RESUMO
Connexin 43, an astroglial gap junction protein, is enriched in perisynaptic astroglial processes and plays major roles in synaptic transmission. We have previously found that astroglial Cx43 controls synaptic glutamate levels and allows for activity-dependent glutamine release to sustain physiological synaptic transmissions and cognitiogns. However, whether Cx43 is important for the release of synaptic vesicles, which is a critical component of synaptic efficacy, remains unanswered. Here, using transgenic mice with a glial conditional knockout of Cx43 (Cx43-/-), we investigate whether and how astrocytes regulate the release of synaptic vesicles from hippocampal synapses. We report that CA1 pyramidal neurons and their synapses develop normally in the absence of astroglial Cx43. However, a significant impairment in synaptic vesicle distribution and release dynamics were observed. In particular, the FM1-43 assays performed using two-photon live imaging and combined with multi-electrode array stimulation in acute hippocampal slices, revealed a slower rate of synaptic vesicle release in Cx43-/- mice. Furthermore, paired-pulse recordings showed that synaptic vesicle release probability was also reduced and is dependent on glutamine supply via Cx43 hemichannel (HC). Taken together, we have uncovered a role for Cx43 in regulating presynaptic functions by controlling the rate and probability of synaptic vesicle release. Our findings further highlight the significance of astroglial Cx43 in synaptic transmission and efficacy.
Assuntos
Conexina 43 , Vesículas Sinápticas , Camundongos , Animais , Conexina 43/metabolismo , Vesículas Sinápticas/metabolismo , Astrócitos/metabolismo , Glutamina/metabolismo , Sinapses/metabolismo , Hipocampo/metabolismo , Camundongos TransgênicosRESUMO
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.
Assuntos
Ácido Glutâmico , Receptores de N-Metil-D-Aspartato , Ratos , Animais , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Ácido Glutâmico/metabolismo , N-Metilaspartato/metabolismo , Córtex Somatossensorial/metabolismo , Elétrons , Receptores de Glutamato/metabolismo , Sinapses/metabolismoRESUMO
Presynaptic glutamate replenishment is fundamental to brain function. In high activity regimes, such as epileptic episodes, this process is thought to rely on the glutamate-glutamine cycle between neurons and astrocytes. However the presence of an astroglial glutamine supply, as well as its functional relevance in vivo in the healthy brain remain controversial, partly due to a lack of tools that can directly examine glutamine transfer. Here, we generated a fluorescent probe that tracks glutamine in live cells, which provides direct visual evidence of an activity-dependent glutamine supply from astroglial networks to presynaptic structures under physiological conditions. This mobilization is mediated by connexin43, an astroglial protein with both gap-junction and hemichannel functions, and is essential for synaptic transmission and object recognition memory. Our findings uncover an indispensable recruitment of astroglial glutamine in physiological synaptic activity and memory via an unconventional pathway, thus providing an astrocyte basis for cognitive processes.
Assuntos
Astrócitos/metabolismo , Glutamina/metabolismo , Hipocampo/fisiologia , Reconhecimento Psicológico , Transmissão Sináptica , Animais , Cognição , Corantes Fluorescentes/química , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Glutamina/química , Hipocampo/citologia , Microscopia Intravital , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Sondas Moleculares , Neurônios/metabolismo , Rodaminas/química , Técnicas EstereotáxicasRESUMO
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.
