Dopamine Secretion Is Mediated by Sparse Active Zone-like Release Sites.

Dopamine controls essential brain functions through volume transmission. Different from fast synaptic transmission, where neurotransmitter release and receptor activation are tightly coupled by an active zone, dopamine transmission is widespread and may not necessitate these organized release sites. Here, we determine whether striatal dopamine secretion employs specialized machinery for release. Using super resolution microscopy, we identified co-clustering of the active zone scaffolding proteins bassoon, RIM and ELKS in ∼30% of dopamine varicosities. Conditional RIM knockout disrupted this scaffold and, unexpectedly, abolished dopamine release, while ELKS knockout had no effect. Optogenetic experiments revealed that dopamine release was fast and had a high release probability, indicating the presence of protein scaffolds for coupling Ca2+ influx to vesicle fusion. Hence, dopamine secretion is mediated by sparse, mechanistically specialized active zone-like release sites. This architecture supports spatially and temporally precise coding for dopamine and provides molecular machinery for regulation.

Ultrastructural localization of calcium homeostasis modulator 1 in mouse taste buds.

Taste receptor cells are morphologically classified as types II and III. Type II cells form a unique type of synapses referred to as channel synapses where calcium homeostasis modulator 1 (CALHM1) together with CALHM3 forms voltage-gated channels that release the neurotransmitter, adenosine triphosphate (ATP). To validate the proposed structural model of channel synapses, the ultrastructural localization of CALHM1 in type II cells of both fungiform and circumvallate taste buds was examined. A monoclonal antibody against CALHM1 was developed and its localization was evaluated via immunofluorescence and immunoelectron microscopy using the immunogold-silver labeling technique. CALHM1 was detected as puncta using immunofluorescence and along the presynaptic membrane of channel synapses facing atypical mitochondria, which provide ATP, by immunoelectron microscopy. In addition, it was detected along the plasma membrane lined by subsurface cisternae at sites apposed to afferent nerve fibers. Our results support the validity of a previously proposed structural model for channel synapses and provide insights into the function of subsurface cisternae whose function in taste receptor cells is unknown. We also examined the localization of CALHM1 in hybrid synapses of type III cells, which are conventional chemical synapses accompanied by mitochondria similar to atypical mitochondria of channel synapses. CALHM1 was not detected in the six hybrid synapses examined using immunoelectron microscopy. We further performed double immunolabeling for CALHM1 and Bassoon, which is detected as puncta corresponding to conventional vesicular synapses in type III cells. Our observations suggest that at least some, and probably most, hybrid synapses are not accompanied by CALHM1.

Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP.

Neuronal presynaptic terminals contain hundreds of neurotransmitter-filled synaptic vesicles (SVs). The morphologically uniform SVs differ in their release competence segregating into functional pools that differentially contribute to neurotransmission. The presynaptic scaffold bassoon is required for neurotransmission, but the underlying molecular mechanisms are unknown. We report that glutamatergic synapses lacking bassoon feature decreased SV release competence and increased resting pool of SVs as assessed by imaging of SV release in cultured neurons. CDK5/calcineurin and cAMP/PKA presynaptic signalling are dysregulated, resulting in an aberrant phosphorylation of their downstream effectors synapsin1 and SNAP25, well-known regulators of SV release competence. An acute pharmacological restoration of physiological CDK5 and cAMP/PKA activity fully normalises the SV pools in neurons lacking bassoon. Finally, we demonstrate that CDK5-dependent regulation of PDE4 activity interacts with cAMP/PKA signalling and thereby controls SV release competence. These data reveal that bassoon organises SV pools in glutamatergic synapses via regulation of presynaptic phosphorylation and cAMP homeostasis and indicate a role of CDK5/PDE4/cAMP axis in the control of neurotransmitter release.

Linking epileptic phenotypes and neural extracellular matrix remodeling signatures in mouse models of epilepsy.

