Abstract
RIM-Binding Protein 2 (RIM-BP2) is a multi-domain protein of the presynaptic active zone (AZ). By binding to Rab-interacting protein (RIM), bassoon and voltage-gated Ca²⁺ channels (CaV), it is considered to be a central organizer of the topography of CaV and release sites of synaptic vesicles (SVs) at the AZ. Here, we investigated the role of RIM-BP2 at the endbulb of Held synapse of auditory nerve fibers with bushy cells of the cochlear nucleus, a fast relay of the auditory pathway with high release probability. Disruption of RIM-BP2 lowered release probability altering short-term plasticity and reduced evoked excitatory postsynaptic currents (EPSCs). Analysis of SV pool dynamics during high frequency train stimulation indicated a reduction of SVs with high release probability but an overall normal size of the readily releasable SV pool (RRP). The Ca2+-dependent fast component of SV replenishment after RRP depletion was slowed. Ultrastructural analysis by super-resolution light and electron microscopy revealed an impaired topography of presynaptic CaV and a reduction of docked and membrane-proximal SVs at the AZ. We conclude that RIM-BP2 organizes the topography of CaV, and promotes SV tethering and docking. This way RIM-BP2 is critical for establishing a high initial release probability as required to reliably signal sound onset information that we found to be degraded in bushy cells of RIM-BP2-deficient mice in vivo.
Significance Statement RIM-binding proteins (RIM-BPs) are key organizers of the active zone (AZ). Using a multidisciplinary approach to the calyceal endbulb of Held synapse that transmit auditory information at rates of up to hundreds of Hertz with sub-millisecond precision we demonstrate a requirement for RIM-BP2 for normal auditory signaling. Endbulb synapses lacking RIM-BP2 show a reduced release probability despite normal whole-terminal Ca2+ influx and abundance of the key priming protein Munc13-1, a reduced rate of SV replenishment, as well as an altered topography of CaV2.1 Ca2+ channels, and fewer docked and membrane proximal synaptic vesicles. This hampers transmission of sound onset information likely affecting downstream neural computations such as of sound localization.
Introduction
Active zones (AZs) are specialized regions at the presynaptic terminals where neurotransmitter release occurs. AZs employ a sophisticated machinery to enable ultrafast coupling of the incoming action potential to the release of transmitter via Ca2+-triggered SV fusion. Voltage-gated Ca2+ channels (CaV) and SV release sites represent the core machinery, and their relative topography at the AZ co-determines the release probability (recent reviews in: Kaeser and Regehr, 2014; Schneggenburger and Rosenmund, 2015; Walter et al., 2018; Dittman and Ryan, 2019). The function and abundance of CaV (recent reviews in: Pangrsic et al., 2018; Dolphin and Lee, 2020) is positively regulated by auxiliary subunits and multi-domain proteins of the AZ such as RIM-BP, RIM, piccolo, bassoon, CAST and ELKS.
Several of these proteins promote the clustering of Ca2+ channels at the AZ and/or their interaction with the SV release sites (Gundelfinger and Fejtova, 2012; Südhof, 2012; Moser et al., 2019). RIM and RIM-BPs, in particular, have been considered as molecular linkers of CaV and vesicular release sites. Direct and indirect interactions of RIMs with CaV are well established (Kiyonaka et al., 2007; Gebhart et al., 2010; Kaeser et al., 2011; Picher et al., 2017). Proline-rich sequences of RIMs have been shown to bind the SH3-domains of RIM-BP that directly interacts with CaV (Hibino et al., 2002; Kaeser et al., 2011). Deletion of Drosophila RIM-BP disrupted CaV clustering at the AZs of larval neuromuscular junctions consequently impairing their functional coupling to SV release: altered short-term plasticity demonstrating a reduced release probability (Liu et al., 2011). Of the three mammalian RIM-BP isoforms, RIM-BP1 and 2 are neuron-specific, whereby RIM-BP2 seems to be the isoform that is most relevant for transmission at conventional synapses (Acuna et al., 2015; Grauel et al., 2016). Disruption of RIM-BP1 and 2 did not alter the Ca2+ current at the calyx of Held synapse (Acuna et al., 2015), while lack of RIM-BP2 (Krinner et al., 2017; Luo et al., 2017) and of RIM-BP1 (Luo et al., 2017) reduced the CaV number at ribbon-type AZs. An alteration of the CaV topography at the AZs has been reported based on super-resolution immunofluorescence microscopy (Grauel et al., 2016; Krinner et al., 2017).
A loosening of the otherwise tight spatial relationship of CaV and SV release sites was indicated by the reduced release probability and a greater sensitivity to the intracellular presence of the “slow” Ca2+ chelator EGTA in the absence of RIM-BP(1)/2 (Acuna et al., 2015; Grauel et al., 2016; Luo et al., 2017). A similar conclusion was reached in sensory hair cells where, unlike in CNS synapses, SV replenishment was impaired in the absence of RIM-BP2 (Krinner et al., 2017). However, in contrast to the dramatic impairment of synaptic transmission at RIM-BP deficient neuromuscular junctions in Drosophila (Liu et al., 2011), transmission was affected more subtly by RIM-BP deletion at mammalian synapses (Acuna et al., 2015; Grauel et al., 2016; Krinner et al., 2017; Luo et al., 2017). This might relate to partial compensation by other candidate linkers of CaV and SV release sites such as long RIM isoforms (Acuna et al, 2016). Recently, two alternative actions of RIM-BPs were indicated based on molecular perturbations studies in hippocampal neurons: i) binding to CaV enabling their tight coupling to SVs or ii) promoting SV priming by interaction with Munc13-1 (Brockmann et al., 2020). Here we aimed to further characterize the function of RIM-BP2 at the endbulb of Held synapse, the first central auditory synapse (von Gersdorff and Borst, 2002; Yu and Goodrich, 2014), that employs high vesicular release probability at its more than 100 AZs for reliable and temporally precise signal transmission from auditory nerve fibers (ANFs) to bushy cells (BCs) at frequencies of hundreds of Hertz (Trussell, 1999; Wang et al., 2011). We combined electrophysiological analysis with studies of the molecular composition and ultrastructure of the AZ in RIM-BP2-deficient endbulbs.
Materials and Methods
Animals
The constitutive RIM-BP2 knockout (RIM-BP2 KO) mice were described in (Grauel et al., 2016) and kindly provided to us by Drs. Christian Rosenmund, Katharina Grauel and Stephan Sigrist. All experiments were performed in compliance with the guidelines of the German animal welfare act and were approved by the board for animal welfare of the University Medical Center Göttingen and the animal welfare office of the state of Lower Saxony.
Slice electrophysiology
Slice preparation: Acute parasagittal slices (150µm) from the anteroventral cochlear nucleus (aVCN) were obtained as described previously (Mendoza Schulz et al., 2014). Briefly, after sacrifice by decapitation, brains were dissected out and quickly immersed in ice-cold low Na+ and low Ca2+ cutting solution containing (in mM): 50 NaCl, 26 NaHCO3, 120 sucrose, 1.25 NaH2PO4.H2O, 2.5 KCl, 20 glucose, CaCl2, 6 MgCl2, 0.7 Na L-ascorbate, 2 Na pyruvate, 3 myo-inositol, 3 Na L-lactate with pH adjusted to 7.4 and osmolarity of around 310 mOsm/l. After removal of the meninges from the ventral face of the brainstem, the two hemispheres were separated by a midsagittal cut and the forebrain was removed at the pons-midbrain junction. The brain blocks containing brain stem and cerebellum were then glued (cyanoacrylate glue; Loctite 401, Henkel) to the stage of a VT 1200S vibratome (Leica microsystems, Wetzlar, Germany) such that the medial side was glued on, the ventral side was facing the blade and the lateral side was facing upwards, submerged in ice-cold cutting solution. For sectioning, the blade was positioned at the height of cerebellar flocculus and sections were cut at a blade feed rate of 0.02 mm/s with an amplitude of 1.50 mm. Slices were incubated for 30min in artificial cerebrospinal fluid (aCSF) maintained at 35°C, and then kept at room temperature (22-24°C) until recording. Composition of aCSF was identical to the cutting solution except (in mM): 125 NaCl, 13 glucose, 2 CaCl2 and 1 MgCl2. The pH of the solution was adjusted to 7.4 and osmolarity was around 310 mOsm/l. All solutions were continuously aerated with carbogen (95% O2, 5% CO2). For presynaptic endbulb recordings, coronal sections were made instead of parasagittal as described above. The only difference was that after the forebrain was removed at the pons-midbrain junction, the dissected brain was not cut midsagitally and the brain block containing the brain stem and the cerebellum was then glued to the vibratome stage with the caudal aspect facing upwards and the ventral side towards the blade.
