A biologically inspired repair mechanism for neuronal reconstructions with a focus on human dendrites.

Investigating and modelling the functionality of human neurons remains challenging due to the technical limitations, resulting in scarce and incomplete 3D anatomical reconstructions. Here we used a morphological modelling approach based on optimal wiring to repair the parts of a dendritic morphology that were lost due to incomplete tissue samples. In Drosophila, where dendritic regrowth has been studied experimentally using laser ablation, we found that modelling the regrowth reproduced a bimodal distribution between regeneration of cut branches and invasion by neighbouring branches. Interestingly, our repair model followed growth rules similar to those for the generation of a new dendritic tree. To generalise the repair algorithm from Drosophila to mammalian neurons, we artificially sectioned reconstructed dendrites from mouse and human hippocampal pyramidal cell morphologies, and showed that the regrown dendrites were morphologically similar to the original ones. Furthermore, we were able to restore their electrophysiological functionality, as evidenced by the recovery of their firing behaviour. Importantly, we show that such repairs also apply to other neuron types including hippocampal granule cells and cerebellar Purkinje cells. We then extrapolated the repair to incomplete human CA1 pyramidal neurons, where the anatomical boundaries of the particular brain areas innervated by the neurons in question were known. Interestingly, the repair of incomplete human dendrites helped to simulate the recently observed increased synaptic thresholds for dendritic NMDA spikes in human versus mouse dendrites. To make the repair tool available to the neuroscience community, we have developed an intuitive and simple graphical user interface (GUI), which is available in the TREES toolbox (www.treestoolbox.org).

Key morphological features of human pyramidal neurons.

The basic building block of the cerebral cortex, the pyramidal cell, has been shown to be characterized by a markedly different dendritic structure among layers, cortical areas, and species. Functionally, differences in the structure of their dendrites and axons are critical in determining how neurons integrate information. However, within the human cortex, these neurons have not been quantified in detail. In the present work, we performed intracellular injections of Lucifer Yellow and 3D reconstructed over 200 pyramidal neurons, including apical and basal dendritic and local axonal arbors and dendritic spines, from human occipital primary visual area and associative temporal cortex. We found that human pyramidal neurons from temporal cortex were larger, displayed more complex apical and basal structural organization, and had more spines compared to those in primary sensory cortex. Moreover, these human neocortical neurons displayed specific shared and distinct characteristics in comparison to previously published human hippocampal pyramidal neurons. Additionally, we identified distinct morphological features in human neurons that set them apart from mouse neurons. Lastly, we observed certain consistent organizational patterns shared across species. This study emphasizes the existing diversity within pyramidal cell structures across different cortical areas and species, suggesting substantial species-specific variations in their computational properties.

Quantitative analysis of the GABAergic innervation of the soma and axon initial segment of pyramidal cells in the human and mouse neocortex.

Perisomatic GABAergic innervation in the cerebral cortex is carried out mostly by basket and chandelier cells, which differentially participate in the control of pyramidal cell action potential output and synchronization. These cells establish multiple synapses with the cell body (and proximal dendrites) and the axon initial segment (AIS) of pyramidal neurons, respectively. Using multiple immunofluorescence, confocal microscopy and 3D quantification techniques, we have estimated the number and density of GABAergic boutons on the cell body and AIS of pyramidal neurons located through cortical layers of the human and mouse neocortex. The results revealed, in both species, that there is clear variability across layers regarding the density and number of perisomatic GABAergic boutons. We found a positive linear correlation between the surface area of the soma, or the AIS, and the number of GABAergic terminals in apposition to these 2 neuronal domains. Furthermore, the density of perisomatic GABAergic boutons was higher in the human cortex than in the mouse. These results suggest a selectivity for the GABAergic innervation of the cell body and AIS that might be related to the different functional attributes of the microcircuits in which neurons from different layers are involved in both human and mouse.

3D synaptic organization of layer III of the human anterior cingulate and temporopolar cortex.

