern suggests that it may contribute to specialized cellular function in the rat CNS such as neuronal metabolism, neurotransmission, and control of brain amino acid homeostasis.

Amino Acid Transport Systems↗
PubMed2003

Synaptic structure and diffusion dynamics of synaptic receptors.

Neurotransmitter receptors are concentrated at synaptic sites through interactions with specific scaffolding proteins and cytoskeletal elements, but can also be found in intracellular compartments or dispersed in the membrane. The notion has emerged in recent years that this distribution results from a dynamic equilibrium between the different pools of receptors. This equilibrium is regulated by neuronal activity and interactions with scaffolding proteins. A change in its set point can result in rapid variations in the number of functional receptors at synapses such as seen during plasticity. Trafficking of receptors in and out of synapses has up to now mainly been viewed within the framework of endo/exocytotic processes. After a brief presentation of the structure of excitatory and inhibitory synapses, we review data indicating that the lateral diffusion of receptors in the plane of the membrane is also a key step to regulate receptor stabilization and accumulation at synapses.

Animals↗
PubMed2003

Association of gephyrin with synaptic and extrasynaptic GABAA receptors varies during development in cultured hippocampal neurons.

Several studies have reported extrasynaptic clusters of GABAA receptors in hippocampal neurons. Yet their functional relevance as well as their evolution in relation with gephyrin during synaptogenesis remain unknown. We have analyzed the expression pattern of the main proteins of the GABAergic synapses during synaptogenesis in cultured hippocampal neurons. We found that GABAergic terminals, characterized by VIAAT and GAD-65 expression, differentiated 3 to 7 days after the glutamatergic endings. At the postsynaptic side, the GABAAR- beta3 subunit was first diffuse and then clustered when GABAergic terminals differentiated and gephyrin formed large clusters. Colocalization of these proteins was high and increased with development. At later stages, GABAAR beta3 clusters colocalized with gephyrin at synaptic but also at extrasynaptic sites. GABAAR gamma2 subunits were directly expressed as clusters which were first extrasynaptic and not associated with gephyrin. Subsequently, the GABAAR gamma2 subunits associated with gephyrin at synaptic and/or extrasynaptic sites. Our data indicate that formation of GABAAR gamma2 subunit clusters is gephyrin independent.

Amino Acid Transport Systems↗
PubMed2003

Herpes simplex virus type 1 glycoprotein B sorting in hippocampal neurons.

Herpes simplex virus type 1 (HSV-1) is a neuroinvasive human pathogen that spreads in the nervous system in functionally connected neurons. Determining how HSV-1 components are sorted in neurons is critical to elucidate the mechanisms of virus neuroinvasion. By using recombinant viruses expressing glycoprotein B (gB) tagged with green fluorescent protein (GFP), the subcellular localization of this envelope protein was visualized in infected hippocampal neurons in culture. Results obtained using a fully infectious recombinant virus containing GFP inserted into the ectodomain of gB support the view that capsids and gB are transported separately in neuron processes. Moreover, they show that during infection gB is sorted to the dendritic tree and the axons of polarized hippocampal neurons. However, GFP insertion into the cytoplasmic tail of gB impaired the maturation of the resulting fusion protein and caused its retention in the endoplasmic reticulum. The defective protein did not gain access to axons of infected neurons. These results suggest that the cytoplasmic tail of gB plays a role in maturation and transport and subsequently in axonal sorting in differentiated hippocampal neurons.

Animals↗
PubMed2002

Involvement of survival motor neuron (SMN) protein in cell death.

Infantile spinal muscular atrophy (SMA) is caused by mutations in the survival motor neuron (SMN)1 gene. We investigated the role of human (h) SMN protein on cell death in PC12 and Rat-1 cells. hSMN prolonged cell survival in PC12 cells deprived of trophic support and in Rat-1 cells induced to die by activation of the proto-oncogene c-Myc, to similar magnitude as Bcl-2 or IAP-2. While hSMN was ineffective in inhibiting apoptosis induced by ultraviolet light (UV) or etoposide treatment in proliferating PC12 or Rat-1 cells, a protective effect was observed in terminally NGF/dBcAMP-differentiated PC12 cells. hSMN inhibited the onset of apoptosis in NGF/dBcAMP-deprived or UV-treated co-differentiated PC12 cells by preventing cytochrome c release and caspase-3 activation, indicating that its effects are through suppression of the mitochondrial apoptotic pathway. Expressing hSMN deleted for exon 7 (Delta7) or for exons 6 and 7 (Delta6/7), or with the SMA point mutant Y272C, resulted in loss of survival function. Moreover, these mutants also exhibited pro-apoptotic effects in Rat-1 cells. The localization pattern of full-length hSMN in PC12 and Rat-1 cells was similar to that of endogenous SMN: granular labelling in the cytoplasm and discrete fluorescence spots in the nucleus, some of which co-localized with p80 coilin, the characteristic marker of Cajal bodies. However, cytoplasmic and nuclear aggregates were often seen with hSMNDelta7, whereas the hSMNDelta6/7 mutant showed homogenous nuclear labelling that excluded the nucleolus. Thus, our results show that the C-terminal region is critical in suppression of apoptosis by SMN.