Assuntos
Neocórtex , Terminações Pré-Sinápticas , Adulto , Humanos , Neocórtex/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Sinapses/ultraestrutura , Transmissão Sináptica , Vesículas Sinápticas/ultraestrutura , Lobo Temporal/ultraestruturaRESUMO
Modern electron microscopy (EM) such as fine-scale transmission EM, focused ion beam scanning EM, and EM tomography have enormously improved our knowledge about the synaptic organization of the normal, developmental, and pathologically altered brain. In contrast to various animal species, comparably little is known about these structures in the human brain. Non-epileptic neocortical access tissue from epilepsy surgery was used to generate quantitative 3D models of synapses. Beside the overall geometry, the number, size, and shape of active zones and of the three functionally defined pools of synaptic vesicles representing morphological correlates for synaptic transmission and plasticity were quantified. EM tomography further allowed new insights in the morphological organization and size of the functionally defined readily releasable pool. Beside similarities, human synaptic boutons, although comparably small (approximately 5 µm), differed substantially in several structural parameters, such as the shape and size of active zones, which were on average 2 to 3-fold larger than in experimental animals. The total pool of synaptic vesicles exceeded that in experimental animals by approximately 2 to 3-fold, in particular the readily releasable and recycling pool by approximately 2 to 5-fold, although these pools seemed to be layer-specifically organized. Taken together, synaptic boutons in the human temporal lobe neocortex represent unique entities perfectly adapted to the "job" they have to fulfill in the circuitry in which they are embedded. Furthermore, the quantitative 3D models of synaptic boutons are useful to explain and even predict the functional properties of synaptic connections in the human neocortex.
Assuntos
Neocórtex/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Sinapses/ultraestrutura , Lobo Temporal/ultraestrutura , Animais , Tomografia com Microscopia Eletrônica , Humanos , Imageamento Tridimensional , Camundongos , Microscopia Eletrônica , Neocórtex/diagnóstico por imagem , Plasticidade Neuronal/fisiologia , Ratos , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/ultraestrutura , Lobo Temporal/diagnóstico por imagemRESUMO
Thalamocortical posterior nucleus (Po) axons innervating the vibrissal somatosensory (S1) and motor (MC) cortices are key links in the brain neuronal network that allows rodents to explore the environment whisking with their motile snout vibrissae. Here, using fine-scale high-end 3D electron microscopy, we demonstrate in adult male C57BL/6 wild-type mice marked differences between MC versus S1 Po synapses in (1) bouton and active zone size, (2) neurotransmitter vesicle pool size, (3) distribution of mitochondria around synapses, and (4) proportion of synapses established on dendritic spines and dendritic shafts. These differences are as large, or even more pronounced, than those between Po and ventro-posterior thalamic nucleus synapses in S1. Moreover, using single-axon transfection labeling, we demonstrate that the above differences actually occur on the MC versus the S1 branches of individual Po cell axons that innervate both areas. Along with recently-discovered divergences in efficacy and plasticity, the synaptic structure differences reported here thus reveal a new subcellular level of complexity. This is a finding that upends current models of thalamocortical circuitry, and that might as well illuminate the functional logic of other branched projection axon systems.SIGNIFICANCE STATEMENT Many long-distance brain connections depend on neurons whose branched axons target separate regions. Using 3D electron microscopy and single-cell transfection, we investigated the mouse Posterior thalamic nucleus (Po) cell axons that simultaneously innervate motor and sensory areas of the cerebral cortex involved in whisker movement control. We demonstrate significant differences in the size of the boutons made in each area by individual Po axons, as well as in functionally-relevant parameters in the composition of their synapses. In addition, we found similarly large differences between the synapses of Po versus ventral posteromedial thalamic nucleus axons in the whisker sensory cortex. Area-specific synapse structure in individual axons implies a new, unsuspected level of complexity in long-distance brain connections.
Assuntos
Axônios/fisiologia , Rede Nervosa/fisiologia , Neurônios/fisiologia , Córtex Somatossensorial/fisiologia , Sinapses/fisiologia , Tálamo/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vias Neurais/fisiologia , Vibrissas/fisiologiaRESUMO
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.