Electrophysiology: Patch-clamp recordings were made from BCs of aVCN using EPC10 USB Patch clamp amplifier controlled by the Patchmaster software (HEKA Elektronik, Lambrecht/Pfalz, Germany). Sampling interval and filter settings were 25 µs and 7.3 kHz respectively. Cells were visualized by differential interference contrast (DIC) microscopy through a 40x water-immersion objective (NA 0.8; Zeiss, Oberkochen, Germany) using an Axioscope 2 FS plus microscope (Zeiss). All experiments were conducted at a temperature of 33-35°C, maintained by constant superfusion (flow rate 3-4 ml/min) of aCSF, heated by an inline solution heater (SH-27B with TC-324B controller; Warner Instruments, Hamden, CT, USA) and monitored by a thermistor placed between the inflow site and the slice, in the recording chamber.
Patch pipettes were pulled with P-87 micropipette puller (Sutter Instruments Co., Novato, CA, USA) from borosilicate glass capillaries with filament (GB150F, 0.86×1.50×80mm; Science Products, Hofheim, Germany). Open tip pipette resistance was 1.5-3 MΩ when filled with intracellular solution containing (in mM): 115 K-gluconate, 10 HEPES, 8 EGTA, 10 Na2Phosphocreatine, 4 ATP-Mg, 0.3 GTP-Na, 4.5 MgCl2, 10 NaCl and 1 N-(2,6-dimethylphenyl carbamoylmethyl) triethylammonium chloride (QX-314; Alomone Labs, Jerusalem, Israel) to block sodium channels, with a pH of 7.35 and an osmolarity of 300 mOsm/l. Additionally, 1 mM of fluorescent dye Alexa-488 (Invitrogen) was added to the recording pipette and cell structure was examined during experiments using a HXP 120 mercury lamp, with FITC filter (Semrock hardcoat). Cells were voltage-clamped at a holding potential of –70 mV, after correction for a liquid junction potential of 12 mV. Mean series resistance was around 5 MΩ and was compensated up to 70% with a 10 µs lag. Presynaptic ANF were minimally stimulated with a monopolar electrode in a patch pipette filled with aCSF, placed at a distance of at least three cell diameters from the cell being recorded. Stimulating currents of 10-20 µA were delivered through a stimulus isolator (A360 World Precision Instruments, Sarasota, FL, USA). For the main set of recordings, bath solution (aCSF) was supplemented with: 1 mM kynurenic acid sodium salt (abcam Biochemicals, Cambride, UK), a low-affinity AMPAR antagonist, to prevent receptor saturation/desensitization, 100µM Cyclothiazide to prevent AMPAR desensitization, 10 µM Bicuculline methchloride, a GABAA receptor antagonist and 2 µM Strychnine hydrochloride, a glycine receptor antagonist.
For patch-clamp experiments at the endbulbs of Held patch-pipettes were coated with either dental wax or Sylgard to minimize fast capacitive transients and stray capacitance during voltage clamp experiments. Open tip pipette resistance was 4-5 MΩ with an intracellular solution containing (in mM): 130 Cs-methanesulfonate, 20 TEA-Cl, 10 HEPES, 0.5 EGTA, 5 Na2Phosphocreatine, 4 ATP-Mg, 0.2 GTP-Na, with pH adjusted to 7.3 with CsOH osmolarity of 320 mOsm/l. For anatomical confirmation 1 mM of fluorescent dye Alexa-488 (Invitrogen) was added to the recording pipette. The bath solution differed from the aCSF normally used (in mM): 85 NaCl, 25 glucose, 2 CaCl2 and 1 MgCl2. The pH of the solution was adjusted to 7.4 and osmolarity was around 310mOsm/l. Additionally, the bath solution was supplemented with 1 µM TTX, 1 mM 4-AP, and 40 mM TEA-Cl to suppress voltage-gated Na+ and K+ currents. Presynaptic terminals were voltage-clamped at a holding potential of –80 mV, a liquid junction potential of 3 mV was ignored. Series resistance was <30 MΩ and was compensated up to 50% with a 10 µs lag.
Systems physiology: Extracellular recordings from single bushy cells
Extracellular recordings from single units of bushy cells (BCs) in the aVCN were performed as described before (Jing et al., 2013; Strenzke et al., 2016) on 9 to 10-week-old mice. After anesthetizing the mice with i.p. injection of urethane (1.32 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.1 mg/kg), a tracheostomy was performed, their cartilaginous ear canals were removed and then they were positioned in a custom-designed head-holder and stereotactic system. After partial removal of the occipital bone and cerebellum to expose the surface of the cochlear nucleus, a glass microelectrode was advanced through the anterior portion of the aVCN to avoid the auditory nerve fibers and instead target the area with a higher fraction of spherical BCs. Acoustic stimulation was provided by an open field Avisoft ScanSpeak Ultrasonic Speaker (Avisoft Bioacoustics). “Putative” spherical BCs were identified and differentiated from other cell types in the cochlear nucleus by their characteristic ‘Primary-like’ peristimulus time histogram (PSTH) (Taberner and Liberman, 2005), irregular firing pattern demonstrated by a ≥ 0.5 coefficient of variation of inter-spike intervals of adapted responses, and a first spike latency of ≤ 5 ms. Bushy cells units were distinguished from auditory nerve fiber (ANFs, also having ‘Primary-like’ PSTH) based on their stereotactic position (<1.1 mm below the surface of the cochlear nucleus). Recordings were performed using TDT system III hardware and an ELC-03XS amplifier (NPI electronics).
Immunohistochemistry and confocal imaging
Mice at postnatal day 20-24 were deeply and terminally anesthetized with Xylazin (5 mg/kg) and Ketamin (10 mg/ml) in 0.9% saline and then transcardially perfused with 2% freshly prepared ice-cold paraformaldehyde with pH adjusted to 7.4. The fixed brain was then removed and brainstem was dissected with a coronal cut few millimetres nasal to the junction between occipital cortex and cerebellum. The brain block was washed overnight in 30% sucrose solution in PBS. For sectioning, the brain block was embedded in Tissue Tek Cryomatrix (Thermo Fisher Scientific, Waltham, MA, USA) and then fixed on the stage of the cryostat (Figocut E cryotome, Reichert-Jung, Depew, NY, USA) such that the caudal aspect was facing upwards and the dorsal side was towards the blade. Advancing from caudal to nasal, 30 µm coronal sections were cut (chamber temperature: −20°C, object temperature: −22°C) and discarded until the appearance of the 7th cranial nerve. Subsequent sections containing aVCN were collected onto electrostatically charged microscope slides (SuperFrost Plus, ThermoFisher Scientific, MA, USA). For parallel processing, one slice of each genotype was collected per slide.
