RESUMEN

Synaptotagmin-9 (Syt9) is a Ca2+ sensor mediating fast synaptic release expressed in various parts of the brain. The presence and role of Syt9 in retina is unknown. We found evidence for Syt9 expression throughout the retina and created mice to conditionally eliminate Syt9 in a cre-dependent manner. We crossed Syt9fl/fl mice with Rho-iCre, HRGP-Cre, and CMV-Cre mice to generate mice in which Syt9 was eliminated from rods (rodSyt9CKO), cones (coneSyt9CKO), or whole animals (CMVSyt9). CMVSyt9 mice showed an increase in scotopic electroretinogram (ERG) b-waves evoked by bright flashes with no change in a-waves. Cone-driven photopic ERG b-waves were not significantly different in CMVSyt9 knockout mice and selective elimination of Syt9 from cones had no effect on ERGs. However, selective elimination from rods decreased scotopic and photopic b-waves as well as oscillatory potentials. These changes occurred only with bright flashes where cone responses contribute. Synaptic release was measured in individual rods by recording anion currents activated by glutamate binding to presynaptic glutamate transporters. Loss of Syt9 from rods had no effect on spontaneous or depolarization-evoked release. Our data show that Syt9 acts at multiple sites in the retina and suggest that it may play a role in regulating transmission of cone signals by rods.

Asunto(s)

RESUMEN

Optomotor responses are a popular way to assess sub-cortical visual responses in mice. We studied photoreceptor inputs into optomotor circuits using genetically-modified mice lacking the exocytotic calcium sensors synaptotagmin 1 (Syt1) and 7 (Syt7) in rods or cones. We also tested mice that in which cone transducin, GNAT2, had been eliminated. We studied spatial frequency sensitivity under mesopic conditions by varying the spatial frequency of a grating rotating at 12 deg/s and contrast sensitivity by varying luminance contrast of 0.2c/deg gratings. We found that eliminating Syt1 from rods reduced responses to a low spatial frequency grating (0.05c/deg) consistent with low resolution in this pathway. Conversely, eliminating the ability of cones to respond to light (by eliminating GNAT2) or transmit light responses (by selectively eliminating Syt1) showed weaker responses to a high spatial frequency grating (3c/deg). Eliminating Syt7 from the entire optomotor pathway in a global knockout had no significant effect on optomotor responses. We isolated the secondary rod pathway involving transmission of rod responses to cones via gap junctions by simultaneously eliminating Syt1 from rods and GNAT2 from cones. We found that the secondary rod pathway is sufficient to drive robust optomotor responses under mesopic conditions. Finally, eliminating Syt1 from both rods and cones almost completely abolished optomotor responses, but we detected weak responses to large, bright rotating gratings that are likely driven by input from intrinsically photosensitive retinal ganglion cells.

RESUMEN

With the development of the social economy, we are exposed to increasing noise in our daily lives. Our previous work found an ABCC1(NM_004996.3:c.A1769G, NP_004987.2:p.N590S) variant which cosegregated with the patients in an autosomal dominant non-syndromic hearing loss family. At present, the specific mechanism of deafness caused by ABCC1 mutation is still not clear. Using the knock-in mouse model simulating human ABCC1 mutation, we found that the occurrence of family-related phenotypes was likely attributed to the combination of the mouse genotype and low-intensity noise. GSH and GSSG are important physiological substrates of ABCC1. The destruction of GSH-GSSG balance in the cochleae of both Abcc1N591S/+ mice and Abcc1N591S/N591S mice during low-intensity noise exposure may result in irreversible damage to the hair cells of the cochleae, consequently leading to hearing loss in mice. The findings offered a potential novel idea for the prevention and management of hereditary hearing loss within this family.

