Oligodendrocytes secrete vesicles into the extracellular space, where they might play a role in neuron–glia communication. These exosomes are small vesicles with a diameter of 50–100 nm that are formed within multivesicular bodies and are released after fusion with the plasma membrane. The intracellular pathways that generate exosomes are poorly defined. Because Rab family guanosine triphosphatases (GTPases) together with their regulators are important membrane trafficking organizers, we investigated which Rab GTPase-activating proteins interfere with exosome release. We find that TBC1D10A–C regulate exosome secretion in a catalytic activity–dependent manner. We show that Rab35 is the target of TBC1D10A–C and that the inhibition of Rab35 function leads to intracellular accumulation of endosomal vesicles and impairs exosome secretion. Rab35 localizes to the surface of oligodendroglia in a GTP-dependent manner, where it increases the density of vesicles, suggesting a function in docking or tethering. These findings provide a basis for understanding the biogenesis and function of exosomes in the central nervous system.
Optogenetics revolutionizes basic research in neuroscience and cell biology and bears potential for medical applications. We develop mutants leading to a unifying concept for the construction of various channelrhodopsins with fast closing kinetics. Due to different absorption maxima these channelrhodopsins allow fast neural photoactivation over the whole range of the visible spectrum. We focus our functional analysis on the fast-switching, red light-activated Chrimson variants, because red light has lower light scattering and marginal phototoxicity in tissues. We show paradigmatically for neurons of the cerebral cortex and the auditory nerve that the fast Chrimson mutants enable neural stimulation with firing frequencies of several hundred Hz. They drive spiking at high rates and temporal fidelity with low thresholds for stimulus intensity and duration. Optical cochlear implants restore auditory nerve activity in deaf mice. This demonstrates that the mutants facilitate neuroscience research and future medical applications such as hearing restoration.
For sounds of a given frequency, spiral ganglion neurons (SGNs) with different thresholds and dynamic ranges collectively encode the wide range of audible sound pressures. Heterogeneity of synapses between inner hair cells (IHCs) and SGNs is an attractive candidate mechanism for generating complementary neural codes covering the entire dynamic range. Here, we quantified active zone (AZ) properties as a function of AZ position within mouse IHCs by combining patch clamp and imaging of presynaptic Ca 2+ influx and by immunohistochemistry. We report substantial AZ heterogeneity whereby the voltage of half-maximal activation of Ca 2+ influx ranged over ∼20 mV. Ca 2+ influx at AZs facing away from the ganglion activated at weaker depolarizations. Estimates of AZ size and Ca 2+ channel number were correlated and larger when AZs faced the ganglion. Disruption of the deafness gene GIPC3 in mice shifted the activation of presynaptic Ca 2+ influx to more hyperpolarized potentials and increased the spontaneous SGN discharge. Moreover, Gipc3 disruption enhanced Ca 2+ influx and exocytosis in IHCs, reversed the spatial gradient of maximal Ca 2+ influx in IHCs, and increased the maximal firing rate of SGNs at sound onset. We propose that IHCs diversify Ca 2+ channel properties among AZs and thereby contribute to decomposing auditory information into complementary representations in SGNs.auditory system | spiral ganglion neuron | dynamic range | synaptic strength | presynaptic heterogeneity T he auditory system enables us to perceive sound pressures that vary over six orders of magnitude. This is achieved by active amplification of cochlear vibrations at low sound pressures and compression at high sound pressures. The receptor potential of inner hair cells (IHCs) represents the full range (1), whereas each postsynaptic type I spiral ganglion neuron (hereafter termed SGN) encodes only a fraction (2-6). SGNs with comparable frequency tuning but different spontaneous spike rates and sound responses are thought to emanate from neighboring, if not the same, IHC at a given tonotopic position of the organ of Corti (2,5,7,8). Even in silence, IHC active zones (AZs) release glutamate at varying rates, evoking "spontaneous" spiking in SGNs. SGNs with greater spontaneous spike rates respond to softer sounds (highspontaneous rate, low-threshold SGNs), than those with lower spontaneous spike rates (low-spontaneous rate, high-threshold SGNs) (2, 9). This diversity likely underlies the representation of sounds across all audible sound pressure levels in the auditory nerve, to which neural adaptation also contributes (10).How SGN diversity arises is poorly understood. Candidate mechanisms include the heterogeneity of ribbon synapses that differ in pre-and/or postsynaptic properties even within individual IHCs (7,(11)(12)(13)(14). IHC AZs vary in the number (11, 15) and voltage dependence of gating (11) of their Ca 2+ channels regardless of tonotopic position (16). Lateral olivocochlear efferent projections to the SGNs regulate postsynaptic exc...
During CNS development, oligodendrocytes wrap their plasma membrane around axons to generate multilamellar myelin sheaths. To drive growth at the leading edge of myelin at the interface with the axon, mechanical forces are necessary, but the underlying mechanisms are not known. Using an interdisciplinary approach that combines morphological, genetic, and biophysical analyses, we identified a key role for actin filament network turnover in myelin growth. At the onset of myelin biogenesis, F-actin is redistributed to the leading edge, where its polymerization-based forces push out non-adhesive and motile protrusions. F-actin disassembly converts protrusions into sheets by reducing surface tension and in turn inducing membrane spreading and adhesion. We identified the actin depolymerizing factor ADF/cofilin1, which mediates high F-actin turnover rates, as an essential factor in this process. We propose that F-actin turnover is the driving force in myelin wrapping by regulating repetitive cycles of leading edge protrusion and spreading.
