Astroglial Connexin 43 Regulates Synaptic Vesicle Release at Hippocampal Synapses.

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.

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.

Neocortical pyramidal neurons with axons emerging from dendrites are frequent in non-primates, but rare in monkey and human.

The canonical view of neuronal function is that inputs are received by dendrites and somata, become integrated in the somatodendritic compartment and upon reaching a sufficient threshold, generate axonal output with axons emerging from the cell body. The latter is not necessarily the case. Instead, axons may originate from dendrites. The terms 'axon carrying dendrite' (AcD) and 'AcD neurons' have been coined to describe this feature. In rodent hippocampus, AcD cells are shown to be functionally 'privileged', since inputs here can circumvent somatic integration and lead to immediate action potential initiation in the axon. Here, we report on the diversity of axon origins in neocortical pyramidal cells of rodent, ungulate, carnivore, and primate. Detection methods were Thy-1-EGFP labeling in mouse, retrograde biocytin tracing in rat, cat, ferret, and macaque, SMI-32/ßIV-spectrin immunofluorescence in pig, cat, and macaque, and Golgi staining in macaque and human. We found that in non-primate mammals, 10-21% of pyramidal cells of layers II-VI had an AcD. In marked contrast, in macaque and human, this proportion was lower and was particularly low for supragranular neurons. A comparison of six cortical areas (being sensory, association, and limbic in nature) in three macaques yielded percentages of AcD cells which varied by a factor of 2 between the areas and between the individuals. Unexpectedly, pyramidal cells in the white matter of postnatal cat and aged human cortex exhibit AcDs to much higher percentages. In addition, interneurons assessed in developing cat and adult human cortex had AcDs at type-specific proportions and for some types at much higher percentages than pyramidal cells. Our findings expand the current knowledge regarding the distribution and proportion of AcD cells in neocortex of non-primate taxa, which strikingly differ from primates where these cells are mainly found in deeper layers and white matter.

Physiological synaptic activity and recognition memory require astroglial glutamine.

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.

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.

Neurotrophic factors in Alzheimer's disease: pathogenesis and therapy.

Alzheimer's disease (AD) is a common neurodegenerative disease with a prevalence estimated to reach 115 million by 2050. It is characterized by abnormal extracellular accumulation of amyloid­beta (Aß) peptide and intracellular neurofibrillary tangles (NFTs) that result in neuro­inflammation, synaptic dysfunction, neurotransmitter imbalance, neuronal loss, and dendritic changes. A hypothesis of neurotrophic factor (NTF) involvement in neurodegenerative diseases and their potential as a therapeutic tool has emerged. There are wide information gaps on this topic. However, consistent with this hypothesis, AD may be caused by a deficiency in neurotrophin proteins or receptors expression. In AD brains, an increase in nerve growth factor and a decrease in brain-derived neurotrophic factor in the hippocampus and certain neocortical regions, and a decrease in TrkA in the cortex and nucleus basalis has been observed. Thus, comparative data relating to recent hypotheses addressing NTF content and receptors in experimental animals and human brains, along with their potential roles in the treat ment of AD, are discussed in this review.

Synaptic Organization of the Human Temporal Lobe Neocortex as Revealed by High-Resolution Transmission, Focused Ion Beam Scanning, and Electron Microscopic Tomography.

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.

Area-Specific Synapse Structure in Branched Posterior Nucleus Axons Reveals a New Level of Complexity in Thalamocortical Networks.

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.

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.

Bmal1-deficiency affects glial synaptic coverage of the hippocampal mossy fiber synapse and the actin cytoskeleton in astrocytes.

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.

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.

Posterior thalamic nucleus axon terminals have different structure and functional impact in the motor and somatosensory vibrissal cortices.