The human anterior cingulate and temporopolar cortices have been proposed as highly connected nodes involved in high-order cognitive functions, but their synaptic organization is still basically unknown due to the difficulties involved in studying the human brain. Using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) to study the synaptic organization of the human brain obtained with a short post-mortem delay allows excellent results to be obtained. We have used this technology to analyze layer III of the anterior cingulate cortex (Brodmann area 24) and the temporopolar cortex, including the temporal pole (Brodmann area 38 ventral and dorsal) and anterior middle temporal gyrus (Brodmann area 21). Our results, based on 6695 synaptic junctions fully reconstructed in 3D, revealed that Brodmann areas 24, 21 and ventral area 38 showed similar synaptic density and synaptic size, whereas dorsal area 38 displayed the highest synaptic density and the smallest synaptic size. However, the proportion of the different types of synapses (excitatory and inhibitory), the postsynaptic targets, and the shapes of excitatory and inhibitory synapses were similar, regardless of the region examined. These observations indicate that certain aspects of the synaptic organization are rather homogeneous, whereas others show specific variations across cortical regions.

Microanatomical study of pyramidal neurons in the contralesional somatosensory cortex after experimental ischemic stroke.

At present, many studies support the notion that after stroke, remote regions connected to the infarcted area are also affected and may contribute to functional outcome. In the present study, we have analyzed possible microanatomical alterations in pyramidal neurons from the contralesional hemisphere after induced stroke. We performed intracellular injections of Lucifer yellow in pyramidal neurons from layer III in the somatosensory cortex of the contralesional hemisphere in an ischemic stroke mouse model. A detailed 3-dimensional analysis of the neuronal complexity and morphological alterations of dendritic spines was then performed. Our results demonstrate that pyramidal neurons from layer III in the somatosensory cortex of the contralesional hemisphere show selective changes in their dendritic arbors, namely, less dendritic complexity of the apical dendritic arbor-but no changes in the basal dendritic arbor. In addition, we found differences in spine morphology in both apical and basal dendrites comparing the contralesional hemisphere with the lesional hemisphere. Our results show that pyramidal neurons of remote areas connected to the infarct zone exhibit a series of selective changes in neuronal complexity and morphological distribution of dendritic spines, supporting the hypothesis that remote regions connected to the peri-infarcted area are also affected after stroke.

Strong and reliable synaptic communication between pyramidal neurons in adult human cerebral cortex.

Synaptic transmission constitutes the primary mode of communication between neurons. It is extensively studied in rodent but not human neocortex. We characterized synaptic transmission between pyramidal neurons in layers 2 and 3 using neurosurgically resected human middle temporal gyrus (MTG, Brodmann area 21), which is part of the distributed language circuitry. We find that local connectivity is comparable with mouse layer 2/3 connections in the anatomical homologue (temporal association area), but synaptic connections in human are 3-fold stronger and more reliable (0% vs 25% failure rates, respectively). We developed a theoretical approach to quantify properties of spinous synapses showing that synaptic conductance and voltage change in human dendritic spines are 3-4-folds larger compared with mouse, leading to significant NMDA receptor activation in human unitary connections. This model prediction was validated experimentally by showing that NMDA receptor activation increases the amplitude and prolongs decay of unitary excitatory postsynaptic potentials in human but not in mouse connections. Since NMDA-dependent recurrent excitation facilitates persistent activity (supporting working memory), our data uncovers cortical microcircuit properties in human that may contribute to language processing in MTG.

Three-dimensional analysis of synaptic organization in the hippocampal CA1 field in Alzheimer's disease.

Alzheimer's disease is the most common form of dementia, characterized by a persistent and progressive impairment of cognitive functions. Alzheimer's disease is typically associated with extracellular deposits of amyloid-ß peptide and accumulation of abnormally phosphorylated tau protein inside neurons (amyloid-ß and neurofibrillary pathologies). It has been proposed that these pathologies cause neuronal degeneration and synaptic alterations, which are thought to constitute the major neurobiological basis of cognitive dysfunction in Alzheimer's disease. The hippocampal formation is especially vulnerable in the early stages of Alzheimer's disease. However, the vast majority of electron microscopy studies have been performed in animal models. In the present study, we performed an extensive 3D study of the neuropil to investigate the synaptic organization in the stratum pyramidale and radiatum in the CA1 field of Alzheimer's disease cases with different stages of the disease, using focused ion beam/scanning electron microscopy (FIB/SEM). In cases with early stages of Alzheimer's disease, the synapse morphology looks normal and we observed no significant differences between control and Alzheimer's disease cases regarding the synaptic density, the ratio of excitatory and inhibitory synapses, or the spatial distribution of synapses. However, differences in the distribution of postsynaptic targets and synaptic shapes were found. Furthermore, a lower proportion of larger excitatory synapses in both strata were found in Alzheimer's disease cases. Individuals in late stages of the disease suffered the most severe synaptic alterations, including a decrease in synaptic density and morphological alterations of the remaining synapses. Since Alzheimer's disease cases show cortical atrophy, our data indicate a reduction in the total number (but not the density) of synapses at early stages of the disease, with this reduction being much more accentuated in subjects with late stages of Alzheimer's disease. The observed synaptic alterations may represent a structural basis for the progressive learning and memory dysfunctions seen in Alzheimer's disease cases.