Assuntos
Neocórtex/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Córtex Somatossensorial/ultraestrutura , Vesículas Sinápticas/ultraestrutura , Animais , Imageamento Tridimensional , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Microscopia EletrônicaRESUMO
Bmal1 is an essential component of the molecular clockwork, which drives circadian rhythms in cell function. In Bmal1-deficient (Bmal1-/-) mice, chronodisruption is associated with cognitive deficits and progressive brain pathology including astrocytosis indicated by increased expression of glial fibrillary acidic protein (GFAP). However, relatively little is known about the impact of Bmal1-deficiency on astrocyte morphology prior to astrocytosis. Therefore, in this study we analysed astrocyte morphology in young (6-8 weeks old) adult Bmal1-/- mice. At this age, overall GFAP immunoreactivity was not increased in Bmal1-deficient mice. At the ultrastructural level, we found a decrease in the volume fraction of the fine astrocytic processes that cover the hippocampal mossy fiber synapse, suggesting an impairment of perisynaptic processes and their contribution to neurotransmission. For further analyses of actin cytoskeleton, which is essential for distal process formation, we used cultured Bmal1-/- astrocytes. Bmal1-/- astrocytes showed an impaired formation of actin stress fibers. Moreover, Bmal1-/- astrocytes showed reduced levels of the actin-binding protein cortactin (CTTN). Cttn promoter region contains an E-Box like element and chromatin immunoprecipitation revealed that Cttn is a potential Bmal1 target gene. In addition, the level of GTP-bound (active) Rho-GTPase (Rho-GTP) was reduced in Bmal1-/- astrocytes. In summary, our data demonstrate that Bmal1-deficiency affects morphology of the fine astrocyte processes prior to strong upregulation of GFAP, presumably because of impaired Cttn expression and reduced Rho-GTP activation. These morphological changes might result in altered synaptic function and, thereby, relate to cognitive deficits in chronodisruption.
Assuntos
Fatores de Transcrição ARNTL/metabolismo , Citoesqueleto de Actina/metabolismo , Astrócitos/metabolismo , Fibras Musgosas Hipocampais/metabolismo , Sinapses/metabolismo , Fatores de Transcrição ARNTL/genética , Animais , Cortactina/genética , Cortactina/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Camundongos , Camundongos Knockout , Transmissão Sináptica/fisiologiaRESUMO
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.
Assuntos
Neocórtex/ultraestrutura , Sinapses/ultraestrutura , Vesículas Sinápticas/ultraestrutura , Lobo Temporal/ultraestrutura , Adulto , Animais , Tomografia com Microscopia Eletrônica/métodos , Epilepsia do Lobo Temporal/fisiopatologia , Feminino , Humanos , Imageamento Tridimensional/métodos , Masculino , Pessoa de Meia-Idade , Transmissão Sináptica/fisiologiaRESUMO
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.
Assuntos
Imageamento Tridimensional/métodos , Neocórtex/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Lobo Temporal/ultraestrutura , Adulto , Feminino , Humanos , Processamento de Imagem Assistida por Computador/métodos , Masculino , Microscopia Eletrônica de Transmissão/métodos , Pessoa de Meia-IdadeRESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
Córtex Somatossensorial/ultraestrutura , Sinapses/ultraestrutura , Animais , Imageamento Tridimensional/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Núcleos Ventrais do Tálamo/ultraestruturaRESUMO
In many vertebrates, postnatally generated neurons often migrate long distances to reach their final destination, where they help shape local circuit activity. Concerted action of extrinsic stimuli is required to regulate long-distance migration. Some migratory principles are evolutionarily conserved, whereas others are species and cell type specific. Here we identified a serotonergic mechanism that governs migration of postnatally generated neurons in the mouse brain. Serotonergic axons originating from the raphe nuclei exhibit a conspicuous alignment with subventricular zone-derived neuroblasts. Optogenetic axonal activation provides functional evidence for serotonergic modulation of neuroblast migration. Furthermore, we show that the underlying mechanism involves serotonin receptor 3A (5HT3A)-mediated calcium influx. Thus, 5HT3A receptor deletion in neuroblasts impaired speed and directionality of migration and abolished calcium spikes. We speculate that serotonergic modulation of postnatally generated neuroblast migration is evolutionarily conserved as indicated by the presence of serotonergic axons in migratory paths in other vertebrates.