Thereafter, the slices were washed for 10 min in PBS and incubated in Goat Serum Dilution Buffer (GSDB; 16% normal goat serum, 450 mM NaCl, 0.3% Triton X-100, 20 mM phosphate buffer, pH 7.4) for 1 h, followed by incubation in primary antibodies diluted in GSDB, for 3 h, in a wet chamber at room temperature. After washing 2×10 min with wash buffer (450 mM NaCl, 0.3% Triton X-100, 20 mM phosphate buffer) and 2×10 min with PBS, the slices were incubated with secondary antibodies diluted in GSDB, for 1 h, in a light-protected wet chamber at room temperature. The slices were then washed 2×10 min with wash buffer, 2×10 min with PBS and 1×10 min in 5 mM phosphate buffer, and finally mounted with a drop of fluorescence mounting medium based on Mowiol 4-88 (Carl Roth, Karlsruhe, Germany) and covered with a thin glass coverslip. The above described perfusion fixation method was used to stain for RIM-BP2 (Figure 4 A, B). The remaining immunofluorescence experiments (Munc13-1, RIM2, CAST, bassoon) were done on samples taken from RIM-BP2 KO mice aged between p15 – p23 and WT littermates. After obtaining coronal sections from the cryostat the slices were maintained frozen at −20°C until immersion fixation or fixed directly on ice in a solution of PBS containing 3% w/v heat depolymerized PFA (70°C) for 3 minutes. This alternative method of fixation showed robust labelling with less background than perfusion fixed samples labelled against the same markers. For the comparison of CaV2.1 immunofluorescence levels in confocal microscopy and the STED analysis of CaV2.1 and bassoon clusters, we used very brief fixation with 3% PFA for less than one minute (30-40 seconds) as CaV2.1 labelling was impeded by stronger fixation. Thereafter, the blocking and immunolabelling protocols were followed exactly as described above for the perfusion fixed samples.
Primary antibodies used were: rabbit anti-RIM-BP2 (1:200), guinea pig anti-VGLUT1 (1:500), rabbit anti-VGLUT1 (1:1000), mouse anti-gephyrin (1:500), mouse anti-Sap7f407 to bassoon (1:500; Abcam, Cambridge, UK), guinea pig anti-bassoon (1:500), rabbit anti-Munc13-1 (1:200), rabbit anti-RIM1 (1:200), rabbit anti-RIM2 (1:200), rabbit anti-CAST (1:200), rabbit anti-P/Q Ca2+ channel (1:500), chicken anti homer1 (1:200). Unless stated otherwise, primary antibodies were purchased from Synaptic Systems, Göttingen, Germany. Secondary antibodies used were: AlexaFluor488-, AlexaFluor568- and AlexaFluor647-labeled antibodies (1:200, Invitrogen), goat anti-guinea pig STAR580 and goat anti-rabbit STAR 635p (1:200, Abberior GmbH, Goettingen, Germany).
Confocal images were acquired using a laser-scanning confocal microscope (Leica TCS SP5; Leica Microsystems) equipped with 488 nm (Ar) and 561/633 nm (He-Ne) lasers and 63x/1.4 NA oil-immersion objective. STED and confocal images (CaV2.1 and bassoon analyses) were acquired using a 2-color STED microscope (Abberior Instruments, Göttingen, Germany) equipped with 561 and 640 nm excitation lasers, a 775 nm laser for STED (1.2W) and a 100x oil immersion objective (1.4NA, Olympus). Confocal z-stacks were processed with Imaris (Bit-plane, Zurich, Switzerland) for spot detection, co-localization analysis and fluorescence intensity calculation using custom Matlab scripts. STED images of CaV2.1 and bassoon were analyzed using Igor Pro7 (Wavemetrics, Lake Oswego, OR, USA). Samples of both genotypes: RIM-BP2 WT and RIM-BP2 KO were processed and imaged in parallel, using same laser power, gain and microscope settings.
SDS – Freeze-fracture Replica Immuno-Labelling (SDS-FRIL)
Mice at postnatal day 20-24 were deeply and terminally anesthetized with Xylazin (5 mg/kg) and Ketamin (10 mg/ml) in 0.9% saline, and perfused transcardially with ice-cold PBS followed by perfusion with freshly-prepared 2% PFA with 15% saturated picric acid solution in 0.1 M phosphate buffer (PB) with pH adjusted to 7.3. The fixed brain was then removed and brainstem was dissected with a coronal cut few millimetres nasal to the junction between occipital cortex and cerebellum. The brain block was washed over night in 30% sucrose solution in PBS. Coronal slices (130 µm thick) were cut from the fixed brain block fusing a vibratome microslicer (Linear-Pro7, Dosaka) in ice-cold PBS. The rostral anterior ventral cochlear nuclei were trimmed by hand from the slices. The trimmed sections were then immersed in graded glycerol concentrations of 10%-20% at room temperature for 20 min each, followed by 30% at 4°C overnight. The trimmed sections were sandwiched between two metal carriers and then rapidly frozen by a high-pressure freezing machine (HPM010, BAL-TEC, Balzers). Using a freeze etching device (BAF060, BAL-TEC), frozen samples were then fractured into two parts at −115°C, and the fractured faces were replicated by sequential deposition of carbon (thickness: 5 nm from 90° angle), platinum (thickness: 2 nm from 60° angle, unidirectional), and carbon again (thickness: 20 nm from 90° angle). After thawing, the tissue debris attached to the replica was digested with gentle shaking at 80°C for 18 h, in a solution containing 2.5 % SDS, 20% sucrose, and 15 mM of Tris-HCl with pH set to 8.3. The replicas were washed 3 x 10 min in wash buffer (0.1% Tween-20, 0.05% BSA, 0.05% NaN3 in TBS, pH 7.4), and then the non-specific binding sites were blocked with 5% BSA in wash buffer for 1 h at 4°C.
For multiple immunolabelling against CaV2.1 and AZ proteins, replicas were first incubated with guinea pig anti-CaV2.1 antibody (8 µg/ml in 1% BSA; Frontier Institute AB_2571851) at 15°C for 3 days, then with anti-guinea pig secondary antibodies conjugated with 10 nm gold particles (1:30 diluted in 5% BSA; British Biocell International) at 15°C overnight, followed by incubation with a cocktail of rabbit anti-AZ proteins antibodies (anti-ELKS at 2 µg/ml in 1% BSA; gift from Prof. Ohtsuka raised against rat ELKS aa117-142, anti-Neurexin at 4 µg/ml in 1% BSA; gift from Prof. Watanabe raised against aa1499-1507 and anti-RIM at 4 µg/ml in 1% BSA; Synaptic Systems 140203) at 15°C overnight, and finally with anti-rabbit secondary antibodies conjugated with 5 nm gold particles (1:30 diluted in 5% BSA; British Biocell International) at 15°C overnight. After immunolabelling, replicas were rinsed three times with 0.05% BSA in TBS, washed with TBS and distilled water, and mounted on formvar-coated copper grids.
The labelled replicas were imaged using a Tecnai-12 transmission electron microscope (FEI; AV 120 kV). To obtain a planar view for quantitative measurement of immuno-gold particle number and densities, profile of synaptic structures (AZ and PSD) were tilted in the electron beam. IMP clusters representing PSDs were manually demarcated and the area was measured using ImageJ software (Rubio et al., 2017). Active zone areas were marked by hand with the experimenter being blinded to the identity of the two genotypes. Quantitative analysis of immune-gold particles was done using an in-house software tool – Gold Particle Detection and Quantification (Luján et al., 2018). To define clusters of gold particles the threshold for the distance between particles belonging to the same cluster was calculated as µ + 2σ, where µ and σ are the mean and standard deviation obtained from a Gaussian fit to the distribution of nearest neighbour distances (NND) between particles. µ + 2σ was 43.02 nm and 43.92 nm for CaV2.1 gold particles in RIM-BP2 WT and KO respectively. We set the threshold at 40 nm to match the value used in a previous analysis (Miki et al., 2017). The distances between particles were measured from their centers of mass, and the minimum number of particles required to form a cluster was set to three. We additionally compared our ‘real’ distribution of NNDs between gold particles and their clustering to 500 random and fitted simulations (keeping the NNDs between the simulated particles similar to the ones between the ‘real’ gold particles) generated by Monte-Carlo simulations (as described in (Luján et al., 2018; Kleindienst et al., 2020)) to confirm that the clusters visualized through our analysis are not generated by chance.