Epilepsies are multifaceted neurological disorders characterized by abnormal brain activity, e.g. caused by imbalanced synaptic excitation and inhibition. The neural extracellular matrix (ECM) is dynamically modulated by physiological and pathophysiological activity and critically involved in controlling the brain's excitability. We used different epilepsy models, i.e. mice lacking the presynaptic scaffolding protein Bassoon at excitatory, inhibitory or all synapse types as genetic models for rapidly generalizing early-onset epilepsy, and intra-hippocampal kainate injection, a model for acquired temporal lobe epilepsy, to study the relationship between epileptic seizures and ECM composition. Electroencephalogram recordings revealed Bassoon deletion at excitatory or inhibitory synapses having diverse effects on epilepsy-related phenotypes. While constitutive Bsn mutants and to a lesser extent GABAergic neuron-specific knockouts (BsnDlx5/6cKO) displayed severe epilepsy with more and stronger seizures than kainate-injected animals, mutants lacking Bassoon solely in excitatory forebrain neurons (BsnEmx1cKO) showed only mild impairments. By semiquantitative immunoblotting and immunohistochemistry we show model-specific patterns of neural ECM remodeling, and we also demonstrate significant upregulation of the ECM receptor CD44 in null and BsnDlx5/6cKO mutants. ECM-associated WFA-binding chondroitin sulfates were strongly augmented in seizure models. Strikingly, Brevican, Neurocan, Aggrecan and link proteins Hapln1 and Hapln4 levels reliably predicted seizure properties across models, suggesting a link between ECM state and epileptic phenotype.

Genetic disruption of bassoon in two mutant mouse lines causes divergent retinal phenotypes.

Bassoon (BSN) is a presynaptic cytomatrix protein ubiquitously present at chemical synapses of the central nervous system, where it regulates synaptic vesicle replenishment and organizes voltage-gated Ca2+ channels. In sensory photoreceptor synapses, BSN additionally plays a decisive role in anchoring the synaptic ribbon, a presynaptic organelle and functional extension of the active zone, to the presynaptic membrane. In this study, we functionally and structurally analyzed two mutant mouse lines with a genetic disruption of Bsn-Bsngt and Bsnko -using electrophysiology and high-resolution microscopy. In both Bsn mutant mouse lines, full-length BSN was abolished, and photoreceptor synaptic function was similarly impaired, yet synapse structure was more severely affected in Bsngt/gt than in Bsnko/ko photoreceptors. The synaptic defects in Bsngt/gt retina coincide with remodeling of the outer retina-rod bipolar and horizontal cell sprouting, formation of ectopic ribbon synaptic sites-and death of cone photoreceptors, processes that did not occur in Bsnko/ko retina. An analysis of Bsngt/ko hybrid mice revealed that the divergent retinal phenotypes of Bsngt/gt and Bsnko/ko mice can be attributed to the expression of the Bsngt allele, which triggers cone photoreceptor death and neurite sprouting in the outer retina. These findings shed new light on the existing Bsn mutant mouse models and might help to understand mechanisms that drive photoreceptor death.

The Presynaptic Scaffold Protein Bassoon in Forebrain Excitatory Neurons Mediates Hippocampal Circuit Maturation: Potential Involvement of TrkB Signalling.

A presynaptic active zone organizer protein Bassoon orchestrates numerous important functions at the presynaptic active zone. We previously showed that the absence of Bassoon exclusively in forebrain glutamatergic presynapses (BsnEmx1cKO) in mice leads to developmental disturbances in dentate gyrus (DG) affecting synaptic excitability, morphology, neurogenesis and related behaviour during adulthood. Here, we demonstrate that hyperexcitability of the medial perforant path-to-DG (MPP-DG) pathway in BsnEmx1cKO mice emerges during adolescence and is sustained during adulthood. We further provide evidence for a potential involvement of tropomyosin-related kinase B (TrkB), the high-affinity receptor for brain-derived neurotrophic factor (BDNF), mediated signalling. We detect elevated TrkB protein levels in the dorsal DG of adult mice (~3-5 months-old) but not in adolescent (~4-5 weeks-old) mice. Electrophysiological analysis reveals increased field-excitatory-postsynaptic-potentials (fEPSPs) in the DG of the adult, but not in adolescent BsnEmx1cKO mice. In line with an increased TrkB expression during adulthood in BsnEmx1cKO, blockade of TrkB normalizes the increased synaptic excitability in the DG during adulthood, while no such effect was observed in adolescence. Accordingly, neurogenesis, which has previously been found to be increased in adult BsnEmx1cKO mice, was unaffected at adolescent age. Our results suggest that Bassoon plays a crucial role in the TrkB-dependent postnatal maturation of the hippocampus.

Neuronal LRP4 regulates synapse formation in the developing CNS.