Asunto(s)

RESUMEN

Deafness is the prevailing sensory impairment among humans, impacting every aspect of one's existence. Half of congenital deafness cases are attributed to genetic factors. Studies have shown that Luzp2 is expressed in hair cells (HCs) and supporting cells of the inner ear, but its specific role in hearing remains unclear. To determine the importance of Luzp2 in auditory function, we generated mice deficient in Luzp2. Our results revealed that Luzp2 has predominant expression within the HCs and pillar cells. However, the loss of Luzp2 did not result in any changes in auditory threshold. HCs or synapse number and HC stereocilia morphology in Luzp2 knockout mice did not show any notable distinctions. This was the first study of the role of Luzp2 in hearing in mice, and our results provide important guidance for the screening of deafness genes.

RESUMEN

Ribbon synapses in the cochlear hair cells are subject to extensive pruning and maturation processes before hearing onset. Previous studies have highlighted the pivotal role of thyroid hormone (TH) in this developmental process, yet the detailed mechanisms are largely unknown. In this study, we found that the thyroid hormone receptor α (Thrα) is expressed in both sensory epithelium and spiral ganglion neurons in mice. Hypothyroidism, induced by Pax8 gene knockout, significantly delays the synaptic pruning during postnatal development in mice. Detailed spatiotemporal analysis of ribbon synapse distribution reveals that synaptic maturation involves not only ribbon pruning but also their migration, both of which are notably delayed in the cochlea of Pax8 knockout mice. Intriguingly, postnatal hyperthyroidism, induced by intraperitoneal injections of liothyronine sodium (T3), accelerates the pruning of ribbon synapses to the mature state without affecting the auditory functions. Our findings suggest that thyroid hormone does not play a deterministic role but rather controls the timing of cochlear ribbon synapse maturation.

Asunto(s)

RESUMEN

We generated a novel Cre mouse strain for cell-specific deletion of floxed genes in ribbon synapse-forming retinal neurons. Previous studies have shown that the RIBEYE promotor targets the expression of recombinant proteins such as fluorescently tagged RIBEYE to photoreceptors and retinal bipolar cells and generates fluorescent synaptic ribbons in situ in these neurons. Here, we used the same promotor to generate a novel transgenic mouse strain in which the RIBEYE promotor controls the expression of a Cre-ER(T2) recombinase (RIBEYE-Cre). To visualize Cre expression, the RIBEYE-Cre animals were crossed with ROSA26 tau-GFP (R26-τGFP) reporter mice. In the resulting RIBEYE-Cre/R26 τGFP animals, Cre-mediated removal of a transcriptional STOP cassette results in the expression of green fluorescent tau protein (tau-GFP) that binds to cellular microtubules. We detected robust tau-GFP expression in retinal bipolar cells. Surprisingly, we did not find fluorescent tau-GFP expression in mouse photoreceptors. The lack of tau-GFP reporter protein in these cells could be based on the previously reported absence of tau protein in mouse photoreceptors which could lead to the degradation of the recombinant tau protein. Consistent with this, we detected Cre and tau-GFP mRNA in mouse photoreceptor slices by RT-PCR. The transgenic RIBEYE-Cre mouse strain provides a new tool to study the deletion of floxed genes in ribbon synapse-forming neurons of the retina and will also allow for analyzing gene deletions that are lethal if globally deleted in neurons.

Asunto(s)

RESUMEN

In congenital stationary night blindness type 2 (CSNB2)-a disorder involving the Cav1.4 (L-type) Ca2+ channel-visual impairment is mild considering that Cav1.4 mediates synaptic release from rod and cone photoreceptors. Here, we addressed this conundrum using a Cav1.4 knockout (KO) mouse and a knock-in (G369i KI) mouse expressing a non-conducting Cav1.4. Surprisingly, Cav3 (T-type) Ca2+ currents were detected in cones of G369i KI mice and Cav1.4 KO mice but not in cones of wild-type mouse, ground squirrel, and macaque retina. Whereas Cav1.4 KO mice are blind, G369i KI mice exhibit normal photopic (i.e., cone-mediated) visual behavior. Cone synapses, which fail to form in Cav1.4 KO mice, are present, albeit enlarged, and with some errors in postsynaptic wiring in G369i KI mice. While Cav1.4 KO mice lack evidence of cone synaptic responses, electrophysiological recordings in G369i KI mice revealed nominal transmission from cones to horizontal cells and bipolar cells. In CSNB2, we propose that Cav3 channels maintain cone synaptic output provided that the nonconducting role of Cav1.4 in cone synaptogenesis remains intact. Our findings reveal an unexpected form of homeostatic plasticity that relies on a non-canonical role of an ion channel.

