Layer-specific distribution and expression pattern of AMPA- and NMDA-type glutamate receptors in the barrel field of the adult rat somatosensory cortex: a quantitative electron microscopic analysis.

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and NMDA (N-methyl-d-aspartate) glutamate receptors are driving forces for synaptic transmission and plasticity at neocortical synapses. However, their distribution pattern in the adult rat neocortex is largely unknown and was quantified using freeze fracture replication combined with postimmunogold-labeling. Both receptors were co-localized at layer (L)4 and L5 postsynaptic densities (PSDs). At L4 dendritic shaft and spine PSDs, the number of gold grains detecting AMPA was similar, whereas at L5 shaft PSDs AMPA-receptors outnumbered those on spine PSDs. Their number was significantly higher at L5 vs. L4 PSDs. At L4 and L5 dendritic shaft PSDs, the number of gold grains detecting GluN1 was ~2-fold higher than at spine PSDs. The number of gold grains detecting the GluN1-subunit was higher for both shaft and spine PSDs in L5 vs. L4. Both receptors showed a large variability in L4 and L5. A high correlation between the number of gold grains and PSD size for both receptors and targets was observed. Both receptors were distributed over the entire PSD but showed a layer- and target-specific distribution pattern. The layer- and target-specific distribution of AMPA and GluN1 glutamate receptors partially contribute to the observed functional differences in synaptic transmission and plasticity in the neocortex.

Effects of the clathrin inhibitor Pitstop-2 on synaptic vesicle recycling at a central synapse in vivo.

Four modes of endocytosis and subsequent synaptic vesicle (SV) recycling have been described at the presynapse to ensure the availability of SVs for synaptic release. However, it is unclear to what extend these modes operate under physiological activity patterns in vivo. The coat protein clathrin can regenerate SVs either directly from the plasma membrane (PM) via clathrin-mediated endocytosis (CME), or indirectly from synaptic endosomes by SV budding. Here, we examined the role of clathrin in SV recycling under physiological conditions by applying the clathrin inhibitor Pitstop-2 to the calyx of Held, a synapse optimized for high frequency synaptic transmission in the auditory brainstem, in vivo. The effects of clathrin-inhibition on SV recycling were investigated by serial sectioning scanning electron microscopy (S3EM) and 3D reconstructions of endocytic structures labeled by the endocytosis marker horseradish peroxidase (HRP). We observed large endosomal compartments as well as HRP-filled, black SVs (bSVs) that have been recently recycled. The application of Pitstop-2 led to reduced bSV but not large endosome density, increased volumes of large endosomes and shifts in the localization of both types of endocytic compartments within the synapse. These changes after perturbation of clathrin function suggest that clathrin plays a role in SV recycling from both, the PM and large endosomes, under physiological activity patterns, in vivo.

Sublamina-Specific Dynamics and Ultrastructural Heterogeneity of Layer 6 Excitatory Synaptic Boutons in the Adult Human Temporal Lobe Neocortex.

Synapses "govern" the computational properties of any given network in the brain. However, their detailed quantitative morphology is still rather unknown, particularly in humans. Quantitative 3D-models of synaptic boutons (SBs) in layer (L)6a and L6b of the temporal lobe neocortex (TLN) were generated from biopsy samples after epilepsy surgery using fine-scale transmission electron microscopy, 3D-volume reconstructions and electron microscopic tomography. Beside the overall geometry of SBs, the size of active zones (AZs) and that of the three pools of synaptic vesicles (SVs) were quantified. SBs in L6 of the TLN were middle-sized (~5 µm2), the majority contained only a single but comparatively large AZ (~0.20 µm2). SBs had a total pool of ~1100 SVs with comparatively large readily releasable (RRP, ~10 SVs L6a), (RRP, ~15 SVs L6b), recycling (RP, ~150 SVs), and resting (~900 SVs) pools. All pools showed a remarkably large variability suggesting a strong modulation of short-term synaptic plasticity. In conclusion, L6 SBs are highly reliable in synaptic transmission within the L6 network in the TLN and may act as "amplifiers," "integrators" but also as "discriminators" for columnar specific, long-range extracortical and cortico-thalamic signals from the sensory periphery.

Quantitative Three-Dimensional Reconstructions of Excitatory Synaptic Boutons in the "Barrel Field" of the Adult "Reeler" Mouse Somatosensory Neocortex: A Comparative Fine-Scale Electron Microscopic Analysis with the Wild Type Mouse.

