Agnès Hémar

A Single In Vivo Exposure to Cocaine Abolishes Endocannabinoid-Mediated Long-Term Depression in the Nucleus Accumbens

In the nucleus accumbens (NAc), a key structure to the effects of all addictive drugs, presynaptic cannabinoid CB1 receptors (CB1Rs) and postsynaptic metabotropic glutamate 5 receptors (mGluR5s) are the principal effectors of endocannabinoid (eCB)-mediated retrograde long-term depression (LTD) (eCB-LTD) at the prefrontal cortex-NAc synapses. Both CB1R and mGluR5 are involved in cocaine-related behaviors; however, the impact of in vivo cocaine exposure on eCB-mediated retrograde synaptic plasticity remains unknown. Electrophysiological and biochemical approaches were used, and we report that a single in vivo cocaine administration abolishes eCB-LTD. This effect of cocaine was not present in D1 dopamine receptor (D1R) -/- mice and was prevented when cocaine was coadministered with the selective D1R antagonist 8-chloro-2,3,4,5-tetrahydro-3-5-1h-3-benzazepin-7-ol (0.5 mg/kg) or with the NMDA receptor (NMDAR) blocker (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate (1 mg/kg), suggesting the involvement of D1R and NMDAR. We found that the cocaine-induced blockade of retrograde signaling was correlated with enhanced expression levels of Homer scaffolding proteins containing the coiled-coil domain and accompanied by a strong reduction of mGluR5 surface expression. The results suggest that cocaine-induced loss of eCB retrograde signaling is caused by a reduction in the ability of mGluR5 to translate anterograde glutamate transmission into retrograde eCB signaling.

The Metabotropic Glutamate Receptor mGluR5 Is Endocytosed by a Clathrin-independent Pathway

Metabotropic glutamate receptors 5 (mGluR5) are members of the growing group C G protein-coupled receptor family. Widely expressed in mammalian brain, they are involved in modulation of the glutamate transmission. By means of transfection of mGluR5 receptors in COS-7 cells and primary hippocampal neurons in culture followed by immunocytochemistry and quantitative image analysis and by a biochemical assay, we have studied the internalization of mGluR5 splice variants. mGluR5a and -5b were endocytosed in COS-7 cells as well as in axons and dendrites of cultured neurons. Endocytosis occurred even in the absence of receptor activity, because receptors mutated in the glutamate binding site were still internalized as well as receptors in which endogenous activity had been inhibited by an inverse agonist. We have measured a constitutive rate of endocytosis of 11.7%/min for mGluR5a. We report for the first time the endocytosis pathway of mGluR5. Internalization of mGluR5 is not mediated by clathrin-coated pits. Indeed, inhibition of this pathway by Eps15 dominant negative mutants did not disturb their endocytosis. However, the large GTPase dynamin 2 is implicated in the endocytosis of mGluR5 in COS-7. mGluR5 is the first shown member of the group C G-protein coupled receptor family internalized by a nonconventional pathway.

Acute stress facilitates hippocampal CA1 metabotropic glutamate receptor-dependent long-term depression.

Acute stress affects NMDA receptor (NMDAR)-dependent synaptic plasticity in the CA1 region of the hippocampus, with long-term potentiation and long-term depression (LTD) being, respectively, diminished and facilitated by acute exposure to stress. Here, we examined whether this facilitatory effect of stress on NMDAR-dependent LTD extends to metabotropic glutamate receptor (mGluR)-dependent LTD at Schaffer collateral–CA1 synapses. Application of a low dose (50 μm) of the selective group 1 mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) promoted LTD in slices from stressed, but not from control, rats. Pretreatment of stressed rats with the glucocorticoid receptor (GR) antagonist RU38486 prevented the facilitation of DHPG-induced LTD (DHPG-LTD), indicating the involvement of corticosterone secretion and, in turn, stimulation of GRs. Finally, pretreatment of slices with an mGluR1, but not an mGluR5, antagonist blunted the sensitizing effect of stress on DHPG-LTD. These results indicate that acute stress, through corticosterone stimulation of GRs, facilitates the expression of mGluR1-dependent DHPG-LTD in the hippocampal CA1 region.

