Neuroligin-3 confines AMPA receptors into nanoclusters, thereby controlling synaptic strength at the calyx of Held synapses.
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ABSTRACT:
SUBMITTER: Han Y
PROVIDER: S-EPMC9200272 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We examined the effect of glutamate transporter blockade at the calyx of Held synapse. In immature synapses [defined as postnatal day 8 (P8) to P10 rats], transporter blockade causes tonic activation of NMDA receptors and strong inhibition of the AMPA receptor-mediated EPSC amplitude. EPSC inhibition was blocked with a metabotropic glutamate receptor (mGluR) antagonist [1 microm LY341495 (2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3-(xanth-9-yl)propanoic acid)], suggesting that elevated resting glutamate concentration specifically activates group II and group III mGluRs. Using mGluR subtype-specific agonists and antagonists, we determined that increased glutamate activates presynaptic mGluR2/3 and mGluR8 receptors but not mGluR4, although this receptor is present. Surprisingly, in older animals (P16-P18), transporter blockade had no effect on EPSC amplitude because of a developmental downregulation of group II/III mGluR activation in rats and mice. In contrast to other CNS synapses, we observed no effect of transporter blockade on EPSC decay kinetics, although expression of glutamate transporters was strong in nearby glial processes at both P9 and P17. Finally, using a low-affinity AMPA receptor antagonist (gamma-D-glutamylglycine), we show that desensitization occurs at P8-P10 but is absent at P16-P18, even during trains of high-frequency (100-300 Hz) stimulation. We suggest that diffusion and transporter activation are insufficient to clear synaptically released glutamate at immature calyces, resulting in significant desensitization. Thus, mGluRs may be expressed in the immature calyx to help limit glutamate release. In the more mature calyx, there is a far smaller diffusional barrier attributable to the highly fenestrated synaptic terminal morphology, so AMPA receptor desensitization is avoided and mGluR-mediated inhibition is not necessary.
| S-EPMC3375655 | biostudies-literature
Project description:The alpha-amino-3-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) is an ionotropic receptor mediating excitatory synaptic transmission, but it can also interact with intracellular messengers. Here we report that, at the calyx of Held in the rat auditory brainstem, activation of AMPARs induced inward currents in the nerve terminal and inhibited presynaptic Ca2+ currents (I(pCa)), thereby attenuating glutamatergic synaptic transmission. The AMPAR-mediated I(pCa) inhibition was disinhibited by a strong depolarizing pulse and occluded by the nonhydrolyzable GTP analog GTPgammaS loaded into the terminal. We conclude that functional AMPARs are expressed at the calyx of Held nerve terminal and that their activation inhibits voltage-gated Ca2+ channels by an interaction with heterotrimeric GTP-binding proteins (G proteins). Thus, at a central glutamatergic synapse, presynaptic AMPARs have a metabotropic nature and regulate transmitter release by means of G proteins.
| S-EPMC1129093 | biostudies-literature
Project description:Neuroligins are postsynaptic cell-adhesion molecules that bind to presynaptic neurexins. Mutations in neuroligin-3 predispose to autism, but how such mutations affect synaptic function remains incompletely understood. Here we systematically examined the effect of three autism-associated mutations, the neuroligin-3 knockout, the R451C knockin, and the R704C knockin, on synaptic transmission in the calyx of Held, a central synapse ideally suited for high-resolution analyses of synaptic transmission. Surprisingly, germline knockout of neuroligin-3 did not alter synaptic transmission, whereas the neuroligin-3 R451C and R704C knockins decreased and increased, respectively, synaptic transmission. These puzzling results prompted us to ask whether neuroligin-3 mutant phenotypes may be reshaped by developmental plasticity. Indeed, conditional knockout of neuroligin-3 during late development produced a marked synaptic phenotype, whereas conditional knockout of neuroligin-3 during early development caused no detectable effect, mimicking the germline knockout. In canvassing potentially redundant candidate genes, we identified developmentally early expression of another synaptic neurexin ligand, cerebellin-1. Strikingly, developmentally early conditional knockout of cerebellin-1 only modestly impaired synaptic transmission, whereas in contrast to the individual single knockouts, developmentally early conditional double knockout of both cerebellin-1 and neuroligin-3 severely decreased synaptic transmission. Our data suggest an unanticipated mechanism of developmental compensation whereby cerebellin-1 and neuroligin-3 functionally occlude each other during development of calyx synapses. Thus, although acute manipulations more likely reveal basic gene functions, developmental plasticity can be a major factor in shaping the overall phenotypes of genetic neuropsychiatric disorders.