Synapses are key structural determinants for information processing and computations in the normal and pathologically altered brain. Here, the quantitative morphology of excitatory synaptic boutons in the "reeler" mutant, a model system for various neurological disorders, was investigated and compared with wild-type (WT) mice using high-resolution, fine-scale electron microscopy (EM) and quantitative three-dimensional (3D) models of synaptic boutons. Beside their overall geometry, the shape and size of presynaptic active zones (PreAZs) and postsynaptic densities (PSDs) forming the active zones and the three pools of synaptic vesicles (SVs), namely the readily releasable pool (RRP), the recycling pool (RP), and the resting pool, were quantified. Although the reeler mouse neocortex is severely disturbed, no significant differences were found in most of the structural parameters investigated: the size of boutons (~3 µm2), size of the PreAZs and PSDs (~0.17 µm2), total number of SVs, and SVs within a perimeter (p) of 10 nm and p20 nm RRP; the p60 nm, p100 nm, and p60-p200 nm RP; and the resting pool, except the synaptic cleft width. Taken together, the synaptic organization and structural composition of synaptic boutons in the reeler neocortex remain comparably "normal" and may thus contribute to a "correct" wiring of neurons within the reeler cortical network.

Ultrastructural heterogeneity of layer 4 excitatory synaptic boutons in the adult human temporal lobe neocortex.

Synapses are fundamental building blocks controlling and modulating the 'behavior' of brain networks. How their structural composition, most notably their quantitative morphology underlie their computational properties remains rather unclear, particularly in humans. Here, excitatory synaptic boutons (SBs) in layer 4 (L4) of the temporal lobe neocortex (TLN) were quantitatively investigated. Biopsies from epilepsy surgery were used for fine-scale and tomographic electron microscopy (EM) to generate 3D-reconstructions of SBs. Particularly, the size of active zones (AZs) and that of the three functionally defined pools of synaptic vesicles (SVs) were quantified. SBs were comparatively small (~2.50 µm2), with a single AZ (~0.13 µm2); preferentially established on spines. SBs had a total pool of ~1800 SVs with strikingly large readily releasable (~20), recycling (~80) and resting pools (~850). Thus, human L4 SBs may act as 'amplifiers' of signals from the sensory periphery, integrate, synchronize and modulate intra- and extracortical synaptic activity.

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.

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.

Structural determinants underlying the high efficacy of synaptic transmission and plasticity at synaptic boutons in layer 4 of the adult rat 'barrel cortex'.

Excitatory layer 4 (L4) neurons in the 'barrel field' of the rat somatosensory cortex represent an important component in thalamocortical information processing. However, no detailed information exists concerning the quantitative geometry of synaptic boutons terminating on these neurons. Thus, L4 synaptic boutons were investigated using serial ultrathin sections and subsequent quantitative 3D reconstructions. In particular, parameters representing structural correlates of synaptic transmission and plasticity such as the number, size and distribution of pre- and postsynaptic densities forming the active zone (AZ) and of the three functionally defined pools of synaptic vesicles were analyzed. L4 synaptic boutons varied substantially in shape and size; the majority had a single, but large AZ with opposing pre- and postsynaptic densities that matched perfectly in size and position. More than a third of the examined boutons showed perforations of the postsynaptic density. Synaptic boutons contained on average a total pool of 561 ± 108 vesicles, with ~5% constituting the putative readily releasable, ~23% the recycling, and the remainder the reserve pool. These pools are comparably larger than other characterized central synapses. Synaptic complexes were surrounded by a dense network of fine astrocytic processes that reached as far as the synaptic cleft, thus regulating the temporal and spatial glutamate concentration, and thereby shaping the unitary EPSP amplitude. In summary, the geometry and size of AZs, the comparably large readily releasable and recycling pools, together with the tight astrocytic ensheathment, may explain and contribute to the high release probability, efficacy and modulation of synaptic transmission at excitatory L4 synaptic boutons. Moreover, the structural variability as indicated by the geometry of L4 synaptic boutons, the presence of mitochondria and the size and shape of the AZs strongly suggest that synaptic reliability, strength and plasticity is governed and modulated individually at excitatory L4 synaptic boutons.

Endocytic structures and synaptic vesicle recycling at a central synapse in awake rats.