High-pressure freezing, Freeze substitution and Electron tomography
Parasagittal slices from cochlear nuclei were obtained as described for slice electrophysiology. Slices containing the cochlear nucleus were trimmed and mounted onto type A specimen carriers (Leica Microsystems, Wetzlar, Germany) filled with cutting solution. The flat side of the type B carriers (Leica Microsystems, Wetzlar, Germany) was dipped in 1-hexadecene (Sigma-Aldrich, Wetzlar, Germany) and placed onto the type A carriers. Samples were frozen immediately using a HPM100 (Leica Microsystems, Wetzlar, Germany) and transferred into liquid nitrogen. Freeze-substitution was performed in an EM AFS2 (Leica Microsystems, Wetzlar, Germany) according to Wong et al. (Wong et al., 2014). The slices were incubated in 0.1% (w/v) tannic acid in acetone at −90°C for 4 days and afterward washed three times for 1 h each in acetone at −90°C. 2% (w/v) osmium tetroxide in acetone was applied and incubated for 40.4 h. During that time the temperature was raised slowly to 4°C (10°C/h). At 4°C, osmium tetroxide was removed, and the samples were washed with acetone three times and brought to room temperature. Slices were infiltrated in epoxy resin (Agar-100 kit, Plano, Germany; epoxy/acetone 1:1 3-6 h; 100% epoxy overnight). Finally, samples were further incubated in fresh 100% epoxy and placed in embedding molds.
After polymerization for 48 h at 70°C, excess resin was removed with a fine file (DiAtome, Switzerland) and the block was trimmed to a pyramid using a razor blade. To check the region and the structural preservation, 65 nm ultrathin sections were cut with a diamond knife (DiAtome, Switzerland) using an EM UC7 (Leica Microsystems, Wetzlar, Germany) ultramicrotome. Sections were collected on formvar-coated copper slot grids (Athene, Plano, Wetzlar, Germany, for ultrathin sections). For electron tomography, 250 nm semi-thin sections were obtained and collected on mesh grids (100 mesh; Athene, Plano, Wetzlar, Germany, for semi-thin sections). Post-staining was performed with Uranyless (EMS, Hatfield, PA) for 20 min.
The region and quality of the tissue was checked at 80 kV using a JEM1011 transmission electron microscope (JEOL, Freising, Germany) equipped with a GatanOrius 1200A camera (Gatan, Munich, Germany). Electron tomography was performed as described previously (Wong et al., 2014). 10 nm gold beads (British Bio Cell/Plano, Germany) were applied to both sides of the stained grids. Big synaptic terminals on bushy cells were identified and tilt series from endbulb AZs were acquired at 200 kV using a JEM2100 transmission electron microscope (JEOL, Freising, Germany) mostly from −60° to +60° with a 1° increment at 15,000× using the Serial-EM software package with an image pixel size of 0.95 nm (Mastronarde, 2005). Tomograms were generated using the IMOD package etomo (Kremer et al., 1996).
Only asymmetric synapses with clearly identifiable PSDs were analyzed. However, in high-pressure frozen samples, PSDs appear less electron-dense compared to chemical fixed synapses. Only AZs that showed a PSD and a clear synaptic cleft, originating from large presynaptic terminals were analyzed to exclude inhibitory synapses.
Tomograms were segmented semi-automatically using 3dmod (Kremer et al., 1996). The AZ membrane was manually segmented every 15 virtual sections for five consecutive virtual sections and then interpolated across the Z-stack, following the extent of the PSD and the parallel synaptic cleft. Moreover, virtual sections were corrected manually after interpolation. The total surface area of this object was then divided by two to calculate the AZ area.
Synaptic vesicles (SVs) were reconstructed at their maximum projection and the sphere size was adjusted for each vesicle. The smallest distances from the outer leaflet of the SV membrane to the inner leaflet of the AZ membrane were measured and SVs in contact with the AZ membrane were defined as morphological docked SVs (0-2 nm distance). Moreover, all vesicles within 200 nm of the AZ were quantified and categorized in 20 nm bins. The radii of the SVs were determined with the program “imodinfo” of the IMOD software package and the diameters were calculated. For quantification of lateral distances of docked SVs, models of tomogram top-views with docked SVs were imported in ImageJ and the center of the captured AZ area was defined by setting two diagonal lines from respective edges of the AZ. The crossing point was defined as the center and the distances were measured from the outer membrane of the SV towards the center point.
Data analysis
Electrophysiology data were analyzed using Igor Pro (Wavemetrics, Lake Oswego, OR, USA), Mini Analysis (Synaptosoft Inc., Fort Lee, NJ, USA) and GraphPad Prism software (La Jolla, CA, USA). Synaptic delay was calculated as the time between the start of stimulus (voltage output of the amplifier as dictated by the experiment protocol) and the time when the respective EPSC response reached 10% of its peak amplitude.
Confocal images were analyzed using ImageJ software, Imaris (Bitplane AG, Zurich, Switzerland) and Matlab (Mathworks). STED images of CaV2.1 channels were analysed using custom Igor pro scripts. Single plane slices of the top (coverslip proximal) and/or bottom (distal from coverslip) membranes of BCs were imaged to capture the CaV2.1 spots on a flat surface top view and enable 2D Gaussian fitting. The CaV2.1 spots that were simultaneously closely juxtaposed to puncta of both homer1 and bassoon were fitted with Igor Pro’s 2D Gaussian function. Figures were assembled for display using Adobe Illustrator (Adobe Systems, Munich, Germany). Unless reported otherwise, statistical significance between groups was determined by either unpaired Student’s t-test (in case of normally distributed data with comparable variances between the groups) or Wilcoxon rank sum test (when data distribution did not satisfy the criteria). Normality of distribution was tested with Jarque-Bera test and variances were compared with F-test. Data were presented as mean ± S.E.M. when compared using Student’s t-test. In case of Wilcoxon’s rank sum test, data were presented as box and whisker plots showing grand median (of the means of all recordings), lower/upper quartiles, 10-90th percentiles). *, **, ***, **** indicate p < 0.05, 0.01, 0.001 and 0.0001 respectively.
Results
Deletion of RIM-BP2 impairs synchronous transmitter release at the endbulb of Held synapse
To determine the functional role of RIM-BP2, we studied synaptic transmission at the endbulb of Held synapse in acute parasagittal slices of the brainstem of constitutive RIM-BP2 knockout mice (RIM-BP2 KO, (Grauel et al., 2016)) recording spontaneous (no stimulation, no TTX applied) and evoked excitatory postsynaptic currents (sEPSCs and eEPSCs, respectively) from bushy cells (BCs) of the anteroventral cochlear nucleus (aVCN, Fig. 1A) at postnatal days 15-21. eEPSCs were elicited by minimal electrical stimulation of the presynaptic ANF by a monopolar electrode placed in the proximity (∼3 cell diameters away) of the recorded BC, whereby each stimulus is aimed to elicit one action potential in one endbulb (Yang and Xu-Friedman, 2008).