| S-EPMC5687809 | biostudies-literature
Project description:The calyx of Held, a large axo-somatic relay synapse containing hundreds of presynaptic active zones, is possibly the largest nerve terminal in the mammalian CNS. Studying its initial growth in-vitro might provide insights into the specification of synaptic connection size in the developing brain. However, attempts to maintain calyces of Held in organotypic cultures have not been fruitful in past studies. Here, we describe an organotypic slice culture method in which calyces of Held form in-vitro. We made coronal brainstem slices with an optimized slice angle using newborn mice in which calyces have not yet formed; the presynaptic bushy cells were genetically labeled using the Math5 promoter. After six to nine days of culturing, we readily observed large Math5-positive nerve terminals in the medial nucleus of the trapezoid body (MNTB), but not in the neighboring lateral superior olive nucleus (LSO). These calyx-like synapses expressed the Ca2+- sensor Synaptotagmin-2 (Syt-2) and the Ca2+ binding protein Parvalbumin (PV), two markers of developing calyces of Held in vivo. Application of the BMP inhibitor LDN-193189 significantly inhibited the growth of calyx synapses, demonstrating the feasibility of long-term pharmacological manipulation using this organotypic culture method. These experiments provide a method for organotypic culturing of calyces of Held, and show that the formation of calyx-like synapses onto MNTB neurons can be preserved in-vitro. Furthermore, our study adds pharmacological evidence for a role of BMP-signaling in the formation of large calyx of Held synapses.
| S-EPMC5395213 | biostudies-literature
Project description:Efficient endocytosis is crucial for maintaining synaptic transmission because of its role in retrieving constituent membrane and associated proteins. In the past three decades three modes of endocytosis have been proposed involving the central nervous system: clathrin-mediated endocytosis, kiss-and-run endocytosis and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. Here, we review the existence and initiation of all three modes of endocytosis at a giant glutamatergic synapse, the calyx of Held. The possibility of direct electrophysiology recording in this synapse allows for accurate tracking of exocytosis and endocytosis via capacitance measurements. Future aims will be focused on identifying the molecules that undergo the different mechanisms of endocytosis and the conditions under which different forms of endocytosis predominate.
| S-EPMC4002768 | biostudies-other
Project description:Vesicle recycling is pivotal for maintaining reliable synaptic signaling, but its basic properties remain poorly understood. Here, we developed an approach to quantitatively analyze the kinetics of vesicle recycling with exquisite signal and temporal resolution at the calyx of Held synapse. The combination of this electrophysiological approach with electron microscopy revealed that ?80% of vesicles (?270,000 out of ?330,000) in the nerve terminal are involved in recycling. Under sustained stimulation, recycled vesicles start to be reused in tens of seconds when ?47% of the preserved vesicles in the recycling pool (RP) are depleted. The heterogeneity of vesicle recycling as well as two kinetic components of RP depletion revealed the existence of a replenishable pool of vesicles before the priming stage and led to a realistic kinetic model that assesses the size of the subpools of the RP. Thus, our study quantified the kinetics of vesicle recycling and kinetically dissected the whole vesicle pool in the calyceal terminal into the readily releasable pool (?0.6%), the readily priming pool (?46%), the premature pool (?33%), and the resting pool (?20%).
| S-EPMC4403166 | biostudies-literature
Project description:The calyx of Held, a large glutamatergic terminal in the mammalian auditory brainstem has been extensively employed to study presynaptic structure and function in the central nervous system. Nevertheless, the nanoarchitecture of presynaptic proteins and subcellular components in the calyx terminal and its relation to functional properties of synaptic transmission is only poorly understood. Here, we use stimulated emission depletion (STED) nanoscopy of calyces in thin sections of aldehyde-fixed rat brain tissue to visualize immuno-labeled synaptic proteins including VGluT1, synaptophysin, Rab3A and synapsin with a lateral resolution of approximately 40 nm. Excitation multiplexing of suitable fluorescent dyes deciphered the spatial arrangement of the presynaptic phospho-protein synapsin relative to synaptic vesicles labeled with anti-VGluT1. Both predominantly occupied the same focal volume, yet may exist in exclusive domains containing either VGluT1 or synapsin immunoreactivity. While the latter have been observed with diffraction-limited fluorescence microscopy, STED microscopy for the first time revealed VGluT1-positive domains lacking synapsins. This observation supports the hypothesis that molecularly and structurally distinct synaptic vesicle pools operate in presynaptic nerve terminals.
