Astrid Rollenhagen

The morphology of excitatory central synapses: from structure to function

Synapses are the key elements for signal transduction and plasticity in the brain. For a better understanding of the functional signal cascades underlying synaptic transmission, a quantitative morphological analysis of the pre- and postsynaptic structures that represent morphological correlates for synaptic transmission is important. In particular, realistic values of the number, distribution, and geometry of synaptic contacts and the organization of the pool of synaptic vesicles provide important constraints for realistic models and numerical simulations of those parameters of synaptic transmission that, at present, are still not accessible to experiment. Although all synapses are composed of almost the same structural elements, the composition of these elements within a given synapse and the microcircuit in which they are embedded are the deciding factors determining its function. One possible way to analyze these structures is by computer-assisted three-dimensional reconstructions of synapses and their subsequent quantitative analysis based on ultrathin serial sections. The present review summarizes and discusses the morphology of five central excitatory synapses that are quantitatively well described: (1) a giant synapse, the so-called Calyx of Held, in the medial nucleus of the trapezoid body in the auditory brain stem, (2) the mossy fiber terminal establishing synapses with multiple cerebellar granule cell dendrites, (3) the mossy fiber bouton in the hippocampus predominantly terminating on proximal dendrites of CA3 pyramidal neurons, (4) the climbing fiber-Purkinje cell synapse in the cerebellum, and (5) cortical input synapses on the basal dendrites of layer 5 pyramidal cells. The detailed morphological description of these synaptic structures may help to define the morphological correlates of the functional parameters of synaptic transmission, such as the readily releasable pool of synaptic vesicles, of release, and of the variability of quantal size and might therefore explain the existing differences in the function between individual synapses embedded in different microcircuits.

The mossy fiber bouton: the "common" or the "unique" synapse?

Synapses are the key elements for signal processing and plasticity in the brain. They are composed of nearly the same structural subelements, an apposition zone including a pre- and postsynaptic density, a cleft and a pool of vesicles. It is, however, their actual composition that determines their different behavior in synaptic transmission and plasticity. Here, we describe and discuss the structural factors underlying the unique functional properties of the hippocampal mossy fiber (MF) synapse. Two membrane specializations, active zones (AZs; transmitter release sites), and puncta adherentia (PA), putative adhesion complexes were found. On average, individual boutons had ∼20 AZs with a mean surface area of 0.1 μm2 and a short distance of 0.45 μm between individual AZs. Mossy fiber boutons (MFBs) and their target structures were isolated from each other by astrocytes, but fine glial processes never reached the AZs. Therefore, two structural factors are likely to promote synaptic cross-talk: the short distance and the absence of fine glial processes between individual AZs. Thus, synaptic crosstalk may contribute to the high efficacy of hippocampal MF synapses. On average, an adult bouton contained ∼16,000 synaptic vesicles; ∼600 vesicles were located within 60 nm from the AZ, ∼4000 between 60 nm and 200 nm, and the remaining beyond 200 nm, suggesting large readily releasable, recycling, and reserve pools. Thus, the size of the three pools together with the number and distribution of AZs underlie the unique extent of synaptic efficacy and plasticity of the hippocampal MF synapse.

Quantitative 3D Ultrastructure of Thalamocortical Synapses from the “Lemniscal” Ventral Posteromedial Nucleus in Mouse Barrel Cortex