Experiments of Figure 1 were performed in the presence of 1 mM kynurenic acid and 100 µM Cyclothiazide (CTZ) to avoid saturation and desensitization of AMPA receptors (Chanda and Xu-Friedman, 2010), respectively. The concentration of CTZ used here, was adopted from previous reports at the calyx of Held (Sakaba and Neher, 2001; Thanawala and Regehr, 2013). The former study (Sakaba & Neher, 2001) examined the NMDA current in the presence of 100 µM CTZ and found no evidence for a presynaptic CTZ effect at the calyx of Held. The eEPSC amplitude was reduced in RIM-BP2 KO BCs as compared to littermate wildtype (WT, Fig. 1B-C, 3.50 ± 0.37 nA (KO) vs. 5.48 ± 0.73 nA (WT), p = 0.022, Mann-Whitney U-test). There was a tendency towards slower eEPSC kinetics, but this did not reach significance for either synaptic delay (Fig. 1D) or rise and decay time (Fig. 1E-G), (p-value ≥ 0.05, Mann-Whitney U-test). EPSC recordings in the absence of kynurenic acid and CTZ (Fig. 1-1) confirmed the eEPSC reduction, eliminating the possibility of CTZ obscuring the changes in AMPA receptor composition or inducing membrane potential changes. The eEPSC kinetics was significantly slower for RIM-BP2 KO BCs under these conditions (Fig. 1-1).
In order to test for potential changes in the quantal release properties we recorded spontaneous EPSCs (sEPSCs) from BCs (Fig. 1-2). We did not observe differences in the sEPSC amplitude (Fig. 1-2A-C), kinetics (Fig. 1-2D-F) and frequency (Fig. 1-2G; p-value ≥0.05 for all 3 quantities), which was also the case when recording in the absence of kynurenic acid and CTZ (Table 1). Unaltered sEPSCs suggest that the properties of single SV release and of postsynaptic glutamate response are intact in RIM-BP2 deficient endbulb synapses at BCs.
Quantal size being unaltered, the reduced eEPSC alteration could result from either: (i) an impaired stimulus-secretion coupling due to reduced Ca2+ influx and/or altered topography of Ca2+ channels (Krinner et al., 2017) and fusion competent SVs (Acuna et al., 2015; Grauel et al., 2016; Luo et al., 2017), or (ii) due to an impaired SV priming that has recently been shown to involve RIM-BP2 interaction with Munc13-1 (Brockmann 2020). We first checked for changes in presynaptic Ca2+ influx in the absence of RIM-BP2 using ruptured-patch recordings from the endbulb of Held (Lin et al., 2011) in mice after the hearing onset (postnatal days 13-16, Fig. 2). We did not observe significant changes in the peak Ca2+ current amplitude or peak Ca2+ current density in KO endbulbs of Held (Fig. 2).
Next, we investigated whether eEPSC alteration is caused by a reduction in release probability or altered vesicle pool dynamics. We used high frequency stimulation to assess short-term plasticity, release probability as well as the size and dynamics of the RRP during quasi physiological regimes of synaptic transmission. Fifty consecutive stimuli were delivered at 100, 200, and 333 Hz (Fig. 3: in the presence of 1 mM kynurenic acid and 100 µM CTZ; Fig. 3-1 in the absence of both drugs). Different from the prominent short-term depression typical for the endbulb of Held (Oleskevich and Walmsley, 2002; Yang and Xu-Friedman, 2008), RIM-BP2 deficient endbulb synapses showed an initial facilitation followed by slower depression (Fig. 3, Table 2, Fig. 3-1). Facilitation was evident from the paired pulse ratio (PPR) > 1 in RIM-BP2 deficient endbulb synapses for all inter-stimulus intervals tested, while PPR was consistently ∼ 0.8 in WT (Table 2). The extent of depression assessed as EPSC30-50/EPSCmax (Fig. 3, Table 2, Fig. 3-1) tended to be less in RIM-BP2 deficient endbulb synapses without reaching statistical significance. Release probability (Pr) as well as the size and dynamics of the RRP were estimated by applying two variants of the cumulative analysis to the EPSC trains (Fig. 3): Schneggenburger-Meyer-Neher (SMN) method (Schneggenburger et al., 1999) and Elmqvist & Quastel (EQ) method (Elmqvist and Quastel, 1965); for review see: (Neher, 2015). Both methods revealed a significant reduction of Pr in RIM-BP2 KO synapses while RRP size and the replenishment rate were not significantly altered (Table 2). In an attempt to address the question whether the reduced release probability reflects a smaller complement of high release probability SVs (“tightly docked” or “super-primed” SVs (Neher and Brose, 2018)) we followed a previously described analysis that found a reduction of SV super-primed SVs upon genetic deletion of all rab3 isoforms (Schlüter et al., 2006). We subtracted the quantal content of each RIM-BP2 KO response from the respective RIM-BP2 WT response during train stimulation (Fig. 3M-O). This analysis revealed the strongest difference between the two genotypes occurs at the beginning of the train, where the RIM-BP2 KO endbulbs release 30-40 SVs less than the RIM-BP2 WT, which is consistent with a reduction of high release probability (“super-primed”) SVs in the absence of RIM-BP2. The difference vanishes already after the first two eEPSCs, when the subtraction curves for 200 and 333 Hz actually cross the zero line, indicating that release from KO-synapses is actually slightly larger than that from WT. Possible reasons for this small difference include i) protracted release of low release probability SVs that could result from impaired CaV-release site coupling and ii) residual desensitization that is more prominent for RIM-BP2 WT synapses with larger eEPSCs (despite 100 µM cyclothiazide and 1 mM kynurenic acid). During the steady state response to the train stimulation, the WT synapses tended to release more SVs than the KO (average difference: 6 SVs for 100 Hz, 3 SVs for 200Hz, no difference for 333 Hz).
In order to further scrutinize RRP dynamics, we studied the recovery from short-term depression, by measuring eEPSC amplitudes elicited by single stimuli presented at varying time intervals after a conditioning 100 Hz train of 50 pulses (Fig. 4). Recovery is displayed as the eEPSC amplitudes normalized to the amplitude of the first eEPSC of the conditioning train (Fig. 4A-B): RIM-BP2 deficient endbulb synapses showed a major reduction of fast recovery. The time course was fitted with a double exponential function revealing the following amplitude and tau values for the fast and slow components of recovery: A1 = 0.17 ± 0.05, τ1 = 123 ± 97.6 ms; A2 = 0.69 ± 0.06, τ2 = 6.1 ± 1.7 s for RIM-BP2 KO and A1 = 0.31 ± 0.04, τ1 = 96 ± 30 ms; A2 = 0.4 ± 0.04, τ2 = 4.6 ± 1.4 s for WT.
Next, we evaluated the impact of the impaired synaptic transmission on processing of auditory information using juxtacelluar recordings from putative spherical bushy cells (for simplicity refered to as “BCs”) in vivo (Fig. 5). Glass microelectrodes were stereotactically navigated to the aVCN from an occipital craniotomy, sound stimuli were presented in the open field, and BCs identified based on electrode position, first spike latency, regularity of firing and shape of the peristimulus spike time histogram (see Materials and Methods). The (non-significant) trend toward lower spontaneous firing rate (Fig. 5A, Table 3) is consistent with the reduced spontaneous ANF input (Krinner et al., 2017) and the trend toward a lower frequency of sEPSCs in RIM-BP2 KO BCs (Fig. 1-2). Sound threshold and frequency tuning were unaltered, whereby the RIM-BP2 KO data set contained more BCs with higher characteristic frequency (Fig. 5B-C, Table 3).
The peak firing rate at sound onset was significantly reduced in RIM-BP2 KO BCs (Fig. 5D, by approximately 40%, Table 3) compatible with the reduced initial release probability. The adapted firing rates were not significantly reduced (Fig. 5Dii, Table 3), which is consistent the better maintained EPSC amplitudes during steady state response to train stimulation in BCs (RIM-BP2 WT: 1.2 ± 0.2 nA, RIM-BP2 KO: 0.9 ± 0.1) (Fig. 3A-F, Table 2) and the normal adapted firing rate of ANFs (Krinner et al., 2017).