RESUMEN

Afferent synapses of cochlear inner hair cells (IHCs) employ a unique molecular machinery. Otoferlin is a key player in this machinery, and its genetic defects cause human auditory synaptopathy. We employed site-directed mutagenesis in mice to investigate the role of Ca2+ binding to the C2F domain of otoferlin. Substituting two aspartate residues of the C2F top loops, which are thought to coordinate Ca2+-ions, by alanines (OtofD1841/1842A) abolished Ca2+-influx-triggered IHC exocytosis and synchronous signaling in the auditory pathway despite substantial expression (~60%) of the mutant otoferlin in the basolateral IHC pole. Ca2+ influx of IHCs and their resting membrane capacitance, reflecting IHC size, as well as the number of IHC synapses were maintained. The mutant otoferlin showed a strong apex-to-base abundance gradient in IHCs, suggesting impaired protein targeting. Our results indicate a role of the C2F domain in otoferlin targeting and of Ca2+ binding by the C2F domain for IHC exocytosis and hearing.

RESUMEN

Introduction: Rod bipolar cells (RBCs) faithfully transmit light-driven signals from rod photoreceptors in the outer retina to third order neurons in the inner retina. Recently, significant work has focused on the role of leucine-rich repeat (LRR) proteins in synaptic development and signal transduction at RBC synapses. We previously identified trophoblast glycoprotein (TPBG) as a novel transmembrane LRR protein localized to the dendrites and axon terminals of RBCs. Methods: We examined the effects on RBC physiology and retinal processing of TPBG genetic knockout in mice using immunofluorescence and electron microscopy, electroretinogram recording, patch-clamp electrophysiology, and time-resolved membrane capacitance measurements. Results: The scotopic electroretinogram showed a modest increase in the b-wave and a marked attenuation in oscillatory potentials in the TPBG knockout. No effect of TPBG knockout was observed on the RBC dendritic morphology, TRPM1 currents, or RBC excitability. Because scotopic oscillatory potentials primarily reflect RBC-driven rhythmic activity of the inner retina, we investigated the contribution of TPBG to downstream transmission from RBCs to third-order neurons. Using electron microscopy, we found shorter synaptic ribbons in TPBG knockout axon terminals in RBCs. Time-resolved capacitance measurements indicated that TPBG knockout reduces synaptic vesicle exocytosis and subsequent GABAergic reciprocal feedback without altering voltage-gated Ca2+ currents. Discussion: TPBG is required for normal synaptic ribbon development and efficient neurotransmitter release from RBCs to downstream cells. Our results highlight a novel synaptic role for TPBG at RBC ribbon synapses and support further examination into the mechanisms by which TPBG regulates RBC physiology and circuit function.

RESUMEN

Retinal ribbon synapses undergo functional changes after eye opening that remain uncharacterized. Using light-flash stimulation and paired patch-clamp recordings, we examined the maturation of the ribbon synapse between rod bipolar cells (RBCs) and AII-amacrine cells (AII-ACs) after eye opening (postnatal day 14) in the mouse retina at near physiological temperatures. We find that light-evoked excitatory postsynaptic currents (EPSCs) in AII-ACs exhibit a slow sustained component that increases in magnitude with advancing age, whereas a fast transient component remains unchanged. Similarly, paired recordings reveal a dual-component EPSC with a slower sustained component that increases during development, even though the miniature EPSC (mEPSC) amplitude and kinetics do not change significantly. We thus propose that the readily releasable pool of vesicles from RBCs increases after eye opening, and we estimate that a short light flash can evoke the release of ∼4,000 vesicles onto a single mature AII-AC.