Active surface transport of metabotropic glutamate receptors through binding to microtubules and actin flow

Receptors for neurotransmitters are concentrated and stabilized at given sites such as synapses through interactions with scaffolding proteins and cytoskeletal elements. The transport of receptors first involves directed vesicular trafficking of intracellularly stored receptors followed by their targeting to the plasma membrane. Once expressed at the cell surface, receptors are thought to reach their final location by random Brownian diffusion in the plasma membrane plane. Here, we investigate whether the metabotropic glutamate receptor mGluR5 can also be transported actively on the cell surface. We used single particle tracking to follow mGluR5 movement in real time at the surface of neuronal growth cones or fibroblast lamellipodia, both of which bear a particularly active cytoskeleton. We found that after a certain lag time mGluR5 undergoes directed rearward transport, which depends on actin flow. On actin depolymerization, directed movement was suppressed, but receptors still bound to a rigid structure. By contrast, receptor transport and immobilization was fully suppressed by microtubule depolymerization but favored by microtubule stabilization. Furthermore, mGluR5 could be immunoprecipitated with tubulin from rat brains, confirming the ability of mGluR5 to bind to microtubules. We propose that mGluR5 can be transported on the cell surface through actin-mediated retrograde transport of microtubules. This process may play a role in receptor targeting and organization during synapse formation or during glutamate-mediated growth cone chemotaxis.

The calcium-sensing protein synaptotagmin 7 is expressed on different endosomal compartments in endocrine, neuroendocrine cells or neurons but not on large dense core vesicles

Synaptotagmin (syt) isoforms function as calcium sensor in post-Golgi transport although the precise transport step and compartment(s) concerned are still not fully resolved. As syt7 has been proposed to operate in lysosomal exocytosis and in exocytosis of large dense core vesicles (LDCVs), we have addressed the distribution of endogenous syt7 in insulin-secreting cells. These cells express different syt7 isoforms comparable to neurons. According to subcellular fractionation and quantitative confocal immunocytochemistry, syt7 is not found on LDCVs or on synaptic-like microvesicles but colocalizes with Rab7 on endosomes and to structures near to or at the plasma membrane. Similarly, endogenous syt7 was absent from LDCVs in pheochromocytoma PC12 cells. In contrast, syt7 localised to lysosomes in both, PC12 cells and hippocampal neurons. In conclusion, endogenous syt7 shows a wider distribution than previously reported but does not qualify as vesicular calcium sensor in SLMV or LDCV exocytosis according to its localisation.

A comparison of image deconvolution algorithms applied to the detection of endocytic vesicles in fluorescence images of neural proteins

In this work we present a comparative study of three image deconvolution methods applied to fluorescence images of neural proteins. The purpose of this work is to compare the efficiency of these methods, in order to establish which one performs better the restoration of this type of image. Moreover we show that image deconvolution improve not only image quality, but detection capabilities and thus the counting of endocytic vesicles. Image deconvolution was performed by Gold-Meinel (GM) and Lucy-Richardson Maximum likelihood (LRML) non-blind methods and by Lucy-Richardson Maximum likelihood blind method (LRMLB). These methods were tested in 120 images from two different experiments. Computed theoretical point spread function (psf) was used for non-blind deconcovolution methods. Twenty five iterations were performed to restore each image using GM and LRML algorithms. In the case of LRMLB, 10 cycles were performed with 15 psf iterations and 5 image iterations per cycle to deconvolve each image. Endocytic vessels counting was manually made in deconvolved and non-deconvolved images by a trained observer. Results showed an increase of 22% and 24% in the detection of endocytic vessels using LRML and LRMLB methods respectively and a decrease of 6% using GM method, against detection with non deconvolved images.

Modulation of the metabotropic glutamate receptor mGluR5a anchorage to the cytoskeleton characterised by videomicroscopy and tracking of single particles manipulated with optical tweezers

Glutamate is the main excitatory neurotransmitter of the vertebrate central nervous system. At the synaptic level, it activates ionotropic and metabotropic receptors which can be located in the post-synaptic density, at its surround or on the presynaptic terminal. The stabilisation of this precise distribution and the modulation of receptor localisation during synaptogenesis or synaptic plasticity implicates anchorage mechanisms, to the cytoskeleton or to adhesive proteins. Many aspects of this process remain unknown. For example, receptors could be either inserted directly in the post-synaptic density or at random in the membrane, and then anchored when they arrive at their place during their random diffusing movement. Indeed, a (c free )P membranous protein diffuses in the lipid bilayer with a diffusion coefficient of approximately 0,05pm2/s ; it thus travels of several microns in a minute and can rapidly move from one membranous domain to the other. Anchoring proteins able to stabilise the receptors are being identified and mainly consist of PDZ domain-containing proteins (Komeau et al, 1997), but up to now, the dynamics of receptor movement are unknown. The aim of this study is the analysis of the mobility of the receptor mGluR5a, which is positively coupled to the PLC. We focused our analysis on the regulation of its anchorage to the cytoskeleton and to the Homer protein (Brakeman et al, 1997).