In addition, we found the temporal jitter of the first spike after stimulus onset to be greater in RIM-BP2 KO BCs, while the first spike latency was comparable between the two genotypes (Fig. 5E, Table 3). The stronger reduction of the peak rate and increased first spike latency jitter of BCs in comparison to ANF likely reflects the impaired transmission at the RIM-BP2-deficient endbulb, which degrades information processing in the lower auditory pathway beyond what is caused by the mildly affected synaptic sound encoding in the cochlea. This hypothesis is further supported by the auditory brainstem responses of RIM-BP2 KO mice, which show a more pronounced amplitude decline for the aVCN related wave III than for the auditory nerve related wave I (Krinner et al., 2017)). In conclusion, the reduced release probability of the endbulbs impairs the transmission of sound onset information, which is likely to hamper hearing and auditory tasks such as gap detection and sound localization in particular.
RIM-BP2 disruption appears not to alter the molecular composition of the endbulb active zones
Given the scope of protein interactions of RIM-BP that includes Ca2+ channels, large conductance Ca2+ activated K+ channels (Sclip et al., 2018) and multi-domain proteins of the AZ, we considered the possibility that some of the above described physiological alterations might reflect changes in the abundance of other AZ proteins. In order to test for potential effects of RIM-BP2 disruption on the molecular composition of the AZs, we performed semi-quantitative, confocal immunofluorescence microscopy in coronal brain slices. RIM-BP2 WT and RIM-BP2 KO samples were harvested and processed strictly in parallel. Likewise, images were acquired using the same laser power and gain settings at the same confocal microscope. Excitatory AZs facing the postsynaptic BC were identified by co-localization of immunofluorescence of the targeted AZ protein with the immunofluorescence of the vesicular glutamate transporter VGlut1 or a juxtaposition to the immunofluorescence of homer 1, a scaffold of excitatory synapses and a lack of juxtaposition to immunofluorescence of gephyrin, a scaffold of inhibitory synapses (Fig. 6). We focused our analysis on the spherical or ovoid BC soma which are engaged by a corona of synapses. Staining for RIM-BP2 showed the expected corona of immunofluorescence spots in WT slices, but no obvious synaptic immunofluorescence in RIM-BP2 KO slices (Fig. 6A, Fig. 6B, left: integrated fluorescence within the Vglut1 positive volume, right: lack of spots). We did not observe significant differences in the integrated immunofluorescence or the number of puncta of Munc13-1 (Fig. 6C, D), RIM2 (Fig. 6E, F), CAST (Fig. 6G, H), and bassoon (Fig. 6I, J) in RIM-BP2 KO slices, suggesting an unaltered abundance of these multi-domain proteins at the excitatory AZ facing the BC in the absence of RIM-BP2.
Increased Ca2+ influx improves release probability but unmasks impaired SV replenishment during train stimulation
Unaltered Ca2+ influx and RRP size but reduced Pr led us to focus the analysis on the coupling of Ca2+ channels to SV release. As a first approach we increased the presynaptic Ca2+ influx by elevating the extracellular Ca2+ concentration [Ca2+]e from physiological (2mM) to 4mM. This manipulation abolished the differences in Pr (both time course of depression and PPR were WT-like, Fig. 7, Table 4). This is consistent with a greater diffusional distance between Ca2+ channels and SV release sites that can be overcome when more Ca2+ enters per channel opening. Alternatively, or in addition, greater Ca2+ influx might foster Ca2+ dependent priming or facilitation of release. In addition, increased Ca2+ influx unmasked a slowed SV replenishment during train stimulation in RIM-BP2 deficient endbulbs.
RIM-BP2 disruption alters the topography of Ca2+ channels at endbulb active zones
In order to probe for potential morphological correlates of altered release probability we employed immunofluorescence microscopy and electron microscopy of immunolabeled Ca2+ channels. First, we performed a strictly parallel study of CaV2.1 distribution at excitatory synapses around the BCs of RIM-BP2 KO and WT mice using stimulated emission depletion (STED) nanoscopy (Fig. 8A-K) as well as confocal microscopy (as described above, Fig. 8-1A, B). We used bassoon and homer1 as context markers to analyse the presynaptic CaV2.1 immunofluorescence distribution. Both the integrated CaV2.1 immunofluorescence intensity per AZ and the number of CaV2.1 puncta were unaltered at the confocal level (Fig. 8-1B), which is consistent with finding normal presynaptic Ca2+ influx (Fig. 2). For our 2-colour STED analysis we focused on AZ/PSD appositions (CaV2.1/homer1, bassoon/homer1) of endbulbs of Held.
We interpret the CaV2.1 and bassoon puncta discerned by STED to represent individual AZs. As the PSD was studied at confocal resolution, we assume the larger homer1-spots represent a merger of several small PSDs. We operationally defined the organization of several CaV2.1 or bassoon puncta around a single homer1 spot a synaptic contact assembly (SCA) whereby its center corresponds to the center of the homer1 punctum. Our analysis indicated a wider spatial distribution of CaV2.1 puncta in RIM-BP2 KO which localized further away from the SCA center and from each other (Fig. 8A-K), suggestive of a more dispersed CaV2.1 topography. Similar findings were made for bassoon (Fig. 8-2).
We then turned to immunolabelling of CaV2.1 in SDS-treated freeze-fracture replica (SDS-FRIL, (Nakamura et al., 2015)). To image the endbulbs onto the BCs, we focused on the BC rich rostral-most aVCN sections of the brainstem. Endbulb terminals were prominently distinguishable in our replicas by their large size synapsing on to the BC soma (Fig. 9-1A). We also validated that we were imaging the correct area and cell type, by analysing the intramembrane particle (IMP) clusters representing PSDs on the exoplasmic face (E-face) of the BC soma (Fig. 9-1C-F). Our estimates of PSD areas (Fig. 9-1B, Table 5) were comparable to the ones previously reported for ANF-BC synapses (Rubio et al., 2017). We then assessed the protoplasmic face (P-face) of the replicas for the analysis of AZ proteins and CaV2.1 channel distribution (Fig. 9-2). AZs were located by simultaneous immunolabelling of three characteristic AZ proteins: RIM, neurexin, and ELKS with 5 nm gold particles. The number of AZ particles was less than that previously observed in other types of synapses (Miki et al., 2017) which might reflect lower expression of ELKS in the endbulb synapses. Since the samples from both genotypes were handled simultaneously by the same experimenter, the comparison between the genotypes remains valid. Nonetheless, given the low labelling efficiency for AZ proteins, we used AZ markers primarily to identify the location of AZs. AZ area was characterized by IMPs of distinct shape, number and size compared with those in surrounding areas, and demarcated manually by connecting the outermost IMPs (Fig. 9-2B-C). The estimated AZ area was comparable between RIM-BP2 KO and WT (Fig. 9-2D). When analysing the distribution of CaV2.1 channels labelled with 10 nm gold particles within the AZ area, both the number and density of CaV2.1 particles were significantly reduced in the RIM-BP2 KO (Fig. 9-2G-H, Table 5).