We studied the role of the synaptic ribbon for sound encoding at the synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) in mice lacking RIBEYE (RBEKO/KO). Electron and immunofluorescence microscopy revealed a lack of synaptic ribbons and an assembly of several small active zones (AZs) at each synaptic contact. Spontaneous and sound-evoked firing rates of SGNs and their compound action potential were reduced, indicating impaired transmission at ribbonless IHC-SGN synapses. The temporal precision of sound encoding was impaired and the recovery of SGN-firing from adaptation indicated slowed synaptic vesicle (SV) replenishment. Activation of Ca2+-channels was shifted to more depolarized potentials and exocytosis was reduced for weak depolarizations. Presynaptic Ca2+-signals showed a broader spread, compatible with the altered Ca2+-channel clustering observed by super-resolution immunofluorescence microscopy. We postulate that RIBEYE disruption is partially compensated by multi-AZ organization. The remaining synaptic deficit indicates ribbon function in SV-replenishment and Ca2+-channel regulation.
Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2l (AP-2l) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2l slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane-proximal vesicles and intact endocytic membrane retrieval. Sound-driven postsynaptic spiking was reduced in a use-dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosomelike vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2l-deficient IHCs, indicating a further role of AP-2l in clathrin-dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP-2 sorts its IHC-cargo otoferlin. We propose that binding of AP-2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP-2 in synaptic vesicle reformation.
Ca2+ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)-interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated Ca V 1.3 Ca 2+ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca 2+ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic Ca V 1.3 Ca 2+ channels, resulting in reduced synaptic Ca 2+ influx. Using superresolution microscopy, we found that Ca 2+ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca 2+ current, whereas the apparent Ca 2+ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca 2+ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca 2+ channels at IHC AZs and are required for normal hearing.ens of Ca V 1.3 Ca 2+ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1-4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5-7). The mechanisms governing the number of Ca 2+ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca 2+ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca 2+ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1-RIM4). All isoforms contain a C-terminal C 2 domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca 2+ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca V β) subunits (12, 13) and poreforming Ca V α subun...
Neuroligin-1 is a potent trigger for the de novo formation of synaptic connections, and it has recently been suggested that it is required for the maturation of functionally competent excitatory synapses. Despite evidence for the role of neuroligin-1 in specifying excitatory synapses, the underlying molecular mechanisms and physiological consequences that neuroligin-1 may have at mature synapses of normal adult animals remain unknown. By silencing endogenous neuroligin-1 acutely in the amygdala of live behaving animals, we have found that neuroligin-1 is required for the storage of associative fear memory. Subsequent cellular physiological studies showed that suppression of neuroligin-1 reduces NMDA receptor-mediated currents and prevents the expression of long-term potentiation without affecting basal synaptic connectivity at the thalamo-amygdala pathway. These results indicate that persistent expression of neuroligin-1 is required for the maintenance of NMDAR-mediated synaptic transmission, which enables normal development of synaptic plasticity and long-term memory in the amygdala of adult animals.synaptic plasticity ͉ neuroligin ͉ autism S everal studies have found that synaptically localized cell adhesion molecules not only trigger synapse formation but also play a major role in regulating both basal synaptic transmission and synaptic plasticity (1, 2). Among them, neurexins and neuroligins (NLs), which undergo a heterophilic interaction with each other, have emerged as important organizers of de novo synapse formation (3). Moreover, modifying the interaction of neuroligin-1 and PSD-95 alters the balance of neuronal excitation and inhibition required for normal brain function (4). The indispensable role of neuroligins for proper neuronal connectivity is further supported by the genetic linkage of neuroligin mutations with autism, a disease that is thought to be a disorder in social cognition that critically involves the amygdala (5, 6).Because neuroligins are present both during development and throughout adulthood (7,8), it is likely that neuroligins play roles other than that of an inducer of synaptogenesis in the adult brain. Indeed, a recent study of knockout (KO) mice deficient in neuroligin-1 demonstrated that neuroligin-1 regulates excitatory synaptic responses (9). Although neuroligin-1 has been suggested to be essential for maintaining normal N-methyl-D-aspartate (NMDA)-type glutamate receptor-mediated currents (9), the underlying mechanism and its physiological consequence remain to be identified. Furthermore, because the regulation of NMDA receptor (NMDAR) is critical for long-term synaptic modification (10), alterations of NMDAR-dependent currents regulated by neuroligin-1 are likely to have effects on synaptic plasticity and long-term memory in adult animals.To address the functional role of neuroligin-1 at existing mature synapses, we used virus-mediated RNA interference to deplete endogenous neuroligin-1 in the lateral nucleus of the amygdala (LA) of adult animals. We investigated the actions...
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