Rodents extract information about nearby objects from the movement of their whiskers through dynamic computations that are carried out by a network of forebrain structures that includes the thalamus and the primary sensory (S1BF) and motor (M1wk) whisker cortices. The posterior nucleus (Po), a higher order thalamic nucleus, is a key hub of this network, receiving cortical and brainstem sensory inputs and innervating both motor and sensory whisker-related cortical areas. In a recent study in rats, we showed that Po inputs differently impact sensory processing in S1BF and M1wk. Here, in C57BL/6 mice, we measured Po synaptic bouton layer distribution and size, compared cortical unit response latencies to "in vivo" Po activation, and pharmacologically examined the glutamatergic receptor mechanisms involved. We found that, in S1BF, a large majority (56%) of Po axon varicosities are located in layer (L)5a and only 12% in L2-L4, whereas in M1wk this proportion is inverted to 18% and 55%, respectively. Light and electron microscopic measurements showed that Po synaptic boutons in M1wk layers 3-4 are significantly larger (~ 50%) than those in S1BF L5a. Electrical Po stimulation elicits different area-specific response patterns. In S1BF, responses show weak or no facilitation, and involve both ionotropic and metabotropic glutamate receptors, whereas in M1wk, unit responses exhibit facilitation to repetitive stimulation and involve ionotropic NMDA glutamate receptors. Because of the different laminar distribution of axon terminals, synaptic bouton size and receptor mechanisms, the impact of Po signals on M1wk and S1BF, although simultaneous, is likely to be markedly different.

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 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.

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.

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

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.

Serotonergic Projections Govern Postnatal Neuroblast Migration.

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.

Influence of blood-brain barrier permeability on O-(2-18F-fluoroethyl)-L-tyrosine uptake in rat gliomas.

PURPOSE: O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET) is an established tracer for the diagnosis of brain tumors with PET. This study investigates the influence of blood-brain barrier (BBB) permeability on 18F-FET uptake in two rat glioma models and one human xenograft model. METHODS: F98 glioma, 9L gliosarcoma or human U87 glioblastoma cells were implanted into the striatum of 56 Fischer or RNU rats. Thereafter, animals were divided into a control group and a group receiving injections of the glucocorticoid dexamethasone (Dex). After 12-13 days of tumor growth animals received injection of Evans blue dye (EBD) to visualize BBB disturbance and underwent 18F-FET PET followed by autoradiography. Time activity curves, standardized uptake values (SUV) and Tumor-to-brain ratios (TBR) of 18F-FET uptake [18-61 min post injection (p.i.)] were evaluated using a volume-of-Interest (VOI) analysis. BBB disturbance was quantitatively evaluated by EBD fluorescence. The membrane gaps of blood vessel endothelial tight junctions were measured using electron microscopy to visualize ultrastructural BBB alterations in one untreated and one Dex treated F98 glioma. Data were analyzed by two-way ANOVAs. RESULTS: In Dex treated animals EBD extravasation was significantly reduced in 9L (P < 0.001) and U87 (P = 0.008) models and showed a trend in F98 models (P = 0.053). In contrast, no significant differences of 18F-FET uptake were observed between Dex treated animals and control group except a decrease of the TBR in the 9L tumor model in PET (P < 0.01). Ultrastructural evaluation of tumor blood vessel endothelia revealed significant reduction of the cleft diameter between endothelial cells after Dex treatment in F98 model (P = 0.010). CONCLUSION: Despite a considerable reduction of BBB permeability in rat gliomas after Dex treatment, no relevant changes of 18F-FET uptake were noted in this experimental study. Thus, 18F-FET uptake in gliomas appears to be widely independent of the permeability of the BBB.

Developmental Profile, Morphology, and Synaptic Connectivity of Cajal-Retzius Cells in the Postnatal Mouse Hippocampus.

Cajal-Retzius (CR) cells are early generated neurons, involved in the assembly of developing neocortical and hippocampal circuits. However, their roles in networks of the postnatal brain remain poorly understood. In order to get insights into these latter functions, we have studied their morphological and synaptic properties in the postnatal hippocampus of the CXCR4-EGFP mouse, where CR cells are easily identifiable. Our data indicate that CR cells are nonuniformly distributed along different subfields of the hippocampal formation, and that their postnatal decline is regulated in a region-specific manner. In fact, CR cells persist in distinct areas of fully mature animals. Subclasses of CR cells project and target either local (molecular layers) or distant regions [subicular complex and entorhinal cortex (EC)] of the hippocampal formation, but have similar firing patterns. Lastly, CR cells are biased toward targeting dendritic shafts compared with spines, and produce large-amplitude glutamatergic unitary postsynaptic potentials on γ-aminobutyric acid (GABA) containing interneurons. Taken together, our results suggest that CR cells are involved in a novel excitatory loop of the postnatal hippocampal formation, which potentially contributes to shaping the flow of information between the hippocampus, parahippocampal regions and entorhinal cortex, and to the low seizure threshold of these brain areas.

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.