The low-density lipoprotein receptor-related protein 4 (LRP4) is essential in muscle fibers for the establishment of the neuromuscular junction. Here, we show that LRP4 is also expressed by embryonic cortical and hippocampal neurons, and that downregulation of LRP4 in these neurons causes a reduction in density of synapses and number of primary dendrites. Accordingly, overexpression of LRP4 in cultured neurons had the opposite effect inducing more but shorter primary dendrites with an increased number of spines. Transsynaptic tracing mediated by rabies virus revealed a reduced number of neurons presynaptic to the cortical neurons in which LRP4 was knocked down. Moreover, neuron-specific knockdown of LRP4 by in utero electroporation of LRP4 miRNA in vivo also resulted in neurons with fewer primary dendrites and a lower density of spines in the developing cortex and hippocampus. Collectively, our results demonstrate an essential and novel role of neuronal LRP4 in dendritic development and synaptogenesis in the CNS.

Na⁺/Ca²âº Exchanger 2 (NCX2) in the Circadian Clock of the Rat Suprachiasmatic Nucleus: Colocalization with Neuropeptides and Daily Profiles of Gene Expression and Protein Levels.

The plasmalemmal Na⁺/Ca²âº changer (NCX) regulates intracellular Ca²âº by exchanging 3 Na⁺ for 1 Ca²âº in either the Ca²âº exit or Ca²âº entry mode. All three NCX isoforms NCX1, NCX2, and NCX3 are expressed in the rat brain, with isoform-specific differential distribution. In the central clock of suprachiasmatic nucleus (SCN), intracellular Ca²âº controls the circadian release of major neuropeptides, which are the arginine vasopressin (AVP), vasoactive intestinal peptide (VIP) and gastrin releasing peptide (GRP), and the NCX, most likely NCX1, rapidly clears depolarization-induced somatic Ca²âº influx. However, the role of NCX2 in the SCN remains unknown. This study aimed to investigate the colocalization of NCX2 with neuropeptides and daily expression profiles of NCX2 in mRNA and protein levels. Consistent with the restricted distribution of NCX2 in the retinorecipient ventral SCN, the immunostaining results showed colocalization of NCX2 with VIP, GRP and VIP/GRP in the ventral SCN, but not with AVP in the dorsal SCN, or markers for astrocyte and major input pathways. Importantly, the presynaptic marker Bassoon was found to colocalize with NCX2/GRP and NCX2/ VIP, indicating localization of both VIP/NCX2 and GRP/NCX2 at the presynaptic sites. Furthermore, real-time PCR and western blotting revealed no day-night difference in NCX2 mRNA and protein levels, in contrast to a robust circadian rhythm in the expression of clock genes Per1 and Per2. Together the results suggest a role of NCX2 in the regulation of the release of VIP and GRP.

Formation of cholinergic synapse-like specializations at developing murine muscle spindles.

Muscle spindles are complex stretch-sensitive mechanoreceptors. They consist of specialized skeletal muscle fibers, called intrafusal fibers, which are innervated in the central (equatorial) region by afferent sensory axons and in both polar regions by efferent γ-motoneurons. We show that AChRs are concentrated at the γ-motoneuron endplate as well as in the equatorial region where they colocalize with the sensory nerve ending. In addition to the AChRs, the contact site between sensory nerve ending and intrafusal muscle fiber contains a high concentration of choline acetyltransferase, vesicular acetylcholine transporter and the AChR-associated protein rapsyn. Moreover, bassoon, a component of the presynaptic cytomatrix involved in synaptic vesicle exocytosis, is present in γ-motoneuron endplates but also in the sensory nerve terminal. Finally, we demonstrate that during postnatal development of the γ-motoneuron endplate, the AChR subunit stoichiometry changes from the γ-subunit-containing fetal AChRs to the ε-subunit-containing adult AChRs, similar and approximately in parallel to the postnatal subunit maturation at the neuromuscular junction. In contrast, despite the onset of ε-subunit expression during postnatal development the γ-subunit remains detectable in the equatorial region by subunit-specific antibodies as well as by analysis of muscle spindles from mice with genetically-labeled AChR γ-subunits. These results demonstrate an unusual maturation of the AChR subunit composition at the annulospiral endings and suggest that in addition to the recently described glutamatergic secretory system, the sensory nerve terminals are also specialized for cholinergic synaptic transmission, synaptic vesicle storage and exocytosis.

Resolving the structure of inner ear ribbon synapses with STED microscopy.