Abstract Thalamocortical synapses from “lemniscal” neurons of the dorsomedial portion of the rodent ventral posteromedial nucleus (VPMdm) are able to induce with remarkable efficacy, despite their relative low numbers, the firing of primary somatosensory cortex (S1) layer 4 (L4) neurons. To which extent this high efficacy depends on structural synaptic features remains unclear. Using both serial transmission (TEM) and focused ion beam milling scanning electron microscopy (FIB/SEM), we 3D-reconstructed and quantitatively analyzed anterogradely labeled VPMdm axons in L4 of adult mouse S1. All VPMdm synapses are asymmetric. Virtually all are established by axonal boutons, 53% of which contact multiple (2–4) elements (overall synapse/bouton ratio = 1.6). Most boutons are large (mean 0.47 μm3), and contain 1–3 mitochondria. Vesicle pools and postsynaptic density (PSD) surface areas are large compared to others in rodent cortex. Most PSDs are complex. Most synapses (83%) are established on dendritic spine heads. Furthermore, 15% of the postsynaptic spines receive a second, symmetric synapse. In addition, 13% of the spine heads have a large protrusion inserted into a membrane pouch of the VPMdm bouton. The unusual combination of structural features in VPMdm synapses is likely to contribute significantly to the high efficacy, strength, and plasticity of these thalamocortical synapses.

Development of Synaptic Boutons in Layer 4 of the Barrel Field of the Rat Somatosensory Cortex: A Quantitative Analysis

Understanding the structural and functional mechanisms underlying the development of individual brain microcircuits is critical for elucidating their computational properties. As synapses are the key structures defining a given microcircuit, it is imperative to investigate their development and precise structural features. Here, synapses in cortical layer 4 were analyzed throughout the first postnatal month using high-end electron microscopy to generate realistic quantitative 3D models. Besides their overall geometry, the size of active zones and the pools of synaptic vesicles were analyzed. At postnatal day 2 only a few shaft synapses were found, but spine synapses steadily increased with ongoing corticogenesis. From postnatal day 2 to 30 synaptic boutons significantly decreased in size whereas that of active zones remained nearly unchanged despite a reshaping. During the first 2 weeks of postnatal development, a rearrangement of synaptic vesicles from a loose distribution toward a densely packed organization close to the presynaptic density was observed, accompanied by the formation of, first a putative readily releasable pool and later a recycling and reserve pool. The quantitative 3D reconstructions of synapses will enable the comparison of structural and functional aspects of signal transduction thus leading to a better understanding of networks in the developing neocortex.

Quantitative Three-Dimensional Reconstructions of Excitatory Synaptic Boutons in Layer 5 of the Adult Human Temporal Lobe Neocortex: A Fine-Scale Electron Microscopic Analysis

Studies of synapses are available for different brain regions of several animal species including non-human primates, but comparatively little is known about their quantitative morphology in humans. Here, synaptic boutons in Layer 5 (L5) of the human temporal lobe (TL) neocortex were investigated in biopsy tissue, using fine-scale electron microscopy, and quantitative three-dimensional reconstructions. The size and organization of the presynaptic active zones (PreAZs), postsynaptic densities (PSDs), and that of the 3 distinct pools of synaptic vesicles (SVs) were particularly analyzed. L5 synaptic boutons were medium-sized (~6 μm2) with a single but relatively large PreAZ (~0.3 μm2). They contained a total of ~1500 SVs/bouton, ~20 constituting the putative readily releasable pool (RRP), ~180 the recycling pool (RP), and the remainder, the resting pool. The PreAZs, PSDs, and vesicle pools are ~3-fold larger than those of CNS synapses in other species. Astrocytic processes reached the synaptic cleft and may regulate the glutamate concentration. Profound differences exist between synapses in human TL neocortex and those described in various species, particularly in the size and geometry of PreAZs and PSDs, the large RRP/RP, and the astrocytic ensheathment suggesting high synaptic efficacy, strength, and modulation of synaptic transmission at human synapses.

