Reconstruction and Simulation of Neocortical Microcircuitry.

We present a first-draft digital reconstruction of the microcircuitry of somatosensory cortex of juvenile rat. The reconstruction uses cellular and synaptic organizing principles to algorithmically reconstruct detailed anatomy and physiology from sparse experimental data. An objective anatomical method defines a neocortical volume of 0.29 ± 0.01 mm(3) containing ~31,000 neurons, and patch-clamp studies identify 55 layer-specific morphological and 207 morpho-electrical neuron subtypes. When digitally reconstructed neurons are positioned in the volume and synapse formation is restricted to biological bouton densities and numbers of synapses per connection, their overlapping arbors form ~8 million connections with ~37 million synapses. Simulations reproduce an array of in vitro and in vivo experiments without parameter tuning. Additionally, we find a spectrum of network states with a sharp transition from synchronous to asynchronous activity, modulated by physiological mechanisms. The spectrum of network states, dynamically reconfigured around this transition, supports diverse information processing strategies. PAPERCLIP: VIDEO ABSTRACT.

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.

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.

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.

Facilitation of AMPA receptor synaptic delivery as a molecular mechanism for cognitive enhancement.

Cell adhesion molecules and downstream growth factor-dependent signaling are critical for brain development and synaptic plasticity, and they have been linked to cognitive function in adult animals. We have previously developed a mimetic peptide (FGL) from the neural cell adhesion molecule (NCAM) that enhances spatial learning and memory in rats. We have now investigated the cellular and molecular basis of this cognitive enhancement, using biochemical, morphological, electrophysiological, and behavioral analyses. We have found that FGL triggers a long-lasting enhancement of synaptic transmission in hippocampal CA1 neurons. This effect is mediated by a facilitated synaptic delivery of AMPA receptors, which is accompanied by enhanced NMDA receptor-dependent long-term potentiation (LTP). Both LTP and cognitive enhancement are mediated by an initial PKC activation, which is followed by persistent CaMKII activation. These results provide a mechanistic link between facilitation of AMPA receptor synaptic delivery and improved hippocampal-dependent learning, induced by a pharmacological cognitive enhancer.

Alterations of the microvascular network in the sclerotic hippocampus of patients with temporal lobe epilepsy.

Hippocampal sclerosis is the most frequent pathology encountered in resected tissue obtained from patients with temporal lobe epilepsy. The main hallmarks of hippocampal sclerosis are neuronal loss and gliosis. Several authors have proposed that an increase in blood vessel density is a further indicator, based on interpretations from staining of markers related to both blood-brain barrier disruption and the formation of new blood vessels. However, previous studies performed in our laboratory using correlative light and electron microscopy revealed that many of these "blood vessels" are in fact atrophic vascular structures with a reduced or virtually absent lumen and are often filled with processes of reactive astrocytes. Thus, "normal" vasculature within the sclerotic CA1 field is drastically reduced. Since this decrease is consistently observed in the human sclerotic CA1, this feature can be considered another key pathological indicator of hippocampal sclerosis associated with temporal lobe epilepsy.

Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex.

Neuroscience produces a vast amount of data from an enormous diversity of neurons. A neuronal classification system is essential to organize such data and the knowledge that is derived from them. Classification depends on the unequivocal identification of the features that distinguish one type of neuron from another. The problems inherent in this are particularly acute when studying cortical interneurons. To tackle this, we convened a representative group of researchers to agree on a set of terms to describe the anatomical, physiological and molecular features of GABAergic interneurons of the cerebral cortex. The resulting terminology might provide a stepping stone towards a future classification of these complex and heterogeneous cells. Consistent adoption will be important for the success of such an initiative, and we also encourage the active involvement of the broader scientific community in the dynamic evolution of this project.

Cortical synapses of the world's smallest mammal: An FIB/SEM study in the Etruscan shrew.