Synapses are diverse in form and function; however, the mechanisms underlying this diversity are poorly understood. To illuminate structure/function relationships, robust analysis of molecular composition and morphology is needed. The molecular-anatomical components of synapses-vesicles, clusters of voltage-gated ion channels in presynaptic densities, arrays of transmitter receptors in postsynaptic densities-are only tens to hundreds of nanometers in size. Measuring the topographies of synaptic proteins requires nanoscale resolution of their molecularly specific labels. Super-resolution light microscopy has emerged to meet this need. Achieving 50 nm resolution in thick tissue, we employed stimulated emission depletion (STED) microscopy to image the functionally and molecularly unique ribbon-type synapses in the inner ear that connect mechano-sensory inner hair cells to cochlear nerve fibers. Synaptic ribbons, bassoon protein, voltage-gated Ca(2+) channels, and glutamate receptors are inhomogeneous in their spatial distributions within synapses; the protein clusters assume variations of shapes typical for each protein specifically at cochlear afferent synapses. Heterogeneity of substructure among these synapses may contribute to functional differences among auditory nerve fibers. The morphology of synaptic voltage-gated Ca(2+) channels matures over development in a way that depends upon bassoon protein, which aggregates in similar form. Functional properties of synaptic transmission appear to depend on voltage-gated Ca(2+) channel cluster morphology and position relative to synaptic vesicles. Super-resolution light microscopy is a group of techniques that complement electron microscopy and conventional light microscopy. Although technical hurdles remain, we are beginning to resolve the details of molecular nanoanatomy that relate mechanistically to synaptic function.

Behavioral and histological analyses of the mouse Bassoon p.P3882A mutation corresponding to the human BSN p.P3866A mutation.

Tauopathy is known to be a major pathognomonic finding in important neurodegenerative diseases such as progressive supranuclear palsy (PSP) and corticobasal degeneration. However, the mechanism by which tauopathy is triggered remains to be elucidated. We previously identified the point mutation c.11596C > G, p.Pro3866Ala in the Bassoon gene (BSN) in a Japanese family with PSP-like syndrome. We showed that mutated BSN may have been involved in its own insolubilization and tau accumulation. Furthermore, BSN mutations have also been related to various neurological diseases. In order to further investigate the pathophysiology of BSN mutation in detail, it is essential to study it in mouse models. We generated a mouse model with the mouse Bassoon p.P3882A mutation, which corresponds to the human BSN p.P3866A mutation, knock-in (KI) and we performed systematic behavioral and histological analyses. Behavioral analyses revealed impaired working memory in a Y-maze test at 3 months of age and decreased locomotor activity in the home cage at 3 and 12 months of age in KI mice compared to those in wild-type mice. Although no obvious structural abnormalities were observed at 3 months of age, immunohistochemical studies showed elevation of Bsn immunoreactivity in the hippocampus and neuronal loss without tau accumulation in the substantia nigra at 12 months of age in KI mice. Although our mice model did not show progressive cognitive dysfunction and locomotor disorder like PSP-like syndrome, dopaminergic neuronal loss was observed in the substantia nigra in 12-month-old KI mice. It is possible that BSN mutation may result in dopaminergic neuronal loss without locomotor symptoms due to the early disease stage. Thus, further clinical course can induce cognitive dysfunction and locomotor symptoms.

Microdeletions of 3p21.31 characterized by developmental delay, distinctive features, elevated serum creatine kinase levels, and white matter involvement.

Interstitial deletions of chromosome 3 are rare, and only one patient with a microdeletion of 3p21.31 has been reported to date. We identified two additional cases of patients with microdeletions of 3p21.31. The characteristic clinical features of developmental delay and distinctive facial features (including arched eyebrows, hypertelorism, epicanthus, and micrognathia) were seen both in the previously reported patient and in the two newly identified patients. In these two new cases, additional features, including elevated serum creatine kinase levels and characteristic neuroradiological features with white matter involvement, were seen. These features had not been described in the previous case in which the patient was examined during infancy, suggesting an age-dependent mechanism. The shortest region of overlap among the three deletions narrowed down the candidate genes that may be responsible for the common neurological features to the bassoon (presynaptic cytomatrix protein) gene (BSN), which has an important function in neuronal synapses. In this study, we confirmed common phenotypic features in the patients with microdeletions of 3p21.31 and identified additional features that have not been reported previously. Because the constellation of such characteristic features is quite unique, clinical manifestations of the patients with microdeletions of 3p21.31 would be clinically recognizable as a contiguous gene deletion syndrome.

Presynaptic Cytomatrix Proteins.