Asunto(s)

RESUMEN

Computer models of the individual components of the peripheral auditory system - the outer, middle, and inner ears and the auditory nerve - have been developed in the past, with varying level of detail, breadth, and faithfulness of the underlying parameters. Building on previous work, we advance the modeling of the ear by presenting a complete, physiologically justified, bottom-up computer model based on up-to-date experimental data that integrates all of these parts together seamlessly. The detailed bottom-up design of the present model allows for the investigation of partial hearing mechanisms and their defects, including genetic, molecular, and microscopic factors. Also, thanks to the completeness of the model, one can study microscopic effects in the context of their implications on hearing as a whole, enabling the correlation with neural recordings and non-invasive psychoacoustic methods. Such a model is instrumental for advancing quantitative understanding of the mechanism of hearing, for investigating various forms of hearing impairment, as well as for devising next generation hearing aids and cochlear implants.

Asunto(s)

RESUMEN

Vision is our primary sense, and maintaining it throughout our lifespan is crucial for our well-being. However, the retina, which initiates vision, suffers from an age-related, irreversible functional decline. What causes this functional decline, and how it might be treated, is still unclear. Synapses are the functional hub for signal transmission between neurons, and studies have shown that aging is widely associated with synaptic dysfunction. In this study, we examined the first synapse of the visual system - the rod and cone photoreceptor ribbon synapse - in the mouse retina using light and electron microscopy at 2-3 months, ~1 year, and >2 years of age. We asked, whether age-related changes in key synaptic components might be a driver of synaptic dysfunction and ultimately age-related functional decline during normal aging. We found sprouting of horizontal and bipolar cells, formation of ectopic photoreceptor ribbon synapses, and a decrease in the number of rod photoreceptors and photoreceptor ribbon synapses in the aged retina. However, the majority of the photoreceptors did not show obvious changes in the structural components and protein composition of their ribbon synapses. Noteworthy is the increase in mitochondrial size in rod photoreceptor terminals in the aged retina.

RESUMEN

Retinal neurons that form ribbon-style synapses operate over a wide dynamic range, continuously relaying visual information to their downstream targets. The remarkable signaling abilities of these neurons are supported by specialized presynaptic machinery, one component of which is syntaxin3B. Syntaxin3B is an essential t-SNARE protein of photoreceptors and bipolar cells that is required for neurotransmitter release. It has a light-regulated phosphorylation site in its N-terminal domain at T14 that has been proposed to modulate membrane fusion. However, a direct test of the latter has been lacking. Using a well-controlled in vitro fusion assay, we found that a phosphomimetic T14 syntaxin3B mutation leads to a small but significant enhancement of SNARE-mediated membrane fusion following the formation of the t-SNARE complex. While the addition of Munc18a had only a minimal effect on membrane fusion mediated by SNARE complexes containing wild-type syntaxin3B, a more significant enhancement was observed in the presence of Munc18a when the SNARE complexes contained a syntaxin3B T14 phosphomimetic mutant. Finally, we showed that the retinal-specific complexins (Cpx III and Cpx IV) inhibited membrane fusion mediated by syntaxin3B-containing SNARE complexes in a dose-dependent manner. Collectively, our results establish that membrane fusion mediated by syntaxin3B-containing SNARE complexes is regulated by the T14 residue of syntaxin3B, Munc18a, and Cpxs III and IV.