Three-Dimensional Synaptic Organization of Layer III of the Human Temporal Neocortex.

In the present study, we have used focused ion beam/scanning electron microscopy (FIB/SEM) to perform a study of the synaptic organization of layer III of Brodmann's area 21 in human tissue samples obtained from autopsies and biopsies. We analyzed the synaptic density, 3D spatial distribution, and type (asymmetric/symmetric), as well as the size and shape of each synaptic junction of 4945 synapses that were fully reconstructed in 3D. Significant differences in the mean synaptic density between autopsy and biopsy samples were found (0.49 and 0.66 synapses/µm3, respectively). However, in both types of samples (autopsy and biopsy), the asymmetric:symmetric ratio was similar (93:7) and most asymmetric synapses were established on dendritic spines (75%), while most symmetric synapses were established on dendritic shafts (85%). We also compared several electron microscopy methods and analysis tools to estimate the synaptic density in the same brain tissue. We have shown that FIB/SEM is much more reliable and robust than the majority of the other commonly used EM techniques. The present work constitutes a detailed description of the synaptic organization of cortical layer III. Further studies on the rest of the cortical layers are necessary to better understand the functional organization of this temporal cortical region.

Variation in Pyramidal Cell Morphology Across the Human Anterior Temporal Lobe.

Pyramidal neurons are the most abundant and characteristic neuronal type in the cerebral cortex and their dendritic spines are the main postsynaptic elements of cortical excitatory synapses. Previous studies have shown that pyramidal cell structure differs across layers, cortical areas, and species. However, within the human cortex, the pyramidal dendritic morphology has been quantified in detail in relatively few cortical areas. In the present work, we performed intracellular injections of Lucifer Yellow at several distances from the temporal pole. We found regional differences in pyramidal cell morphology, which showed large inter-individual variability in most of the morphological variables measured. However, some values remained similar in all cases. The smallest and least complex cells in the most posterior temporal region showed the greatest dendritic spine density. Neurons in the temporal pole showed the greatest sizes with the highest number of spines. Layer V cells were larger, more complex, and had a greater number of dendritic spines than those in layer III. The present results suggest that, while some aspects of pyramidal structure are conserved, there are specific variations across cortical regions, and species.

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.

Volume Electron Microscopy Study of the Relationship Between Synapses and Astrocytes in the Developing Rat Somatosensory Cortex.

In recent years, numerous studies have shown that astrocytes play an important role in neuronal processing of information. One of the most interesting findings is the existence of bidirectional interactions between neurons and astrocytes at synapses, which has given rise to the concept of "tripartite synapses" from a functional point of view. We used focused ion beam milling and scanning electron microscopy (FIB/SEM) to examine in 3D the relationship of synapses with astrocytes that were previously labeled by intracellular injections in the rat somatosensory cortex. We observed that a large number of synapses (32%) had no contact with astrocytic processes. The remaining synapses (68%) were in contact with astrocytic processes, either at the level of the synaptic cleft (44%) or with the pre- and/or post-synaptic elements (24%). Regarding synaptic morphology, larger synapses with more complex shapes were most frequently found within the population that had the synaptic cleft in contact with astrocytic processes. Furthermore, we observed that although synapses were randomly distributed in space, synapses that were free of astrocytic processes tended to form clusters. Overall, at least in the developing rat neocortex, the concept of tripartite synapse only seems to be applicable to a subset of synapses.

Differential Structure of Hippocampal CA1 Pyramidal Neurons in the Human and Mouse.