A low labelling efficiency for AZ proteins would overestimate the AZ to AZ and CaV2.1 to AZ particles nearest neighbour distances (NNDs). Hence, we only compared the NND for CaV2.1 to CaV2.1 and AZ to CaV2.1 particles. The value of mean + 2 standard deviations of the Gaussian fit to the distribution of CaV2.1 to CaV2.1 NNDs was considered the threshold for the maximum distance by which two particles can be separated and still belong to the same cluster (Fig 9A-B, see materials and methods): mean + 2 s.d. were 43.09 nm and 43.92 nm for WT and RIM-BP2 KO respectively. The threshold used for defining a cluster in our analysis was 40 nm as used in previous analysis (Miki et al., 2017). In many KO AZ images, there were not even three CaV2.1 gold particles to qualify as a cluster (Fig. 9-2C). Within the qualified clusters, we still found a significantly reduced number of CaV2.1 particles per cluster in the RIM-BP2 KO (Table 5) which is consistent with a more dispersed CaV2.1 topography at the AZ as indicated in the STED analysis. To confirm that the clusters obtained in our analysis were valid and not reflecting chance occurrence, we compared our ‘real’ data to 500 random simulations of cluster arrangements (see materials and methods, and (Luján et al., 2018; Kleindienst et al., 2020)). Table 6 confirms the validity of CaV2.1 clusters and NNDs between CaV2.1 to CaV2.1 and AZ to CaV2.1 particles. The major findings of our FRIL analysis are the significantly lower number and density of CaV2.1 channels as well as the increased AZ to CaV2.1 NND in RIM-BP2 KO AZs (Fig. 9C, Table 5). There was a trend toward larger CaV2.1 to CaV2.1 NND at RIM-BP2 KO AZs which did not reach significance (Fig. 9D). The increased AZ to CaV2.1 NND implies a looser coupling between the Ca2+ channels and the AZ proteins likely marking SV release sites.
RIM-BP2 disruption alters the SV organization at the endbulb active zones
Next, we high pressure froze aVCNs slices, acutely prepared as for physiology in order to best relate structure and function, for electron tomography analysis of SV organization at the endbulb AZ. Following freeze-substitution, embedding, sectioning and tomography, we rigorously analysed and reconstructed AZ of RIM-BP2 deficient and WT endbulb synapses (Fig. 10). The AZ area, approximated from the extent of the postsynaptic density, was significantly larger in RIM-BP2-deficient endbulbs (Fig. 10C & Table 7, p < 0.01, Student’s t-test). The number (Fig. 10D) and density (Fig. 10E) of SVs per AZ were not altered. For a more comprehensive analysis we compared the SV distribution within 200 nm of the presynaptic AZ membrane (perpendicular to the presynaptic membrane into the cytosol of the presynaptic terminal) in 20 nm bins. Morphologically docked SVs (0-2 nm distance), analyzed separately, were significantly fewer in number at RIM-BP2 KO AZs. Significantly fewer SVs were also found for non-docked membrane proximal SVs (2 to 20 nm) and SVs within 40 to 60 nm from the AZ membrane (Fig 10F, p < 0.01, Wilcoxon rank test, Table 7). We also analysed top-views of the AZs showing only the docked SVs in the models generated from the tomograms and quantified the percentage of AZs with zero to eight docked SVs. We found that 50% of the analysed RIM-BP2 KO AZs showed zero docked SVs, while two docked SVs per AZ were most frequently encountered in WT (27% of the AZs, Fig. 10G). We further tested for effects of RIM-BP2 disruption on the lateral distribution of the docked SVs by setting a central point within the generated top views and quantifying the distances of all docked SVs from the center (we note that the AZ area captured in the tomograms might not necessarily allow proper definition of the AZ center). The docked SVs appeared to be further away from the center in RIM-BP2 KO synapses, possibly representing a broader distribution over the whole AZs area (Fig. 10J). To analyse the membrane proximal SVs in more detail, we quantified the proportion of SVs in the 2-20 nm bin and observed a shift towards fewer SVs at mutant AZs. Whereas most of the WT (15%) AZs contained 6 SVs within 2-20 nm from the AZ membrane, most mutant AZs showed only 2 or 5 SVs within this distance (Fig. 10K). Lastly, by measuring the diameter of SVs, we found that they exhibited comparable sizes at RIM-BP2 KO and RIM-BP2 WT endbulb AZs (WT: 51.94 ± 0.55 nm; KO: 52.67 ± 0.54 nm). We conclude that RIM-BP2 contributes to normal SV docking and SV organization in close proximity to the membrane of the presynaptic AZ. These changes seem compatible with the lower number of super-primed SVs, impaired SV replenishment (Figs. 3 and 7) and the reduced release probability (Fig. 3).
Discussion
Priming of SVs, CaV function as well as the topography of CaV and SV release sites at the AZ, co-determine the probability of SV release in response to the action potential invading the presynaptic terminal. Here, we probed the role of RIM-BP2, thought to serve as molecular linker between CaV and release sites, and alternatively in SV priming via Munc13, in synaptic transmission at the endbulb of Held synapse. Using super-resolution immunofluorescence and immuno-electron microscopy we demonstrate that RIM-BP2 disruption alters the topography of CaV2.1 channels at the AZs. Electron tomography revealed fewer docked and membrane-proximal SVs at the AZ. As a physiological corollary of these structural changes, we found a reduction in the amplitude of evoked EPSCs, reduced release probability, and slowed SV replenishment to the RRP. We postulate that RIM-BP2, likely via interaction with Munc13-1, promotes a ‘superprimed’ (Taschenberger et al., 2016) or ‘tightly docked’ (Neher and Brose, 2018) SV state. Moreover, RIM-BP2 organizes the topography of CaV2.1 channels, tightly coupling them to SV release sites.
Synaptic transmission at calyceal synapses of the lower auditory pathway shows impressive temporal fidelity. At the first central relay of the auditory pathway, co-incident transmission from endbulbs formed by ANFs drives the postsynaptic BCs at hundreds of Hz and with microsecond precision as required for time-critical neural computations such as in sound localization (Trussell, 1999; von Gersdorff and Borst, 2002). Such fidelity is enabled by synergistic adaptations on molecular, synaptic and network levels. In the mouse AVCN, BCs receive input from on average 3-4 endbulbs (Cao and Oertel, 2010; Butola et al., 2017) with approximately 400 excitatory AZs (Nicol and Walmsley, 2002; Mendoza Schulz et al., 2014; Butola et al., 2017). Endbulbs feature a large RRP (Lin et al., 2011) and high release probability (estimates range from 0.2 to 0.7: Oleskevich and Walmsley, 2002; Wang and Manis, 2005; Chanda and Xu-Friedman, 2010; Mendoza Schulz et al., 2014; Butola et al., 2017) which enable massive synchronous parallel release for powerful excitation of BCs. RIM-BP2 deletion hampers this reliable and temporally precise transmission of auditory information: firing at sound onset was impaired both in rate and temporal precision (Fig. 5). We mainly attribute the reduced firing at sound onset (40% reduction) to the observed impairment of endbulb transmission (EPSC1 amplitude reduction: 36%, Fig. 1), as the deficit in sound onset coding in RIM-BP2 KO ANF was mild (approximately 10% reduction in peak onset firing rate, (Krinner et al., 2017)) and convergence of ANF inputs to BCs is expected to alleviate consequences of impaired ANF coding for BC firing (Joris et al., 1994; Buran et al., 2010). These results from single-neuron recordings are in good agreement with previously reported auditory brainstem responses (Krinner et al., 2017), in which the amplitude reduction was lower for wave I (approximately 30 %) than for wave III (approximately 70%). Wave I and III are attributed to the synchronized firing of ANFs and cochlear nucleus neurons respectively (Melcher et al., 1996). Future studies, also employing analysis of gap detection or sound localization should evaluate the behavioural consequences of this temporal processing deficit.
