114 Figure S2 The number of docked SVs and the size of the AZ region do not. 131 Table S10 Quantitative analysis of tomograms of adult wild-type and. m) EPSC (miniature) excitatory postsynaptic current.
Chemical synapses
The mammalian nervous system consists of the central nervous system (CNS), which consists of the brain and spinal cord, and the peripheral nervous system, which connects the CNS to the rest of the body, including muscles and internal organs. Neurons are the fundamental units of the nervous system responsible for receiving and transmitting these signals.
Synaptic vesicle formation and cycle
Exocytosis pathway
The vesicular (V-) SNARE protein synaptobrevin thereby interacts with the target (T-) SNARE proteins syntaxin-1 and SNAP25 (synaptosome-associated protein of 25 kDa) on the plasma membrane, forming an α-helical SNARE complex (Poirier et al. ., 1998; Sutton et al., 1998). At the ultrastructural level, it has also been shown that at least the partial formation of SNARE complexes is required for SV docking and priming (Imig et al., 2014).
Endocytosis pathway
The cytomatrix of the active zone
The role of RIM and RIM-BP at synapses
RIM-BPs are involved in recruiting and organizing the topography of VGCCs (Grauel et al., 2016; Krinner et al., 2017). This function has been shown to depend on the interaction of RIM BPs with bassoon (Davydova et al., 2014).
The vertebrate-specific protein Mover
While the lack of the neuron-specific RIM-BP1 and RIM-BP2 did not affect Ca2+ current at Held's calyx (Acuna et al., 2015), the absence of RIM-BP1 and RIM-BP2 reduced the number of VGCCs on the ribbon. -type AZs in the mouse cochlea and retina (Krinner et al., 2017; Luo et al., 2017). Synaptic transmission is only slightly affected by RIM-BP deletion at mammalian synapses (Acuna et al., 2015; Grauel et al., 2016; Krinner et al., 2017; Luo et al., 2017), while in Drosophila larval neuromuscular junctions The influx of Ca2+ is impaired, resulting in a strongly reduced Pr (Liu et al., 2011).
Synaptic vesicle pools at conventional synapses
However, ultrastructural studies in Drosophila larval neuromuscular junctions revealed a strong spatial mixing of the recycling and the reserve pool ( Denker et al., 2009 ). Superprimed SVs show a higher Pr (about 0.5), but they regenerate slowly after fusion ( Taschenberger et al., 2016 ).
The role of presynaptic mitochondria in synaptic activity
The presence of mitochondria further maintains synaptic transmission during prolonged stimulation, while absence of a mitochondrion results in depletion of SV release (Sun et al., 2013). In addition, the mobilization of reserve pool SVs has been shown to be disrupted at synapses lacking mitochondria due to an insufficient supply of ATP (Verstreken et al., 2005).
The auditory system
- The early auditory pathway
- Otoferlin, a hair cell-specific protein involved in SV exocytosis
- The cochlear nucleus
- Developmental maturation of auditory synapses
- Synapses on bushy cells
- The structure of endbulbs
- Influence of neuronal activity on endbulb structure
The exocytosis of neurotransmitters activates postsynaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors on afferent boutons of the spiral ganglion neurons (SGNs) of the auditory nerve (Glowatzki & Fuchs, 2002; Schnee et al. al., 2011). However, only after 48 weeks is a decrease in the number of SGNs observed (Stalmann et al., 2021).
Hypotheses and Aims
In the next part of the thesis, I analyzed the effects of the CAZ protein RIM-BP2 on the organization of SV pools at the endbulb AZ. Mover has been shown to be present in a subset of synapses in different brain regions (Kremer et al., 2007; Wallrafen & Dresbach, 2018) and its function is not fully elucidated.
Animals
Materials
Chemicals
Equipment
Solutions
Sample preparation for electron microscopy
High-pressure freezing (HPF) and freeze-substitution (FS)
- Vibratome slice preparation
- High-pressure freezing (HPF)
- Freeze-substitution (FS)
During the final wash step, the samples were brought to room temperature, infiltrated into epoxy resin (Table 2.6) and embedded in fresh 100% epoxy resin, placed in embedding molds and polymerized at 70°C for 48 hours. During FS, samples were slowly brought to room temperature while incubating with 0.1% tannic acid and 2% (w/v) osmium tetroxide.
Conventional embedding
Slices containing AVCN were trimmed and prepared for high-pressure freezing by assembling a sandwich with sample carriers. Samples were immediately frozen and stored in liquid nitrogen until freeze exchange (FS) was performed.
