Agrin, a synaptic basal lamina protein synthesized by motoneurons is involved in the aggregation ... more Agrin, a synaptic basal lamina protein synthesized by motoneurons is involved in the aggregation of nicotinic acetylcholine receptors (nAchRs) at the neuromuscular junction. Agrin transcripts are broadly expressed in the central nervous system (CNS) including non-cholinergic regions. This wide distribution of agrin mRNAs raises the question of its function in these areas. To approach this question, we analysed the expression and cellular distribution of agrin in primary cultures of rat embryonic dorsal horn neurons. Polymerase chain reaction analysis demonstrated that the four agrin isoform (B0, B8, B11, B19) mRNAs are expressed as early as 4 days in vitro, before the formation of functional synaptic contacts. Western blots also showed that agrin-like proteins are secreted in conditioned medium from 7 days cultures. We analysed the subcellular distribution of agrin by double immunolabeling and fluorescence microscopy. We found that agrin is synthesized by almost all neurons and was ...
Super-resolution imaging of synapses has revealed that key synaptic proteins are dynamically orga... more Super-resolution imaging of synapses has revealed that key synaptic proteins are dynamically organized within sub-synaptic domains (SSDs). At mixed inhibitory synapses in spinal cord neurons, both GlyRs and GABAARs reside at the same post-synaptic density (PSD). To examine how the different inhibitory receptors are organized and regulated, we carried out dual-color direct stochastic optical reconstruction microscopy (dSTORM). We found that endogenous GlyRs and GABAARs as well as their common scaffold protein gephyrin form SSDs that align with pre-synaptic RIM1/2, thus forming trans-synaptic nanocolumns. Strikingly, GlyRs and GABAARs occupy different sub-synaptic spaces, exhibiting only a partial overlap at mixed inhibitory synapses. When network activity was increased by pharmacological treatment using the K+ channel blocker 4-aminopyridine (4-AP), the GABAAR copy numbers of as well as the number of GABAAR SSDs were reduced, while GlyRs remained largely unchanged. This differential ...
Proceedings of the National Academy of Sciences, 2021
Significance Learning depends on synaptic plasticity. The signaling mechanisms that control induc... more Significance Learning depends on synaptic plasticity. The signaling mechanisms that control induction of plasticity determine the learning rules at the specific synapse involved. Moreover, the relationship between the activity patterns of synaptic inputs and the type, direction, and level of plasticity induced may evolve during development. Here, we establish a key link between receptor activation presynaptic to cerebellar Purkinje cells, downstream signaling mechanisms, and the ability of adult animals to learn a cerebellar motor task.
Pyramidal neurons (PNs) are covered by thousands of dendritic spines receiving excitatory synapti... more Pyramidal neurons (PNs) are covered by thousands of dendritic spines receiving excitatory synaptic inputs. The ultrastructure of dendritic spines shapes signal compartmentalization, but ultrastructural diversity is rarely taken into account in computational models of synaptic integration. Here, we developed a 3D correlative light–electron microscopy (3D-CLEM) approach allowing the analysis of specific populations of synapses in genetically defined neuronal types in intact brain circuits. We used it to reconstruct segments of basal dendrites of layer 2/3 PNs of adult mouse somatosensory cortex and quantify spine ultrastructural diversity. We found that 10% of spines were dually innervated and 38% of inhibitory synapses localized to spines. Using our morphometric data to constrain a model of synaptic signal compartmentalization, we assessed the impact of spinous versus dendritic shaft inhibition. Our results indicate that spinous inhibition is locally more efficient than shaft inhibit...
ABSTRACTPyramidal neurons are covered by thousands of dendritic spines receiving excitatory synap... more ABSTRACTPyramidal neurons are covered by thousands of dendritic spines receiving excitatory synaptic inputs. The ultrastructure of dendritic spines shapes signal compartmentalization but ultrastructural diversity is rarely taken into account in computational models of synaptic integration. Here, we developed a 3D correlative light-electron microscopy (3D-CLEM) approach allowing the analysis of specific populations of synapses in genetically defined neuronal types in intact brain circuits. We used it to reconstruct segments of basal dendrites of layer 2/3 pyramidal neurons of adult mouse somatosensory cortex and quantify spine ultrastructural diversity. We found that 10% of spines were dually-innervated and 38% of inhibitory synapses localized to spines. Using our morphometric data to constrain a model of synaptic signal compartmentalization, we assessed the impact of spinous versus dendritic shaft inhibition. Our results indicate that spinous inhibition is locally more efficient tha...