Animals↗
PubMed2002

Motoneuron-derived neurotrophin-3 is a survival factor for PAX2-expressing spinal interneurons.

Rat spinal cord interneurons undergo programmed cell death shortly after birth. We investigated here whether cell death of interneurons could be regulated by trophic factors produced by motoneurons, one of their main targets. To test this hypothesis, we studied the effect of the selective destruction of motoneurons on the survival of interneurons in organotypic cultures of embryonic rat spinal cords. Motoneurons were eliminated by an anti-p75(NTR)-specific immunotoxin (192 IgG-saporin). We then observed a decrease of 28% in the number of ventral spinal interneurons immunoreactive (IR) for the homeoprotein PAX2. This was correlated with an increase in the number of apoptotic nuclei in the same area. Because neurotrophin-3 (NT-3) is specifically produced by motoneurons and because interneurons express the NT-3 high-affinity receptor trkC, we examined the role of NT-3 in the survival of PAX2-IR interneurons. Addition of NT-3 to 192 IgG-saporin-treated explants rescued ventral PAX2-IR interneurons. Depletion of secreted NT-3 by anti-NT-3 antibodies induced 66% loss of ventral PAX2-IR interneurons. We conclude that motoneuron-derived NT-3 is a trophic factor for ventral PAX2-IR interneurons.

Animals↗
PubMed2002

Gephyrin interacts with Dynein light chains 1 and 2, components of motor protein complexes.

The clustering of glycine receptors and major subtypes of GABA(A) receptors at inhibitory synapses is mediated by the tubulin-binding protein gephyrin. In an attempt to identify additional components of inhibitory postsynaptic specializations, we performed a yeast two-hybrid screen using gephyrin as bait. Multiple positive clones encoded either the dynein light chain-1 (Dlc-1), also known as dynein LC8 and protein inhibitor of neuronal nitric oxide synthase, or its homolog Dlc-2. Dlc-1 protein bound efficiently to gephyrin in in vitro binding assays and colocalized with gephyrin during coexpression in HEK293 cells. The binding site for Dlc was mapped to a fragment of 63 amino acids within the central linker domain of gephyrin. In hippocampal neurons, endogenous Dlc protein was enriched at synaptic sites identified by synaptophysin and gephyrin immunostaining. Immunoelectron microscopy in spinal cord sections revealed Dlc immunoreactivity at the edges of postsynaptic differentiations, in close contact with cytoskeletal structures and at the periphery of the Golgi apparatus. Because Dlc-1 and Dlc-2 have been described as stoichiometric components of cytoplasmic dynein and myosin-Va complexes, our results suggest that motor proteins are involved in the subcellular localization of gephyrin.

Amino Acid Sequence↗
PubMed2002

Strychnine-blocked glycine receptor is removed from synapses by a shift in insertion/degradation equilibrium.

The long-term inhibition by strychnine of glycine receptor activity in neurons provokes the receptor's selective intracellular accumulation and disappearance from synapses. This could result either from a disruption of the postsynaptic anchoring of the receptor or from an arrest of its exocytic transport. In this study we combined biochemical and fluorescence microscopy analyses to determine on a short time scale the fate of the strychnine-inactivated glycine receptor. Quantification of the cellular content of receptor showed that the rapid accumulation depends on protein synthesis. Cell surface biotinylation of neurons demonstrated that strychnine did not accelerate the turnover rate of the receptor. Labeling of endosomes indicated that, in strychnine-treated cells, the accumulated receptor is not blocked in the endosomal transport pathway. Taken together, these results indicate that strychnine does not destabilize the postsynaptic receptor but triggers its disappearance from synapses by a nondegradative sequestration of newly synthesized molecules in a nonendocytic compartment.

Animals↗
PubMed2002

Desensitization of homomeric alpha1 glycine receptor increases with receptor density.

Variations in the number of receptors at glycinergic synapses are now established and are believed to contribute to inhibitory synaptic plasticity. However, the relation between glycine receptor (GlyR) kinetics and density is still unclear. We used outside-out patch-clamp recordings and fast-flow application techniques to resolve fast homomeric GlyRalpha1 kinetics and to determine how the functional properties of these receptors depend on their density and on the presence of the anchoring protein gephyrin. The expression of GlyRs in human embryonic kidney cells increased with time and was correlated with an increase in GlyR desensitization at 2 days after transfection. Cotransfection of homomeric GlyRalpha1 bearing the gephyrin-binding site with gephyrin also increased desensitization but at 1 day after transfection compared with transfections of homomeric GlyRalpha1 without gephyrin. This increase results from the occurrence of a fast desensitization component and short applications of a saturating concentration of glycine suffice to promote a rapidly entered desensitized closed state. The level of desensitization changed neither the EC(50) value nor the Hill coefficient of the glycine dose-response curves because the amplitude of the current was measured at the peak of the responses. These results demonstrate that variations in GlyR density during cluster formation result from a change in GlyR efficiency due to modifications in their desensitization properties.

Cell Membrane↗