Asunto(s)

RESUMEN

Considerable evidence of reactive oxygen species (ROS) involvement in cochlear hair cell (HC) loss, leading to acquired sensorineural hearing loss (SNHL), were reported. Cochlear synaptopathy between HCs and spiral ganglion neurons has been gathering attention as a cochlear HC loss precursor not detectable by normal auditory evaluation. However, the molecular mechanisms linking ROS with HC loss, as well as the relationship between ROS and cochlear synaptopathy have not been elucidated. Here, we examined these linkages using NOX4-TG mice, which constitutively produce ROS without stimulation. mRNA levels of Piccolo 1, a major component of the synaptic ribbon (a specialized structure surrounded by synaptic vesicles in HCs), were decreased in postnatal day 6 NOX4-TG mice cochleae compared to those in WT mice; they were also decreased by noise exposure in 2-week-old WT cochleae. As noise exposure induces ROS production, this suggests that the synaptic ribbon is a target of ROS. The level of CtBP2, another synaptic ribbon component, was significantly lower in NOX4-TG cochleae of 1-month-old and 4-month-old mice compared to that in WT mice, although no significant differences were noted at 1.5- and 2-months. The decrease in CtBP2 plateaued in 4-month-old NOX4-TG, while it gradually decreased from 1 to 6 months in WT mice. Furthermore, CtBP2 level in 2-month-old NOX4-TG mice decreased significantly after exposure to cisplatin and noise compared to that in WT mice. These findings suggest that ROS lead to developmental delays and early degeneration of synaptic ribbons, which could be potential targets for novel therapeutics for ROS-induced SNHL.

Asunto(s)

RESUMEN

Neural sound encoding in the mammalian cochlea faces the challenge of representing audible sound pressures that vary over six orders of magnitude. The cochlea meets this demand through the use of active micromechanics as well as the diversity and adaptation of afferent neurons and their synapses. Mechanisms underlying neural diversity likely include heterogeneous presynaptic input from inner hair cells (IHCs) to spiral ganglion neurons (SGNs) as well as differences in the molecular profile of SGNs and in their efferent control. Here, we tested whether glutamate release from IHCs, previously found to be critical for maintaining different molecular SGN profiles, is required for establishing heterogeneity of active zones (AZs) in IHCs. We analyzed structural and functional heterogeneity of IHC AZs in mouse mutants with disrupted glutamate release from IHCs due to lack of a vesicular glutamate transporter (Vglut3) or impaired exocytosis due to defective otoferlin. We found the variance of the voltage-dependence of presynaptic Ca2+ influx to be reduced in exocytosis-deficient IHCs of otoferlin mutants. Yet, the spatial gradients of maximal amplitude and voltage-dependence of Ca2+ influx along the pillar-modiolar IHC axis were maintained in both mutants. Further immunohistochemical analysis showed an intact spatial gradient of ribbon size in Vglut3-/- mice. These results indicate that IHC exocytosis and glutamate release are not strictly required for establishing the heterogeneity of IHC AZs.

RESUMEN

In mammals, spatial orientation is synaptically-encoded by sensory hair cells of the vestibular labyrinth. Vestibular hair cells (VHCs) harbor synaptic ribbons at their presynaptic active zones (AZs), which play a critical role in molecular scaffolding and facilitate synaptic release and vesicular replenishment. With advancing age, the prevalence of vestibular deficits increases; yet, the underlying mechanisms are not well understood and the possible accompanying morphological changes in the VHC synapses have not yet been systematically examined. We investigated the effects of maturation and aging on the ultrastructure of the ribbon-type AZs in murine utricles using various electron microscopic techniques and combined them with confocal and super-resolution light microscopy as well as metabolic imaging up to 1 year of age. In older animals, we detected predominantly in type I VHCs the formation of floating ribbon clusters, mostly consisting of newly synthesized ribbon material. Our findings suggest that VHC ribbon-type AZs undergo dramatic structural alterations upon aging.

RESUMEN

The architecture of the presynaptic release site is exquisitely designed to facilitate and regulate synaptic vesicle exocytosis. With the identification of some of the building blocks of the active zone and the advent of super resolution imaging techniques, we are beginning to understand the morphological and functional properties of synapses in great detail. Presynaptic release sites consist of the plasma membrane, the cytomatrix, and dense projections. These three components are morphologically distinct but intimately connected with each other and with postsynaptic specializations, ensuring the fidelity of synaptic vesicle tethering, docking, and fusion, as well as signal detection. Although the morphology and molecular compositions of active zones may vary among species, tissues, and cells, global architectural design of the release sites is highly conserved.