Zinc has been reported to potentiate glycine receptors (GlyR), but the physiological significance... more Zinc has been reported to potentiate glycine receptors (GlyR), but the physiological significance of this observation has been put in doubt by the relatively high values of the EC50, 0.5–1 μM, since such concentrations may not be attained in the synaptic cleft of glycinergic synapses. We have re-evaluated this observation in the frame of the hypothesis that contaminant heavy metals present in usual solutions may have lead to underestimate the affinity of the zinc binding site, and therefore to underestimate the potential physiological role of zinc. Using chelators either to complex heavy metals or to apply zinc at controlled concentrations, we have examined the action of zinc on GlyR kinetics in outside-out patches from 50-h-old zebrafish Mauthner cells. Chelating contaminating heavy metals with tricine or N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) decreased the duration of the currents evoked by glycine, confirming that traces of heavy metals alter the GlyR respons...
In biological membranes, many factors such as cytoskeleton, lipid composition, crowding, and mole... more In biological membranes, many factors such as cytoskeleton, lipid composition, crowding, and molecular interactions deviate lateral diffusion from the expected random walks. These factors have different effects on diffusion but act simultaneously, so the observed diffusion is a complex mixture of diffusive behaviors (directed, Brownian, anomalous, or confined). Therefore, commonly used approaches to quantify diffusion based on averaging of the displacements such as the mean square displacement, are not adapted to the analysis of this heterogeneity. We introduce a parameter-the packing coefficient Pc, which gives an estimate of the degree of free movement that a molecule displays in a period of time independently of its global diffusivity. Applying this approach to two different situations (diffusion of a lipid probe and trapping of receptors at synapses), we show that Pc detected and localized temporary changes of diffusive behavior both in time and in space. More importantly, it al...
Alzheimer's disease (AD) is associated with a progressive loss of synapses and neurons. Studi... more Alzheimer's disease (AD) is associated with a progressive loss of synapses and neurons. Studies in animal models indicate that morphological alterations of dendritic spines precede synapse loss, increasing the proportion of large and short ("stubby") spines. Whether similar alterations occur in human patients, and what their functional consequences could be, is not known. We analyzed biopsies from AD patients and APP x presenilin 1 knock-in mice that were previously shown to present a loss of pyramidal neurons in the CA1 area of the hippocampus. We observed that the proportion of stubby spines and the width of spine necks are inversely correlated with synapse density in frontal cortical biopsies from non-AD and AD patients. In mice, the reduction in the density of synapses in the stratum radiatum was preceded by an alteration of spine morphology, with a reduction of their length and an enlargement of their neck. Serial sectioning examined with electron microscopy allow...
GABAergic synaptic transmission regulates brain function by establishing the appropriate excitati... more GABAergic synaptic transmission regulates brain function by establishing the appropriate excitation-inhibition (E/I) balance in neural circuits. The structure and function of GABAergic synapses are sensitive to destabilization by impinging neurotransmitters. However, signaling mechanisms that promote the restorative homeostatic stabilization of GABAergic synapses remain unknown. Here, by quantum dot single-particle tracking, we characterize a signaling pathway that promotes the stability of GABAA receptor (GABAAR) postsynaptic organization. Slow metabotropic glutamate receptor signaling activates IP3 receptor-dependent calcium release and protein kinase C to promote GABAAR clustering and GABAergic transmission. This GABAAR stabilization pathway counteracts the rapid cluster dispersion caused by glutamate-driven NMDA receptor-dependent calcium influx and calcineurin dephosphorylation, including in conditions of pathological glutamate toxicity. These findings show that glutamate activ...