The Cytomatrix Assembled at the active Zone (CAZ) of a presynaptic terminal displays electron-dense appearance and defines the center of the synaptic vesicle release. The protein constituents of CAZ are multiple-domain scaffolds that interact extensively with each other and also with an ensemble of synaptic vesicle proteins to ensure docking, fusion, and recycling. Reflecting the central roles of the active zone in synaptic transmission, CAZ proteins are highly conserved throughout evolution. As the nervous system increases complexity and diversity in types of neurons and synapses, CAZ proteins expand in the number of gene and protein isoforms and interacting partners. This chapter summarizes the discovery of the core CAZ proteins and current knowledge of their functions.

Similarity and Diversity of Presynaptic Molecules at Neuromuscular Junctions and Central Synapses.

Synaptic transmission is essential for controlling motor functions and maintaining brain functions such as walking, breathing, cognition, learning, and memory. Neurotransmitter release is regulated by presynaptic molecules assembled in active zones of presynaptic terminals. The size of presynaptic terminals varies, but the size of a single active zone and the types of presynaptic molecules are highly conserved among neuromuscular junctions (NMJs) and central synapses. Three parameters play an important role in the determination of neurotransmitter release properties at NMJs and central excitatory/inhibitory synapses: the number of presynaptic molecular clusters, the protein families of the presynaptic molecules, and the distance between presynaptic molecules and voltage-gated calcium channels. In addition, dysfunction of presynaptic molecules causes clinical symptoms such as motor and cognitive decline in patients with various neurological disorders and during aging. This review focuses on the molecular mechanisms responsible for the functional similarities and differences between excitatory and inhibitory synapses in the peripheral and central nervous systems, and summarizes recent findings regarding presynaptic molecules assembled in the active zone. Furthermore, we discuss the relationship between functional alterations of presynaptic molecules and dysfunction of NMJs or central synapses in diseases and during aging.

Organization of Presynaptic Autophagy-Related Processes.

Brain synapses pose special challenges on the quality control of their protein machineries as they are far away from the neuronal soma, display a high potential for plastic adaptation and have a high energy demand to fulfill their physiological tasks. This applies in particular to the presynaptic part where neurotransmitter is released from synaptic vesicles, which in turn have to be recycled and refilled in a complex membrane trafficking cycle. Pathways to remove outdated and damaged proteins include the ubiquitin-proteasome system acting in the cytoplasm as well as membrane-associated endolysosomal and the autophagy systems. Here we focus on the latter systems and review what is known about the spatial organization of autophagy and endolysomal processes within the presynapse. We provide an inventory of which components of these degradative systems were found to be present in presynaptic boutons and where they might be anchored to the presynaptic apparatus. We identify three presynaptic structures reported to interact with known constituents of membrane-based protein-degradation pathways and therefore may serve as docking stations. These are (i) scaffolding proteins of the cytomatrix at the active zone, such as Bassoon or Clarinet, (ii) the endocytic machinery localized mainly at the peri-active zone, and (iii) synaptic vesicles. Finally, we sketch scenarios, how presynaptic autophagic cargos are tagged and recruited and which cellular mechanisms may govern membrane-associated protein turnover in the presynapse.

Nanoscopical Analysis Reveals an Orderly Arrangement of the Presynaptic Scaffold Protein Bassoon at the Golgi-Apparatus.

Bassoon is a core scaffold protein of the presynaptic active zone. In brain synapses, the C-terminus of Bassoon is oriented toward the plasma membrane and its N-terminus is oriented toward synaptic vesicles. At the Golgi-apparatus, Bassoon is thought to assemble active zone precursor structures, but whether it is arranged in an orderly fashion is unknown. Understanding the topology of this large scaffold protein is important for models of active zone biogenesis. Using stimulated emission depletion nanoscopy in cultured hippocampal neurons, we found that an N-terminal intramolecular tag of recombinant Bassoon, but not C-terminal tag, colocalized with markers of the trans-Golgi network (TGN). The N-terminus of Bassoon was located between 48 and 69 nm away from TGN38, while its C-terminus was located between 100 and 115 nm away from TGN38. Sequences within the first 95 amino acids of Bassoon were required for this arrangement. Our results indicate that, at the Golgi-apparatus, Bassoon is oriented with its N-terminus toward and its C-terminus away from the trans Golgi network membrane. Moreover, they suggest that Bassoon is an extended molecule at the trans Golgi network with the distance between amino acids 97 and 3,938, estimated to be between 46 and 52 nm. Our data are consistent with a model, in which the N-terminus of Bassoon binds to the membranes of the trans-Golgi network, while the C-terminus associates with active zone components, thus reflecting the topographic arrangement characteristic of synapses also at the Golgi-apparatus.