Impaired transmission of sound onset information at RIM-BP2-deficient endbulbs is primarily rooted in a nearly halved initial release probability, which could be restored to wildtype levels upon increasing Ca2+ influx (Fig. 7). We attribute the decreased release probability to i) the altered topography of CaV2.1 and SVs and ii) to the impaired SV docking at RIM-BP2-deficient endbulb AZs. Whole-cell patch-clamp recordings showed normal Ca2+ currents in RIM-BP2-deficient endbulbs (Fig. 2) and confocal imaging of CaV2.1 immunofluorescence semi-quantitatively reported a normal Ca2+ channel complement of the AZs (Fig. 8-1). This suggests that unlike ribbon synapses (Krinner et al., 2017; Luo et al., 2017), RIM-BP2 is not strictly required for establishing a normal presynaptic CaV complement. However, both FRIL and STED imaging of immunolabeled CaV2.1 channels revealed an impaired clustering of CaV2.1 at RIM-BP2-deficient endbulb AZs. The number and density of CaV2.1 immunogold particles was reduced at the AZs and the nearest neighboring CaV2.1 was further away from an immunogold particle marking an AZ protein (Table 5). Nonetheless, distribution of the CaV2.1 immunogold particles was significantly different from random, likely reflecting the presynaptic CaV organization by other multi-domain proteins of the AZ such as RIMs (Han et al., 2011; Kaeser et al., 2011; Jung et al., 2015) and CAST (Dong et al., 2018; Hagiwara et al., 2018). Immunofluorescence of CaV2.1 as well as of bassoon (marking the presynaptic density) was less confined in space and immunofluorescent spots were more oval in shape in the absence of RIM-BP2 as compared to compact, round spots in the WT (Figs. 8 and 8-2). Moreover, the nearest neighboring immunofluorescence spot was further away both for CaV2.1 and bassoon. In summary, our data indicate that RIM-BP2 contributes to orchestrating CaV2.1 channels at the AZ. Based on our data and in line with previous studies on other synapses (Liu et al., 2011; Acuna et al., 2015; Brockmann et al., 2020; Petzoldt et al., 2020), we hypothesize that RIM-BP2 via its interaction with CaV2.1 and AZ proteins, contributes to tight coupling of Ca2+ channels and vesicular release sites also at the endbulb of Held synapse. Future experimental and theoretical studies will be needed to further test this hypothesis.
Recently, an alternative interaction of RIM-BP2 with the C2B domain of Munc13-1 has been reported by which RIM-BP promotes release probability via SV docking/priming (Brockmann et al., 2020). The Munc13-1 C2B domain carrying the KW mutation (Shin et al., 2010), showed the highest affinity for RIM-BP2 binding. Of note, we did not observe a reduction in Munc13-1 or in any other major AZ protein in our semi-quantitative analysis of AZs in RIM-BP2 deficient endbulbs of Held (Fig. 6). This provides confidence in attributing functional and morphological alterations to the lack of RIM-BP2 function rather than to quantitative changes in other AZ proteins. Our functional and electron tomographic analysis of SV pool organization at RIM-BP2-deficient endbulbs now provides evidence for a role of RIM-BP in SV priming, likely via its interaction with Munc13-1. Rigorous electron tomography analysis of docking showed nearly halved numbers of morphologically docked SVs (defined in a distance from 0-2 nm to the AZ membrane) at RIM-BP2-deficient AZs, which might represent a ‘tight docking’ (Neher and Brose, 2018). We speculate this to indicate that Munc13-1-mediated docking that generates primed SVs (Siksou et al., 2009; Imig et al., 2014) partially depends on the interaction with RIM-BP2. The reduction of both docked SVs and those at a distance of 2-20 nm (membrane proximal SVs) from the plasma membrane might then suggest that the upstream loose-docking is rate limiting or less stable in the absence of RIM-BP2. An alternative explanation for retarded recovery from depression might be that RIM-BP2 facilitates release site clearance (Neher and Sakaba, 2008).
Yet, our analysis of pool dynamics in regular [Ca2+]e indicated an unaltered RRP size despite the fact that the numbers of docked and membrane-proximal SVs were nearly halved. Interestingly, we found a trend toward a smaller RRP in RIM-BP2-deficient endbulbs when restoring release probability by enhanced Ca2+ influx. Despite this partial restoration, the first evoked EPSC amplitude in the absence of RIM-BP2 was still only 50% of that in the WT synapses (Fig. 7, table 4). This persisting difference alludes to a deficit in ‘super-primed’ (Fig. 3, table 2) or ‘tightly docked’ SVs (Fig. 10, table 7). It is tempting to speculate that the impaired SV docking is uncovered under conditions that occlude the effect of altered spatial Ca2+ channel-release site coupling on release probability. Our morphological analysis of Ca2+ channel-release site coupling is hampered by i) the low number of docked SVs in electron tomography and ii) the lack of information on the CaV position in electron tomography and of SV docking in FRIL electron micrographs. Nonetheless, there was a trend for docked SVs to be further away from the estimated AZ center, possibly reflecting a more random SV topography due to lack of RIM-BP2-mediated interaction with CaVs. In addition, or alternatively, the RRP estimated by SMN/EQ analyses of responses to train stimulation might contain SVs that undergo tethering, docking and fusion during the train. Recent work by (Pofantis et al., 2021), showed that deleting the presynaptic protein Mover specifically affects the high Pr component of the RRP at the Calyx of Held. Pofantis et al. analysed eEPSC trains with non-negative matrix factorization to reveal components representing sub-pools of SVs with different contributions to transmitter release during the train. Future studies that tackle how such sub-pools deplete and how they recover from depletion in synapses lacking both RIM-BP2 as well as RIM-BP2 and Mover will help to further dissect the contributions of RIM-BP2 to SV super-priming and CaV clustering as determinants of Pr.
Exciting topics for future studies include i) the relative contributions of altered Ca2+ channel-release site coupling and impaired SV docking to the reduced release probability, ii) a potential contribution of release site clearance to the observed deficit in SV docking, and iii) the molecular mechanisms and structure-function relationship of SV replenishment. Our morphological and functional experiments indicate that RIM-BP2 takes a role in tethering SVs near the plasma membrane en route to docking. Functionally, the fast, Ca2+ dependent component of recovery from depression due to trains stimulation was hampered. This could reflect a lower local [Ca2+] due to mislocalization of CaV2.1 channels. Indeed, a key role of Ca2+ in regulating SV replenishment at calyceal synapses has been reported in multiple studies (Wang and Kaczmarek, 1998; Hosoi et al., 2007). Interestingly, enhancing Ca2+ influx by elevated [Ca2+]e uncovered a reduced SV replenishment during train stimulation, which would seem reflect a Ca2+ independent limitation e.g. of SV priming. Future studies on calyceal synapses of mice carrying mutations that target Ca2+ dependent effects on Munc13-1 function and/or the RIM-BP2-Munc13-1 interactions by combined functional and ultrastructural analyses will be required to elucidate this intricate process. To best relate AZ structure and function, future studies will ideally employ optogenetic or electric field stimulation followed by high pressure freezing and EM tomography (Watanabe et al., 2013; Imig et al., 2020 p.202).
Author Contribution
This study was conceived by T.M. and T.B. The experimental work was performed by T.B. (slice electrophysiology, in vivo electrophysiology, electron microscopy in the lab of R.S. with help of P.K., D.K. and R.S.), T.A. (slice electrophysiology, immunohistochemistry) and A.H. (electron microscopy). T.M., T. A., and T.B. prepared the manuscript with contributions of A.H. and C.W.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
We thank S. Gerke, A.J. Goldak and C. Senger-Freitag for expert technical assistance, G. Hoch for developing image analysis routines, and S. Chepurwar and N. Strenzke for technical support and discussion regarding in vivo experiments. We thank Drs. Christian Rosenmund, Katharina Grauel and Stephan Sigrist for providing RIM-BP2 KO mice. We thank J. Neef for help with the STED imaging and image analysis. We thank E. Neher and S. Rizzoli for discussion and comments on the manuscript. Our gratitude also goes to C. H. Huang and J. Neef for constant support and scientific discussion. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through the Collaborative Sensory Research Center 1286 (to C.W. (A4) and T.M. (B5)) and under Germany’s Excellence Strategy - EXC 2067/1-390729940.