Proceedings of the National Academy of Sciences, 1995
Simultaneous pre- and postsynaptic recordings from identified glycinergic inhibitory interneurons... more Simultaneous pre- and postsynaptic recordings from identified glycinergic inhibitory interneurons and the Mauthner cell showed that 25% of the afferents produced no or extremely small postsynaptic responses. Morphological determination of the number of contacts made by these cells on the Mauthner cell revealed a connectivity similar to that of functional neurons which always produce clear inhibitory postsynaptic potentials, suggesting that most of the endings, made by weak interneurons are silent. Intraaxonal injection of 4-aminopyridine or Ca2+ greatly enhanced transmission at functional connections but did not modify those which were ineffective. However, after eighth nerve tetanic stimuli, transmission at the weak connections was unmasked or enhanced for prolonged periods and was twice as likely to be potentiated, with a 6-fold greater mean enhancement than the potent ones. This result provides additional support for long-term potentiation of inhibitory synapses. Furthermore, wea...
Concentration of neurotransmitter receptors at synapses is thought to result from stable binding ... more Concentration of neurotransmitter receptors at synapses is thought to result from stable binding to subsynaptic scaffold proteins. Recent data on synaptic plasticity have shown that changes in synaptic strength derive partly from modification of postsynaptic receptor numbers. This has led to the notion of receptor trafficking into and out of synapses. The proposed underlying mechanisms have under-evaluated the role of extrasynaptic receptors. Recent technological advances have allowed imaging of receptor movements at the single-molecule level, and these experiments demonstrate that receptors switch at unexpected rates between extrasynaptic and synaptic localizations by lateral diffusion. Variation in receptor numbers at postsynaptic sites is therefore likely to depend on regulation of diffusion by modification of the structure of the membrane and/or by transient interactions with scaffolding proteins. This review is part of the TINS Synaptic Connectivity series.
INTRODUCTIONThis protocol describes a highly sensitive approach for tracking the motion of membra... more INTRODUCTIONThis protocol describes a highly sensitive approach for tracking the motion of membrane molecules over extended time periods with single-molecule resolution. This technique uses nanometer-sized quantum dots (QDs) linked to the extracellular part of the proteins to be followed. Single-fluorophore epifluorescence imaging then reveals the membrane diffusion of the particle of interest. Two methods are presented for labeling neurons with the primary antibody of choice along with a secondary anti-Fab antibody that is either biotinylated or directly coupled to the desired QD. The behavior of QD-labeled molecules can then be followed within the cell using epifluorescence imaging.
Agrin, a synaptic basal lamina protein synthesized by motoneurons is involved in the aggregation ... more Agrin, a synaptic basal lamina protein synthesized by motoneurons is involved in the aggregation of nicotinic acetylcholine receptors (nAchRs) at the neuromuscular junction. Agrin transcripts are broadly expressed in the central nervous system (CNS) including non-cholinergic regions. This wide distribution of agrin mRNAs raises the question of its function in these areas. To approach this question, we analysed the expression and cellular distribution of agrin in primary cultures of rat embryonic dorsal horn neurons. Polymerase chain reaction analysis demonstrated that the four agrin isoform (B0, B8, B11, B19) mRNAs are expressed as early as 4 days in vitro, before the formation of functional synaptic contacts. Western blots also showed that agrin-like proteins are secreted in conditioned medium from 7 days cultures. We analysed the subcellular distribution of agrin by double immunolabeling and fluorescence microscopy. We found that agrin is synthesized by almost all neurons and was ...
Super-resolution imaging of synapses has revealed that key synaptic proteins are dynamically orga... more Super-resolution imaging of synapses has revealed that key synaptic proteins are dynamically organized within sub-synaptic domains (SSDs). At mixed inhibitory synapses in spinal cord neurons, both GlyRs and GABAARs reside at the same post-synaptic density (PSD). To examine how the different inhibitory receptors are organized and regulated, we carried out dual-color direct stochastic optical reconstruction microscopy (dSTORM). We found that endogenous GlyRs and GABAARs as well as their common scaffold protein gephyrin form SSDs that align with pre-synaptic RIM1/2, thus forming trans-synaptic nanocolumns. Strikingly, GlyRs and GABAARs occupy different sub-synaptic spaces, exhibiting only a partial overlap at mixed inhibitory synapses. When network activity was increased by pharmacological treatment using the K+ channel blocker 4-aminopyridine (4-AP), the GABAAR copy numbers of as well as the number of GABAAR SSDs were reduced, while GlyRs remained largely unchanged. This differential ...