Trimming of embedded samples
Formvar coating of slot and mesh grids
Sectioning and post-staining
- Electron tomography
- Generation of tomograms
- Quality assessment of tomograms
- Model rendering and three-dimensional (3D) analysis
- Electron tomography of presynaptic mitochondria
No distortion of the tissue due to freezing artefacts can be observed and the pre- and postsynaptic membrane are parallel. CMs, was calculated by dividing the total CM area by the total internal volume of the cristae.
Statistical analysis
Ultrastructural end-bulb maturation of Held's active zones compared with wild-type and otoferlin-deficient mice. As part one, this part of the paper is based on Hintze et al., 2021.
Ultrastructural changes at endbulb active zones upon maturation from
The density, size and distribution of synaptic vesicles are unaltered at endbulb of
Therefore, I measured the sizes of SVs and compared the mean diameters at individual AZs, but did not find a significant change between P10 and P21 (Fig. 3.2H; Table S1). The schema of an AZ depicts the bins used to analyze the distribution of SVs.
Refinement of the SV pool
- The density of docked SVs is unaffected from the transition to hearing
- The pool of membrane-proximal synaptic vesicles is comparable between P10
The density of anchored SVs correlates with the density of all SVs at the AZ (see text for details). Furthermore, the ratio of anchored to membrane-proximal SVs remained the same before and after hearing onset (Fig. 3.4G).
Summary
Presynaptic mitochondria within the AVCN
- Mitochondria are highly variable in size and shape
- Presynaptic mitochondria are larger after the onset of hearing at endbulbs of Held
- Mitochondrial volumes increase at non-endbulb synapses from pre-hearing to young
- Mitochondria are larger at non-endbulb synapses compared to endbulb AZs of P10
- Mitochondria at endbulb synapses display a higher crista membrane density after the
- Summary
The sum of the CM surface area is greater at mitochondria from non-terminal globule synapses. The CM surface area was also comparable between mitochondria in endosphere and nonendosphere mitochondria (Fig. 3.10E).
Ultrastructural changes at endbulb active zones upon adulthood and lack
- The synaptic vesicle pool increases during development of wild-type active zones
- Synaptic vesicle numbers decline in ageing Otof -/- mice
- Synaptic vesicle numbers are comparable between wild-type and mutant mice at
- Active zones of adult Otof -/- endbulbs contain less morphologically docked synaptic
- The density of membrane-proximal vesicles initially increases but declines towards
- Sizes of synaptic vesicles decrease during development at Otof -/- endbulb active
- Summary
The density of SVs within 80 to 200 nm away from the AZ membrane also showed a steady increase towards adulthood (Fig. 3.12E). The total number of membrane-proximal SVs was comparable between the age groups at AZs of wt end bulbs (Fig. 3.14B, Table S3).
RIM-BP2 disruption alters the vesicle organization at endbulb active
RIM-BP2 -/- endbulb active zones exhibit a different distribution of synaptic vesicles
Anchoring density as well as SVs within 2–20 and 40–60 nm is lower in mutant AZs, while SV densities in other bins are comparable.
RIM-BP2 -/- active zones contain less synaptic vesicles in membrane proximity
Furthermore, since RIM-BP2 is involved in the recruitment and organization of VGCCs ( Grauel et al., 2016 ; Krinner et al., 2017 ), the effects of RIM-BP2 deletion on the lateral distribution of the docked SVs were investigated. The quantification revealed that docked SVs were further away from the center at RIM-BP2-/- AZs, which may speak for a broader distribution over the AZs area (Fig. 3.18D).
The lack of Mover has differentially effects on excitatory and inhibitory
The number of SVs per AZ at endbulb and inhibitory synapses is similar in wt compared with Mover−/− mice, but is greater in inhibitory compared with endbulb AZs of wt as well as Mover−/− mice. This difference in the number of anchored SVs resulted in a significantly higher ratio of anchored and membrane-proximal SVs (Fig.
Summary
To better understand the underlying processes of synaptic transmission, a quantitative morphological analysis of synaptic structures is required. In addition, a detailed description of synaptic morphology helps to define structural correlates of functional aspects of synaptic transmission.
Methodology
Sample preparation
At the level of individual synapses, chemical fixation was shown to affect SV shape and distribution: the SVs of chemically fixed inhibitory synapses appeared flattened while they were round after flash freezing and FS (Fig. 1.4; Korogod et al ., 2015; Tatsuoka & Reese, 1989). After HPF/FS they exhibited fine filamentous structures instead of a coarse electron-dense structure (Fig. 1.4; Rostaing et al., 2006; Tatsuoka & Reese, 1989).