Bidirectional Interactions With Humpback Whale Singer Using Concrete Sound Elements.

We describe an art-science project called "Feral Interactions-The Answer of the Humpback Whale" inspired by humpback whale songs and interactions between individuals based on mutual influences, learning process, or ranking in the dominance hierarchy. The aim was to build new sounds that can be used to initiate acoustic interactions with these whales, not in a one-way direction, as playbacks do, but in real interspecies exchanges. Thus, we investigated how the humpback whales generate sounds in order to better understand their abilities and limits. By carefully listening to their emitted vocalizations, we also describe their acoustic features and temporal structure, in a scientific way and also with a musical approach as it is done with musique concrète, in order to specify the types and the morphologies of whale sounds. The idea is to highlight the most precise information to generate our own sounds that will be suggested to the whales. Based on the approach developed in musique concrète, similarities with the sounds produced by bassoon were identified and then were processed to become "concrete sound elements." This analysis also brought us to design a new music interface that allows us to create adapted musical phrases in real-time. With this approach, interactions will be possible in both directions, from and to whales.

In trans neuregulin3-Caspr3 interaction controls DA axonal bassoon cluster development.

Dopamine (DA) transmission is critical to motivation, movement, and emotion. Unlike glutamatergic and GABAergic synapses, the development of DA synapses is less understood. We show that bassoon (BSN) clusters along DA axons in the core of nucleus accumbens (NAcc) were increased in neonatal stages and reduced afterward, suggesting DA synapse elimination. Remarkably, DA neuron-specific ablating neuregulin 3 (NRG3), a protein whose levels correlate with BSN clusters, increased the clusters and impaired DA release and behaviors related to DA transmission. An unbiased screen of transmembrane proteins with the extracellular domain (ECD) of NRG3 identified Caspr3 (contactin associate-like protein 3) as a binding partner. Caspr3 was enriched in striatal medium spiny neurons (MSNs). NRG3 and Caspr3 interact in trans, which was blocked by Caspr3-ECD. Caspr3 null mice displayed phenotypes similar to those in DAT-Nrg3f/f mice in DA axonal BSN clusters and DA transmission. Finally, in vivo disruption of the NRG3-Caspr3 interaction increased BSN clusters. Together, these results demonstrate that DA synapse development is controlled by trans interaction between NRG3 in DA neurons and Caspr3 in MSNs, identifying a novel pair of cell adhesion molecules for brain circuit wiring.

The absence of functional bassoon at cone photoreceptor ribbon synapses affects signal transmission at Off cone bipolar cell contacts in mouse retina.

AIM: Off cone bipolar cells of the mammalian retina connect to cone photoreceptor synaptic terminals via non-invaginating flat contacts at a considerable distance from the only established neurotransmitter release site so far, the synaptic ribbon. Diffusion from the ribbon synaptic active zone is considered the most likely mechanism for the neurotransmitter glutamate to reach postsynaptic receptors on the dendritic tips of Off cone bipolar cells. We used a mutant mouse with functionally impaired photoreceptor ribbon synapses to investigate the importance of intact ribbon synaptic active zones for signal transmission at Off cone bipolar cell contacts. METHODS: Whole-cell patch-clamp recordings from Off cone bipolar cells in a horizontal slice preparation of wildtype (Bsnwt ) and mutant (BsnΔEx4/5 ) mouse retina were applied to investigate signal transmission between cone photoreceptors and Off cone bipolar cells. The distribution of postsynaptic glutamate receptors in Off cone bipolar cell dendrites was studied using multiplex immunocytochemistry. RESULTS: Tonic synaptic activity and evoked release were significantly reduced in mutant animals. Vesicle replenishment rates and the size of the readily releasable pool were likewise decreased. The precisely timed transient current response to light offset changed to a sustained response in the mutant, exemplified by random release events only loosely time-locked to the stimulus. The kainate receptor distribution in postsynaptic Off cone bipolar cell dendritic contacts in BsnΔEx4/5 mice was largely disturbed. CONCLUSION: Our results suggest a major role of functional ribbon synaptic active zones for signal transmission and postsynaptic glutamate receptor organization at flat Off cone bipolar cell contacts.