Pyramidal neurons are the most common cell type and are considered the main output neuron in most mammalian forebrain structures. In terms of function, differences in the structure of the dendrites of these neurons appear to be crucial in determining how neurons integrate information. To further shed light on the structure of the human pyramidal neurons we investigated the geometry of pyramidal cells in the human and mouse CA1 region-one of the most evolutionary conserved archicortical regions, which is critically involved in the formation, consolidation, and retrieval of memory. We aimed to assess to what extent neurons corresponding to a homologous region in different species have parallel morphologies. Over 100 intracellularly injected and 3D-reconstructed cells across both species revealed that dendritic and axonal morphologies of human cells are not only larger but also have structural differences, when compared to mouse. The results show that human CA1 pyramidal cells are not a stretched version of mouse CA1 cells. These results indicate that there are some morphological parameters of the pyramidal cells that are conserved, whereas others are species-specific.

Slow-Wave Activity in the S1HL Cortex Is Contributed by Different Layer-Specific Field Potential Sources during Development.

Spontaneous correlated activity in cortical columns is critical for postnatal circuit refinement. We used spatial discrimination techniques to explore the late maturation of synaptic pathways through the laminar distribution of the field potential (FP) generators underlying spontaneous and evoked activities of the S1HL cortex in juvenile (P14-P16) and adult anesthetized rats. Juveniles exhibit an intermittent FP pattern resembling Up/Down states in adults, but with much reduced power and different laminar distribution. Whereas FPs in active periods are dominated by a layer VI generator in juveniles, in adults a developing multipart generator takes over, displaying current sinks in middle layers (III-V). The blockade of excitatory transmission in upper and middle layers of adults recovered the juvenile-like FP profiles. In addition to the layer VI generator, a gamma-specific generator in supragranular layers was the same in both age groups. While searching for dynamical coupling among generators in juveniles we found significant cross-correlation in ∼one-half of the tested pairs, whereas excessive coherence hindered their efficient separation in adults. Also, potentials evoked by tactile and electrical stimuli showed different short-latency dipoles between the two age groups, and the juveniles lacked the characteristic long latency UP state currents in middle layers. In addition, the mean firing rate of neurons was lower in juveniles. Thus, cortical FPs originate from different intra-columnar segments as they become active postnatally. We suggest that although some cortical segments are active early postnatally, a functional sensory-motor control relies on a delayed maturation and network integration of synaptic connections in middle layers.SIGNIFICANCE STATEMENT Early postnatal activity in the rodent cortex is mostly endogenous, whereas it becomes driven by peripheral input at later stages. The precise schedule for the maturation of synaptic pathways is largely unknown. We explored this in the somatosensory hindlimb cortex at an age when animals begin to use their limbs by uncovering the laminar distribution of the field potential generators underlying the dominant delta waves in juveniles and adults. Our results suggest that field potentials are mostly generated by a pathway in deep layers, whereas other pathways mature later in middle layers and take over in adults. We suggest that a functional sensory-motor control relies on a delayed maturation and network integration of synaptic connections in middle layers.

Ultrastructural, Molecular and Functional Mapping of GABAergic Synapses on Dendritic Spines and Shafts of Neocortical Pyramidal Neurons.

The location of GABAergic synapses on dendrites is likely key for neuronal integration. In particular, inhibitory inputs on dendritic spines could serve to selectively veto or modulate individual excitatory inputs, greatly expanding the computational power of individual neurons. To investigate this, we have undertaken a combined functional, molecular, and ultrastructural mapping of the location of GABAergic inputs onto dendrites of pyramidal neurons from upper layers of juvenile mouse somatosensory cortex. Using two-photon uncaging of GABA, intracellular labeling with gerphyrin intrabodies, and focused ion beam milling with scanning electron microscopy, we find that most (96-98%) spines lack GABAergic synapses, although they still display GABAergic responses, potentially due to extrasynaptic GABA receptors. We conclude that GABAergic inputs, in practice, contact dendritic shafts and likely control clusters of excitatory inputs, defining functional zones on dendrites.

27-Hydroxycholesterol Induces Aberrant Morphology and Synaptic Dysfunction in Hippocampal Neurons.

Hypercholesterolemia is a risk factor for neurodegenerative diseases, but how high blood cholesterol levels are linked to neurodegeneration is still unknown. Here, we show that an excess of the blood-brain barrier permeable cholesterol metabolite 27-hydroxycholesterol (27-OH) impairs neuronal morphology and reduces hippocampal spine density and the levels of the postsynaptic protein PSD95. Dendritic spines are the main postsynaptic elements of excitatory synapses and are crucial structures for memory and cognition. Furthermore, PSD95 has an essential function for synaptic maintenance and plasticity. PSD95 synthesis is controlled by the REST-miR124a-PTBP1 axis. Here, we report that high levels of 27-OH induce REST-miR124a-PTBP1 axis dysregulation in a possible RxRγ-dependent manner, suggesting that 27-OH reduces PSD95 levels through this mechanism. Our results reveal a possible molecular link between hypercholesterolemia and neurodegeneration. We discuss the possibility that reduction of 27-OH levels could be a useful strategy for preventing memory and cognitive decline in neurodegenerative disorders.