Proceedings of the National Academy of Sciences, 2021
Significance Learning depends on synaptic plasticity. The signaling mechanisms that control induc... more Significance Learning depends on synaptic plasticity. The signaling mechanisms that control induction of plasticity determine the learning rules at the specific synapse involved. Moreover, the relationship between the activity patterns of synaptic inputs and the type, direction, and level of plasticity induced may evolve during development. Here, we establish a key link between receptor activation presynaptic to cerebellar Purkinje cells, downstream signaling mechanisms, and the ability of adult animals to learn a cerebellar motor task.
Pyramidal neurons (PNs) are covered by thousands of dendritic spines receiving excitatory synapti... more Pyramidal neurons (PNs) are covered by thousands of dendritic spines receiving excitatory synaptic inputs. The ultrastructure of dendritic spines shapes signal compartmentalization, but ultrastructural diversity is rarely taken into account in computational models of synaptic integration. Here, we developed a 3D correlative light–electron microscopy (3D-CLEM) approach allowing the analysis of specific populations of synapses in genetically defined neuronal types in intact brain circuits. We used it to reconstruct segments of basal dendrites of layer 2/3 PNs of adult mouse somatosensory cortex and quantify spine ultrastructural diversity. We found that 10% of spines were dually innervated and 38% of inhibitory synapses localized to spines. Using our morphometric data to constrain a model of synaptic signal compartmentalization, we assessed the impact of spinous versus dendritic shaft inhibition. Our results indicate that spinous inhibition is locally more efficient than shaft inhibit...
ABSTRACTPyramidal neurons are covered by thousands of dendritic spines receiving excitatory synap... more ABSTRACTPyramidal neurons are covered by thousands of dendritic spines receiving excitatory synaptic inputs. The ultrastructure of dendritic spines shapes signal compartmentalization but ultrastructural diversity is rarely taken into account in computational models of synaptic integration. Here, we developed a 3D correlative light-electron microscopy (3D-CLEM) approach allowing the analysis of specific populations of synapses in genetically defined neuronal types in intact brain circuits. We used it to reconstruct segments of basal dendrites of layer 2/3 pyramidal neurons of adult mouse somatosensory cortex and quantify spine ultrastructural diversity. We found that 10% of spines were dually-innervated and 38% of inhibitory synapses localized to spines. Using our morphometric data to constrain a model of synaptic signal compartmentalization, we assessed the impact of spinous versus dendritic shaft inhibition. Our results indicate that spinous inhibition is locally more efficient tha...
Zinc has been reported to potentiate glycine receptors (GlyR), but the physiological significance... more Zinc has been reported to potentiate glycine receptors (GlyR), but the physiological significance of this observation has been put in doubt by the relatively high values of the EC50, 0.5–1 μM, since such concentrations may not be attained in the synaptic cleft of glycinergic synapses. We have re-evaluated this observation in the frame of the hypothesis that contaminant heavy metals present in usual solutions may have lead to underestimate the affinity of the zinc binding site, and therefore to underestimate the potential physiological role of zinc. Using chelators either to complex heavy metals or to apply zinc at controlled concentrations, we have examined the action of zinc on GlyR kinetics in outside-out patches from 50-h-old zebrafish Mauthner cells. Chelating contaminating heavy metals with tricine or N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) decreased the duration of the currents evoked by glycine, confirming that traces of heavy metals alter the GlyR respons...
In biological membranes, many factors such as cytoskeleton, lipid composition, crowding, and mole... more In biological membranes, many factors such as cytoskeleton, lipid composition, crowding, and molecular interactions deviate lateral diffusion from the expected random walks. These factors have different effects on diffusion but act simultaneously, so the observed diffusion is a complex mixture of diffusive behaviors (directed, Brownian, anomalous, or confined). Therefore, commonly used approaches to quantify diffusion based on averaging of the displacements such as the mean square displacement, are not adapted to the analysis of this heterogeneity. We introduce a parameter-the packing coefficient Pc, which gives an estimate of the degree of free movement that a molecule displays in a period of time independently of its global diffusivity. Applying this approach to two different situations (diffusion of a lipid probe and trapping of receptors at synapses), we show that Pc detected and localized temporary changes of diffusive behavior both in time and in space. More importantly, it al...