| S-EPMC3637247 | biostudies-literature
Project description:Key pointsDuring development the giant, auditory calyx of Held forms a one-to-one connection with a principal neuron of the medial nucleus of the trapezoid body. While anatomical studies described that most of the target cells are temporarily contacted by multiple calyces, multi-calyceal innervation was only sporadically observed in in vivo recordings, suggesting a structure-function discrepancy. We correlated synaptic strength of inputs, identified in in vivo recordings, with post hoc labelling of the recorded neuron and synaptic terminals containing vesicular glutamate transporters (VGluT). During development only one input increased to the level of the calyx of Held synapse, and its strength correlated with the large VGluT cluster contacting the postsynaptic soma. As neither competing strong inputs nor multiple large VGluT clusters on a single cell were observed, our findings did not indicate a structure-function discrepancy.AbstractIn adult rodents, a principal neuron in the medial nucleus of the trapezoid (MNTB) is generally contacted by a single, giant axosomatic terminal called the calyx of Held. How this one-on-one relation is established is still unknown, but anatomical evidence suggests that during development principal neurons are innervated by multiple calyces, which may indicate calyceal competition. However, in vivo electrophysiological recordings from principal neurons indicated that only a single strong synaptic connection forms per cell. To test whether a mismatch exists between synaptic strength and terminal size, we compared the strength of synaptic inputs with the morphology of the synaptic terminals. In vivo whole-cell recordings of the MNTB neurons from newborn Wistar rats of either sex were made while stimulating their afferent axons, allowing us to identify multiple inputs. The strength of the strongest input increased to calyceal levels in a few days across cells, while the strength of the second strongest input was stable. The recorded cells were subsequently immunolabelled for vesicular glutamate transporters (VGluT) to reveal axosomatic terminals with structured-illumination microscopy. Synaptic strength of the strongest input was correlated with the contact area of the largest VGluT cluster at the soma (r = 0.8), and no indication of a mismatch between structure and strength was observed. Together, our data agree with a developmental scheme in which one input strengthens and becomes the calyx of Held, but not with multi-calyceal competition.
| S-EPMC7689866 | biostudies-literature
Project description:Synaptic vesicle (SV) exocytosis is intimately dependent on free local Ca2+ near active zones. Genetically encoded calcium indicators (GECIs) have become an indispensable tool to monitor calcium dynamics during physiological responses, and they are widely used as a proxy to monitor activity in neuronal ensembles and at synaptic terminals. However, GECIs' ability to bind Ca2+ at physiologically relevant concentration makes them strong candidates to affect calcium homeostasis and alter synaptic transmission by exogenously increasing Ca2+ buffering. In the present study, we show that genetically expressed GCaMP6m modulates SV release probability at the mouse calyx of Held synapse. GCaMP6m expression for approximately three weeks decreased initial SV release for both low-frequency stimulation and high-frequency stimulation trains, and slowed presynaptic short-term depression. However, GCaMP6m does not affect quantal events during spontaneous activity at this synapse. This study emphasizes the careful use of GECIs as monitors of neuronal activity and inspects the role of these transgenic indicators which may alter calcium-dependent physiological responses.
| S-EPMC6186185 | biostudies-literature
Project description:Neuroligins enhance synapse formation in vitro, but surprisingly are not required for the generation of synapses in vivo. We now show that in cultured neurons, neuroligin-1 overexpression increases excitatory, but not inhibitory, synaptic responses, and potentiates synaptic NMDAR/AMPAR ratios. In contrast, neuroligin-2 overexpression increases inhibitory, but not excitatory, synaptic responses. Accordingly, deletion of neuroligin-1 in knockout mice selectively decreases the NMDAR/AMPAR ratio, whereas deletion of neuroligin-2 selectively decreases inhibitory synaptic responses. Strikingly, chronic inhibition of NMDARs or CaM-Kinase II, which signals downstream of NMDARs, suppresses the synapse-boosting activity of neuroligin-1, whereas chronic inhibition of general synaptic activity suppresses the synapse-boosting activity of neuroligin-2. Taken together, these data indicate that neuroligins do not establish, but specify and validate, synapses via an activity-dependent mechanism, with different neuroligins acting on distinct types of synapses. This hypothesis reconciles the overexpression and knockout phenotypes and suggests that neuroligins contribute to the use-dependent formation of neural circuits.
| S-EPMC3738748 | biostudies-literature