The advantages of 3D electron tomography
However, problems in preserving large volumes of brain tissue have been described leading to structural changes and the formation of ice crystals comparing regions at the border and in the center of brain slices (Korogod et al., 2015; Rostaing et al., 2006). To rule out such problems, the tomograms I used for the analysis underwent a strict quality control to ensure the following criteria (see also section 2.7.3): a good structural preservation without freezing artefacts, the presence of a PSD to distinguish between excitatory and inhibitory synapses and the distinction between terminal bulbs and other excitatory synapses on BCs.
The model system
BCs receive auditory input from different endbulbs (Spirou et al., 2005), meaning that synaptic integration is necessary (Golding & Oertel, 2012). MNTB chief cells are monoinnervated (Hoffpauir et al., 2006), suggesting a pure sign-inverting relay where the firing characteristics of the GBCs are transmitted to the LSO with high speed and fidelity (Joris & Trussell, 2018).
The onset of hearing is not reflected by morphological changes on the
Consistent with this, the probability of SV release (Pr) was found to decrease during maturation (Iwasaki & Takahashi, 2001; . Taschenberger et al., 2002; Taschenberger & von Gersdorff, 2000) and calculated according to a binomial model (Oertner et al. al., 2002 ) found that 62% of discharge events were multivesicular in rats before hearing but only 14% after hearing onset ( Taschenberger et al., 2002 ). These findings raise the general question, whether the number of anchored SVs is a direct readout for Pr.
Structure of presynaptic mitochondria changes according to energy
Mitochondria become larger during maturation of the auditory system
An increase in mitochondrial volumes was also shown during the development of hippocampal neurons, which was accompanied by an increase in the amount of mitochondria at synaptic boutons (Smith et al., 2016). In cat terminal bulbs, mean mitochondrial size did not change, but an increase in mitochondrial volume fraction was observed with age (Ryugo et al., 2006).
Developmental maturation is associated with ultrastructural remodeling of
However, synaptic mitochondria were reported to have fewer tubular segments compared to mitochondria of other neuronal compartments (Perkins et al., 2001). CMs of MAC mitochondria were found to be lamellar organized and often oriented approximately perpendicular to the mitochondrial plaque (Perkins et al., 2010; Spirou et al., 1998).
Spontaneous activity shapes the auditory pathway
Endbulb active zones of young wild-type and Otof -/- mice display a similar synaptic
On the other hand, the mEPSC amplitudes were larger in the mutant that could speak for postsynaptic enhancement due to a higher number of AMPA receptors in BCs of the deaf mice (Wright et al., 2014). In terminal bulbs of deaf mice without spontaneous auditory nerve activity due to a mutation in the transmembrane cochlear expressed gene 1, the mEPSC frequency was also not significantly different between P7-11 and P13-16.
Alterations of synaptic vesicle pools at endbulb of Held active zones of adult wild-type
Therefore, the similar mEPSC frequencies at BCs from otoferlin mutant and hearing control mice suggest similar properties of endbulb terminals ( Wright et al., 2014 ). Together with the functional impairment in the endbulbs of otoferlin mutant mice aged P11 to P60, where most animals aged P16–22 have stronger depression and higher synaptic error rates (Wright et al., 2014), these results indicate that synaptic transmission may be less reliable in endbulbs of Otof-/- mice.
SV sizes differ between endbulbs of wild-type and Otof -/- mice
For example, in Drosophila mutants lacking the clathrin adapter protein AP180, endocytosis of SVs is impaired, but SVs were found to be larger ( Zhang et al., 1998 ). Further, in P15–P16 mice with reduced otoferlin levels, larger SVs were observed by IHCs ( Strenzke et al., 2016 ).
Endbulb active zones contain a variable number of docked synaptic
The relationship between release probability and number of docked synaptic
However, the quantitative content of wt and deaf otoferlin mutant mice was similar at age P16-P22 and mEPSC analyzes revealed a possible upregulation of AMPA receptors on the postsynaptic membrane of mutant mice (Wright et al., 2014).
Changes of release probability during development of endbulb AZs and lack of
Release probability at endbulbs lacking presynaptic proteins
RIM-BP2 and Mover mediate tethering and docking of synaptic vesicles
Fine structure of nerve endings in the nucleus of the trapezoid body and ventral cochlear nucleus. Morphology of primary axosomatic endings in the cat anteroventral cochlear nucleus: A study of the terminal bulbs of Held.