Alzheimer's disease (AD) is associated with a progressive loss of synapses and neurons. Studi... more Alzheimer's disease (AD) is associated with a progressive loss of synapses and neurons. Studies in animal models indicate that morphological alterations of dendritic spines precede synapse loss, increasing the proportion of large and short ("stubby") spines. Whether similar alterations occur in human patients, and what their functional consequences could be, is not known. We analyzed biopsies from AD patients and APP x presenilin 1 knock-in mice that were previously shown to present a loss of pyramidal neurons in the CA1 area of the hippocampus. We observed that the proportion of stubby spines and the width of spine necks are inversely correlated with synapse density in frontal cortical biopsies from non-AD and AD patients. In mice, the reduction in the density of synapses in the stratum radiatum was preceded by an alteration of spine morphology, with a reduction of their length and an enlargement of their neck. Serial sectioning examined with electron microscopy allow...
GABAergic synaptic transmission regulates brain function by establishing the appropriate excitati... more GABAergic synaptic transmission regulates brain function by establishing the appropriate excitation-inhibition (E/I) balance in neural circuits. The structure and function of GABAergic synapses are sensitive to destabilization by impinging neurotransmitters. However, signaling mechanisms that promote the restorative homeostatic stabilization of GABAergic synapses remain unknown. Here, by quantum dot single-particle tracking, we characterize a signaling pathway that promotes the stability of GABAA receptor (GABAAR) postsynaptic organization. Slow metabotropic glutamate receptor signaling activates IP3 receptor-dependent calcium release and protein kinase C to promote GABAAR clustering and GABAergic transmission. This GABAAR stabilization pathway counteracts the rapid cluster dispersion caused by glutamate-driven NMDA receptor-dependent calcium influx and calcineurin dephosphorylation, including in conditions of pathological glutamate toxicity. These findings show that glutamate activ...
Proceedings of the National Academy of Sciences, 1995
Simultaneous pre- and postsynaptic recordings from identified glycinergic inhibitory interneurons... more Simultaneous pre- and postsynaptic recordings from identified glycinergic inhibitory interneurons and the Mauthner cell showed that 25% of the afferents produced no or extremely small postsynaptic responses. Morphological determination of the number of contacts made by these cells on the Mauthner cell revealed a connectivity similar to that of functional neurons which always produce clear inhibitory postsynaptic potentials, suggesting that most of the endings, made by weak interneurons are silent. Intraaxonal injection of 4-aminopyridine or Ca2+ greatly enhanced transmission at functional connections but did not modify those which were ineffective. However, after eighth nerve tetanic stimuli, transmission at the weak connections was unmasked or enhanced for prolonged periods and was twice as likely to be potentiated, with a 6-fold greater mean enhancement than the potent ones. This result provides additional support for long-term potentiation of inhibitory synapses. Furthermore, wea...
Concentration of neurotransmitter receptors at synapses is thought to result from stable binding ... more Concentration of neurotransmitter receptors at synapses is thought to result from stable binding to subsynaptic scaffold proteins. Recent data on synaptic plasticity have shown that changes in synaptic strength derive partly from modification of postsynaptic receptor numbers. This has led to the notion of receptor trafficking into and out of synapses. The proposed underlying mechanisms have under-evaluated the role of extrasynaptic receptors. Recent technological advances have allowed imaging of receptor movements at the single-molecule level, and these experiments demonstrate that receptors switch at unexpected rates between extrasynaptic and synaptic localizations by lateral diffusion. Variation in receptor numbers at postsynaptic sites is therefore likely to depend on regulation of diffusion by modification of the structure of the membrane and/or by transient interactions with scaffolding proteins. This review is part of the TINS Synaptic Connectivity series.
INTRODUCTIONThis protocol describes a highly sensitive approach for tracking the motion of membra... more INTRODUCTIONThis protocol describes a highly sensitive approach for tracking the motion of membrane molecules over extended time periods with single-molecule resolution. This technique uses nanometer-sized quantum dots (QDs) linked to the extracellular part of the proteins to be followed. Single-fluorophore epifluorescence imaging then reveals the membrane diffusion of the particle of interest. Two methods are presented for labeling neurons with the primary antibody of choice along with a secondary anti-Fab antibody that is either biotinylated or directly coupled to the desired QD. The behavior of QD-labeled molecules can then be followed within the cell using epifluorescence imaging.
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