Asunto(s)

RESUMEN

Ribbon synapses reliably transmit synaptic signals over a broad signalling range. Rod photoreceptor ribbon synapses are capable of transmitting signals generated by the absorption of single photons. The high precision of ribbon synapses emphasizes the need for particularly efficient signalling mechanisms. Synaptic ribbons are presynaptic specializations of ribbon synapses and are anchored to the active zone. Synaptic ribbons bind many synaptic vesicles that are delivered to the active zone for continuous and faithful signalling. In the present study we demonstrate with independent antibodies at the light- and electron microscopic level that rabconnectin-3α (RC3α)-alternative name Dmx-like 2 (DMXL2)-is localized to the synaptic ribbons of rod photoreceptor synapses in the mouse retina. In the brain, RC3α-containing complexes are known to interact with important components of synaptic vesicles, including Rab3-activating/inactivating enzymes, priming proteins and the vesicular H+-ATPase that acidifies the synaptic vesicle lumen to promote full neurotransmitter loading. The association of RC3α/DMXL2 with rod synaptic ribbons of the mouse retina could enable these structures to deliver only fully signalling-competent synaptic vesicles to the active zone thus contributing to reliable synaptic communication.

Asunto(s)

RESUMEN

The cytomatrix at the active zone-associated structural protein (CAST) and its homologue, named ELKS, being rich in glutamate (E), leucine (L), lysine (K), and serine (S), belong to a family of proteins that organize presynaptic active zones at nerve terminals. These proteins interact with other active zone proteins, including RIMs, Munc13s, Bassoon, and the ß subunit of Ca2+ channels, and have various roles in neurotransmitter release. A previous study showed that depletion of CAST/ELKS in the retina causes morphological changes and functional impairment of this structure. In this study, we investigated the roles of CAST and ELKS in ectopic synapse localization. We found that the involvement of these proteins in ribbon synapse distribution is complex. Unexpectedly, CAST and ELKS, in photoreceptors or in horizontal cells, did not play a major role in ribbon synapse ectopic localization. However, depletion of CAST and ELKS in the mature retina resulted in degeneration of the photoreceptors. These findings suggest that CAST and ELKS play critical roles in maintaining neural signal transduction in the retina, but the regulation of photoreceptor triad synapse distribution is not solely dependent on their actions within photoreceptors and horizontal cells.

Asunto(s)

RESUMEN

Our understanding of inner ear hair cell ultrastructure has heretofore relied upon two-dimensional imaging; however, serial block-face scanning electron microscopy (SBFSEM) changes this paradigm allowing for three-dimensional evaluation. We compared inner ear hair cells of the apical cristae in myo7aa-/- null zebrafish, a model of human Usher Syndrome type 1B, to hair cells in wild-type zebrafish by SBFSEM to investigate possible ribbon synapse ultrastructural differences. Previously, it has been shown that compared to wild type, myo7aa-/- zebrafish neuromast hair cells have fewer ribbon synapses yet similar ribbon areas. We expect the recapitulation of these results within the inner ear apical crista hair cells furthering the knowledge of three-dimensional ribbon synapse structure while resolving the feasibility of therapeutically targeting myo7aa-/- mutant ribbons. In this report, we evaluated ribbon synapse number, volume, surface area, and sphericity. Localization of ribbons and their distance from the nearest innervation were also evaluated. We determined that myo7aa-/- mutant ribbon synapses are smaller in volume and surface area; however, all other measurements were not significantly different from wild-type zebrafish. Because the ribbon synapses are nearly indistinguishable between the myo7aa-/- mutant and wild type, it suggests that the ribbons are structurally receptive, supporting that therapeutic intervention may be feasible.

Asunto(s)