Structural Properties of Synaptic Transmission and Temporal Dynamics at Excitatory Layer 5B Synapses in the Adult Rat Somatosensory Cortex

Cortical computations rely on functionally diverse and highly dynamic synapses. How their structural composition affects synaptic transmission and plasticity and whether they support functional diversity remains rather unclear. Here, synaptic boutons on layer 5B (L5B) pyramidal neurons in the adult rat barrel cortex were investigated. Simultaneous patch-clamp recordings from synaptically connected L5B pyramidal neurons revealed great heterogeneity in amplitudes, coefficients of variation (CVs), and failures (F%) of EPSPs. Quantal analysis indicated multivesicular release as a likely source of this variability. Trains of EPSPs decayed with fast and slow time constants, presumably representing release from small readily releasable (RRP; 5.40 ± 1.24 synaptic vesicles) and large recycling (RP; 74 ± 21 synaptic vesicles) pools that were independent and highly variable at individual synaptic contacts (RRP range 1.2–12.8 synaptic vesicles; RP range 3.4–204 synaptic vesicles). Most presynaptic boutons (~85%) had a single, often perforated active zone (AZ) with a ~2 to 5-fold larger pre- (0.29 ± 0.19 μm2) and postsynaptic density (0.31 ± 0.21 μm2) when compared with even larger CNS synaptic boutons. They contained 200–3400 vesicles (mean ~800). At the AZ, ~4 and ~12 vesicles were located within a perimeter of 10 and 20 nm, reflecting docked and readily releasable vesicles of a putative RRP. Vesicles (~160) at 60–200 nm constituting the structural estimate of the presumed RP were ~2-fold larger than our functional estimate of the RP although both with a high variability. The remaining constituted a presumed large resting pool. Multivariate analysis revealed two clusters of L5B synaptic boutons distinguished by the size of their resting pool. Our functional and ultrastructural analyses closely link stationary properties, temporal dynamics and endurance of synaptic transmission to vesicular content and distribution within the presynaptic boutons suggesting that functional diversity of L5B synapses is enhanced by their structural heterogeneity.

Structural and Synaptic Organization of the Adult Reeler Mouse Somatosensory Neocortex: A Comparative Fine-Scale Electron Microscopic Study of Reeler With Wild Type Mice

The reeler mouse has been widely used to study various aspects of cortico- and synaptogenesis, but also as a model for several neurological and neurodegenerative disorders. In contrast to development, comparably little is known about the neuronal composition and synaptic organization of the adult reeler mouse neocortex, in particular at the fine-scale electron microscopic level, which was investigated here and compared with wild type (WT) mice. In this study, the “barrel field” of the adult reeler and WT mouse somatosensory neocortex is used as a model system. In reeler the characteristic six-layered structure is no longer existent, but replaced by a conglomerate of neurons organized in homologous clusters with maintained morphological identity and heterologous clusters between neurons and/or oligodendrocytes. These clusters are loosely scattered throughout the neocortical mass between the pial surface and the white matter. In contrast to WT, layer 1 (L1), if existent, seems to be diluted into the volume of the neocortical mass with no clear boundary. L1 also contains clusters of migrated or persistent neurons, oligodendro- and astrocytes. As in WT, myelinated and unmyelinated axons were found throughout the neocortical mass, but in reeler they were organized in massive fiber bundles with a high fiber packing density. A prominent and massive thalamocortical projection traverses through the neocortical mass, always accompanied by numerous “active” oligodendrocytes whereas in WT no such projections were found and “silent” oligodendrocytes were restricted to the white matter. In the adult reeler mouse neocortex, synaptic boutons terminate on somata, dendritic shafts, spines of different types and axon initial segments with no signs of structural distortion and/or degeneration, indicating a “normal” postsynaptic innervation pattern of neurons. In addition, synaptic complexes between boutons and their postsynaptic targets are tightly ensheathed by fine astrocytic processes, as in WT. In conclusion, the neuronal clusters may represent a possible alternative organization principle in adult reeler mice “replacing” layer formation. If so, these homologous clusters may represent individual “functional units” where neurons are highly interconnected and may function as the equivalent of neurons integrated in a cortical layer. The structural composition and postsynaptic innervation pattern of neurons by synaptic boutons provide the structural basis for the establishment of a functional although altered cortical network in the adult reeler mouse.