Assuntos
Axônios/metabolismo , Encéfalo/crescimento & desenvolvimento , Cálcio/metabolismo , Movimento Celular/genética , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Receptores 5-HT3 de Serotonina/genética , Neurônios Serotoninérgicos/metabolismo , Animais , Southern Blotting , Encéfalo/citologia , Encéfalo/metabolismo , Pré-Escolar , Tentilhões , Humanos , Imuno-Histoquímica , Macaca mulatta , Masculino , Camundongos Knockout , Microscopia Confocal , Microscopia de Vídeo , Células-Tronco Neurais/citologia , Imagem Óptica , Optogenética , Coelhos , Núcleos da Rafe/citologia , Núcleos da Rafe/crescimento & desenvolvimento , Núcleos da Rafe/metabolismo , Receptores 5-HT3 de Serotonina/metabolismo , Neurônios Serotoninérgicos/citologia , Imagem com Lapso de Tempo , Peixe-ZebraRESUMO
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.
Assuntos
Terminações Pré-Sinápticas/fisiologia , Córtex Somatossensorial/citologia , Córtex Somatossensorial/crescimento & desenvolvimento , Sinapses/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica , Rede Nervosa/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Wistar , Sinapses/ultraestrutura , Vesículas Sinápticas/ultraestruturaRESUMO
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.
Assuntos
Córtex Cerebral/anatomia & histologia , Córtex Cerebral/fisiologia , Plasticidade Neuronal/fisiologia , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Transmissão Sináptica/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Vesículas Sinápticas/fisiologiaRESUMO
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 â¼20 AZs with a mean surface area of 0.1 µm(2) 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 â¼16,000 synaptic vesicles; â¼600 vesicles were located within 60 nm from the AZ, â¼4000 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.
RESUMO
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.
Assuntos
Fibras Musgosas Hipocampais/fisiologia , Fibras Musgosas Hipocampais/ultraestrutura , Transmissão Sináptica/fisiologia , Animais , Imageamento Tridimensional , Microscopia Eletrônica , Modelos Animais , Plasticidade Neuronal/fisiologia , Neurotransmissores/fisiologia , Ratos , Ratos Wistar , Sinapses/fisiologia , Sinapses/ultraestrutura , Vesículas Sinápticas/fisiologiaRESUMO
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.
Assuntos
Vias Auditivas/fisiologia , Vias Auditivas/ultraestrutura , Modelos Neurológicos , Membranas Sinápticas/fisiologia , Membranas Sinápticas/ultraestrutura , Transmissão Sináptica/fisiologia , Animais , Humanos , Imageamento Tridimensional , Plasticidade Neuronal/fisiologia , Neurotransmissores/fisiologia , Vesículas Sinápticas/fisiologia , Vesículas Sinápticas/ultraestruturaRESUMO
The extracellular matrix glycoprotein tenascin-C (TNC) has been implicated in neural development and plasticity but many of its functions in vivo remain obscure. Here we addressed the question as to whether the constitutive absence of TNC in mice affects cortical physiology and structure. Defined major cell populations (neurons and inhibitory neuronal subpopulations, astrocytes, oligodendrocytes and microglia) were quantified in the somatosensory and motor cortices of adult TNC deficient (TNC-/-) and wild-type (TNC+/+) mice by immunofluorescence labelling and stereology. In both areas studied we found abnormally high neuronal density, astrogliosis, low density of parvalbumin-positive interneurons and reduced ratios of oligodendrocytes to neurons and of inhibitory to excitatory neurons in the TNC deficient as opposed to the non-deficient animals. Analysis of Golgi-impregnated layer V pyramidal neurons in TNC-/- animals showed aberrant dendrite tortuosity and redistribution of stubby spines within first- to third-order dendritic arbors. Significantly enhanced responses upon whisker stimulation were recorded epicranially over the barrel and the motor cortices of TNC-/- as compared to TNC+/+ animals, and this effect might be associated with the diminished inhibitory circuitry. These results indicate that TNC is essential for normal cortical development and function.