The synaptic vesicle (SV) cycle has been studied extensively in cultured cells and slice preparations, but not much is known about the roles and relative contributions of endocytic pathways and mechanisms of SV recycling in vivo, under physiological patterns of activity. We employed horseradish peroxidase (HRP) as an in vivo marker of endocytosis at the calyx of Held synapse in the awake rat. Ex vivo serial section scanning electron microscopy and 3D reconstructions revealed two categories of labelled structures: HRP-filled SVs and large cisternal endosomes. Inhibition of adaptor protein complexes 1 and 3 (AP-1, AP-3) by in vivo application of Brefeldin A (BFA) disrupted endosomal SV budding while SV recycling via clathrin-mediated endocytosis (CME) remained unaffected. In conclusion, our study establishes cisternal endosomes as an intermediate of the SV cycle and reveals CME and endosomal budding as the predominant mechanisms of SV recycling in a tonically active central synapse in vivo.

Serial section scanning electron microscopy (S3EM) on silicon wafers for ultra-structural volume imaging of cells and tissues.

High resolution, three-dimensional (3D) representations of cellular ultrastructure are essential for structure function studies in all areas of cell biology. While limited subcellular volumes have been routinely examined using serial section transmission electron microscopy (ssTEM), complete ultrastructural reconstructions of large volumes, entire cells or even tissue are difficult to achieve using ssTEM. Here, we introduce a novel approach combining serial sectioning of tissue with scanning electron microscopy (SEM) using a conductive silicon wafer as a support. Ribbons containing hundreds of 35 nm thick sections can be generated and imaged on the wafer at a lateral pixel resolution of 3.7 nm by recording the backscattered electrons with the in-lens detector of the SEM. The resulting electron micrographs are qualitatively comparable to those obtained by conventional TEM. S(3)EM images of the same region of interest in consecutive sections can be used for 3D reconstructions of large structures. We demonstrate the potential of this approach by reconstructing a 31.7 µm(3) volume of a calyx of Held presynaptic terminal. The approach introduced here, Serial Section SEM (S(3)EM), for the first time provides the possibility to obtain 3D ultrastructure of large volumes with high resolution and to selectively and repetitively home in on structures of interest. S(3)EM accelerates process duration, is amenable to full automation and can be implemented with standard instrumentation.

Different fear states engage distinct networks within the intercalated cell clusters of the amygdala.

Although extinction-based therapies are among the most effective treatments for anxiety disorders, the neural bases of fear extinction remain still essentially unclear. Recent evidence suggests that the intercalated cell masses of the amygdala (ITCs) are critical structures for fear extinction. However, the neuronal organization of ITCs and how distinct clusters contribute to different fear states are still entirely unknown. Here, by combining whole-cell patch-clamp recordings and biocytin labeling with full anatomical reconstruction of the filled neurons and ultrastructural analysis of their synaptic contacts, we have elucidated the cellular organization and efferent connections of one of the main ITC clusters in mice. Our data showed an unexpected heterogeneity in the axonal pattern of medial paracapsular ITC (Imp) neurons and the presence of three distinct neuronal subtypes. Functionally, we observed that the Imp was preferentially activated during fear expression, whereas extinction training and extinction retrieval activated the main ITC nucleus (IN), as measured by quantifying Zif268 expression. This can be explained by the IPSPs evoked in the IN after Imp stimulation, most likely through the GABAergic monosynaptic innervation of IN neurons by one subtype of Imp cells, namely the medial capsular-projecting (MCp)-Imp neurons. MCp-Imp neurons also target large ITC cells that surround ITC clusters and express the metabotropic glutamate receptor 1α. These findings reveal a distinctive participation of ITC clusters to different fear states and the underlying anatomical circuitries, hence shedding new light on ITC networks and providing a novel framework to elucidate their role in fear expression and extinction.

Targeted three-dimensional immunohistochemistry reveals localization of presynaptic proteins Bassoon and Piccolo in the rat calyx of Held before and after the onset of hearing.