Metabolomic Study of Hibernating Syrian Hamster Brains: In Search of Neuroprotective Agents.

Syrian hamsters undergo a reversible hyperphosphorylation of protein τ during hibernation, providing a unique natural model that may unveil the physiological mechanisms behind this critical process involved in the development of Alzheimer's disease and other tauopathies. The hibernation cycle of these animals fluctuates between a pair of stages: 3-4 days of torpor bouts interspersed with periods of euthermia called arousals that last several hours. In this study, we investigated for the first time the metabolic changes in brain tissue during hibernation. A total of 337 metabolites showed statistically significant differences during hibernation. Based on these metabolites, several pathways were found to be significantly regulated and, therefore, play a key role in the regulation of hibernation processes. The increase in the levels of ceramides containing more than 20 C atoms was found in torpor animals, reflecting a higher activity of CerS2 during hibernation, linked to neurofibrillary tangle generation and structural changes in the Golgi apparatus. Our results open up the debate about the possible significance of some metabolites during hibernation, which may possibly be related to τ phosphorylation and dephosphorylation events. In general, this study may provide insights into novel neuroprotective agents because the alterations described throughout the hibernation process are reversible.

3D morphology-based clustering and simulation of human pyramidal cell dendritic spines.

The dendritic spines of pyramidal neurons are the targets of most excitatory synapses in the cerebral cortex. They have a wide variety of morphologies, and their morphology appears to be critical from the functional point of view. To further characterize dendritic spine geometry, we used in this paper over 7,000 individually 3D reconstructed dendritic spines from human cortical pyramidal neurons to group dendritic spines using model-based clustering. This approach uncovered six separate groups of human dendritic spines. To better understand the differences between these groups, the discriminative characteristics of each group were identified as a set of rules. Model-based clustering was also useful for simulating accurate 3D virtual representations of spines that matched the morphological definitions of each cluster. This mathematical approach could provide a useful tool for theoretical predictions on the functional features of human pyramidal neurons based on the morphology of dendritic spines.

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.

Towards a supervised classification of neocortical interneuron morphologies.

BACKGROUND: The challenge of classifying cortical interneurons is yet to be solved. Data-driven classification into established morphological types may provide insight and practical value. RESULTS: We trained models using 217 high-quality morphologies of rat somatosensory neocortex interneurons reconstructed by a single laboratory and pre-classified into eight types. We quantified 103 axonal and dendritic morphometrics, including novel ones that capture features such as arbor orientation, extent in layer one, and dendritic polarity. We trained a one-versus-rest classifier for each type, combining well-known supervised classification algorithms with feature selection and over- and under-sampling. We accurately classified the nest basket, Martinotti, and basket cell types with the Martinotti model outperforming 39 out of 42 leading neuroscientists. We had moderate accuracy for the double bouquet, small and large basket types, and limited accuracy for the chandelier and bitufted types. We characterized the types with interpretable models or with up to ten morphometrics. CONCLUSION: Except for large basket, 50 high-quality reconstructions sufficed to learn an accurate model of a type. Improving these models may require quantifying complex arborization patterns and finding correlates of bouton-related features. Our study brings attention to practical aspects important for neuron classification and is readily reproducible, with all code and data available online.

New insights into the classification and nomenclature of cortical GABAergic interneurons.

A systematic classification and accepted nomenclature of neuron types is much needed but is currently lacking. This article describes a possible taxonomical solution for classifying GABAergic interneurons of the cerebral cortex based on a novel, web-based interactive system that allows experts to classify neurons with pre-determined criteria. Using Bayesian analysis and clustering algorithms on the resulting data, we investigated the suitability of several anatomical terms and neuron names for cortical GABAergic interneurons. Moreover, we show that supervised classification models could automatically categorize interneurons in agreement with experts' assignments. These results demonstrate a practical and objective approach to the naming, characterization and classification of neurons based on community consensus.