Bassoon and Piccolo contribute to the cytomatrix of active zones (AZ), the sites of neurotransmitter release in nerve terminals. Here, we examined the 3D localization of Bassoon and Piccolo in the rat calyx of Held between postnatal days 9 and 21, the period of hearing onset characterized by pronounced structural and functional changes. Bassoon and Piccolo were identified by immunohistochemistry (IHC) on slices of the brainstem harboring calyces labeled with membrane-anchored green fluorescent protein (mGFP). By using confocal microscopy and 3D reconstructions, we examined the distribution of Bassoon and Piccolo in calyces delineated by mGFP. This allowed us to discriminate calyceal IHC signals from noncalyceal signals located in the spaces between the calyceal stalks, which could mimic a calyx-like distribution. We found that both proteins were arranged in clusters resembling the size of AZs. These clusters were located along the presynaptic membrane facing the principal cell, close to or overlapping with synaptic vesicle (SV) clusters. Only about 60% of Bassoon and Piccolo clusters overlapped, whereas the remaining clusters contained predominantly Bassoon or Piccolo, suggesting differential targeting of these proteins within a single nerve terminal and potentially heterogeneous AZs functional properties. The total number of Bassoon and Piccolo clusters, which may approximate the number of AZs, was 405 +/- 35 at P9 and 601 +/- 45 at P21 (mean +/- SEM, n = 12). Normalized to calyx volume at P9 and P21, the density of clusters was similar, suggesting that the absolute number of clusters, not density, may contribute to the functional maturation associated with hearing onset.

Structural determinants of transmission at large hippocampal mossy fiber synapses.

Synapses are the key elements for signal processing and plasticity in the brain. To determine the structural factors underlying the unique functional properties of the hippocampal mossy fiber synapse, the complete quantitative geometry was investigated, using electron microscopy of serial ultrathin sections followed by computer-assisted three-dimensional reconstruction. In particular, parameters relevant for transmitter release and synaptic plasticity were examined. Two membrane specializations were found: active zones (AZs), transmitter release sites, and puncta adherentia, putative adhesion complexes. Individual boutons had, on average, 25 AZs (range, 7-45) that varied in shape and size (mean, 0.1 microm2; range, 0.07-0.17 microm2). The mean distance between individual AZs was 0.45 microm. Mossy fiber boutons and their target structures were mostly ensheathed by astrocytes, but fine glial processes never reached the active zones. Two structural factors are likely to promote synaptic cross talk: the short distance between AZs and the absence of fine glial processes at AZs. Thus, synaptic cross talk may contribute to the efficacy of hippocampal mossy fiber synapses. On average, a bouton contained 20,400 synaptic vesicles; approximately 900 vesicles were located within 60 nm from the active zone, approximately 4400 between 60 and 200 nm, and the remaining beyond 200 nm, suggesting large readily releasable, recycling, and reserve pools. The organization of the different pools may be a key structural correlate of presynaptic plasticity at this synapse. Thus, the mossy fiber bouton differs fundamentally in structure and function from the calyx of Held and other central synapses.

Three-dimensional reconstruction of a calyx of Held and its postsynaptic principal neuron in the medial nucleus of the trapezoid body.

The three-dimensional morphology of the axosomatic synaptic structures between a calyx of Held and a principal neuron in the medial nucleus of the trapezoid body (MNTB) in the brainstem of young postnatal day 9 rats was reconstructed from serial ultrathin sections. In the apposition zone between the calyx and the principal neuron two types of membrane specializations were identified: synaptic contacts (SCs) with active zones (AZs) and their associated postsynaptic densities (PSDs) constituted approximately 35% (n = 554) of the specializations; the remaining 65% (n = 1010) were puncta adherentia (PA). Synaptic contacts comprised approximately 5% of the apposition area of presynaptic and postsynaptic membranes. A SC had an average area of 0.100 microm(2), and the nearest neighbors were separated, on average, by 0.59 microm. Approximately one-half of the synaptic vesicles in the calyx were clustered within a distance of 200 nm of the AZ membrane area, a cluster consisting of approximately 60 synaptic vesicles (n = 52 SCs). Approximately two synaptic vesicles per SC were "anatomically docked." Comparing the geometry of the synaptic structure with its previously studied functional properties, we find that during a single presynaptic action potential (AP) (1) approximately 35% of the AZs release a transmitter quantum, (2) the number of SCs and anatomically docked vesicles is comparable with the low estimates of the readily releasable pool (RRP) of quanta, and (3) the broad distribution of PSD areas [coefficient of variation (CV) = 0.9] is likely to contribute to the large variability of miniature EPSC peaks. The geometry of the reconstructed synapse suggests that each of the hundreds of SCs is likely to contribute independently to the size and rising phase of the EPSC during a single AP.