The main aim of the present study was to determine if synapses from the exceptionally small brain of the Etruscan shrew show any peculiarities compared to the much larger human brain. We analyzed the cortical synaptic density and a variety of structural characteristics of 7,239 3D reconstructed synapses, using using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM). We found that some of the general synaptic characteristics are remarkably similar to those found in the human cerebral cortex. However, the cortical volume of the human brain is about 50,000 times larger than the cortical volume of the Etruscan shrew, while the total number of cortical synapses in human is only 20,000 times the number of synapses in the shrew, and synaptic junctions are 35% smaller in the Etruscan shrew. Thus, the differences in the number and size of synapses cannot be attributed to a brain size scaling effect but rather to adaptations of synaptic circuits to particular functions.

Emerging insights into synapse dysregulation in Alzheimer's disease.

Alzheimer's disease is the leading cause of dementia and a growing worldwide problem, with its incidence expected to increase in the coming years. Since synapse loss is a major pathology and is correlated with symptoms in Alzheimer's disease, synapse dysfunction and loss may underlie pathophysiology. In this context, this review focuses on emerging insights into synaptic changes at the ultrastructural level. The three-dimensional electron microscopy technique unequivocally detects all types of synapses, including multi-synapses, which are indicators of synaptic connectivity between neurons. In recent years it has become feasible to perform sophisticated three-dimensional electron microscopy analyses on post-mortem human Alzheimer's disease brain as tissue preservation and electron microscopy techniques have improved. This ultrastructural analysis found that synapse loss does not always precede neuronal loss, as long believed. For instance, in the transentorhinal cortex and area CA1 of the hippocampus, synapse loss does not precede neuronal loss. However, in the entorhinal cortex, synapse loss precedes neuronal loss. Moreover, the ultrastructural analysis provides details about synapse morphology. For example, changes in excitatory synapses' post-synaptic densities, with fragmented postsynaptic densities increasing at the expense of perforated synapses, are seen in Alzheimer's disease brain. Further, multi-synapses also appear to be altered in Alzheimer's disease by doubling the abundance of multi-innervated spines in the transentorhinal cortex of Alzheimer's disease brain. Collectively, these recent ultrastructural analyses highlight distinct synaptic phenotypes in different Alzheimer's disease brain regions and broaden the understanding of synapse alterations, which may unravel some new therapeutic targets.

Neuroanatomical and psychological considerations in temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy and is associated with a variety of structural and psychological alterations. Recently, there has been renewed interest in using brain tissue resected during epilepsy surgery, in particular 'non-epileptic' brain samples with normal histology that can be found alongside epileptic tissue in the same epileptic patients - with the aim being to study the normal human brain organization using a variety of methods. An important limitation is that different medical characteristics of the patients may modify the brain tissue. Thus, to better determine how 'normal' the resected tissue is, it is fundamental to know certain clinical, anatomical and psychological characteristics of the patients. Unfortunately, this information is frequently not fully available for the patient from which the resected tissue has been obtained - or is not fully appreciated by the neuroscientists analyzing the brain samples, who are not necessarily experts in epilepsy. In order to present the full picture of TLE in a way that would be accessible to multiple communities (e.g., basic researchers in neuroscience, neurologists, neurosurgeons and psychologists), we have reviewed 34 TLE patients, who were selected due to the availability of detailed clinical, anatomical, and psychological information for each of the patients. Our aim was to convey the full complexity of the disorder, its putative anatomical substrates, and the wide range of individual variability, with a view toward: (1) emphasizing the importance of considering critical patient information when using brain samples for basic research and (2) gaining a better understanding of normal and abnormal brain functioning. In agreement with a large number of previous reports, this study (1) reinforces the notion of substantial individual variability among epileptic patients, and (2) highlights the common but overlooked psychopathological alterations that occur even in patients who become "seizure-free" after surgery. The first point is based on pre- and post-surgical comparisons of patients with hippocampal sclerosis and patients with normal-looking hippocampus in neuropsychological evaluations. The second emerges from our extensive battery of personality and projective tests, in a two-way comparison of these two types of patients with regard to pre- and post-surgical performance.

Three-dimensional synaptic organization of the human hippocampal CA1 field.

The hippocampal CA1 field integrates a wide variety of subcortical and cortical inputs, but its synaptic organization in humans is still unknown due to the difficulties involved studying the human brain via electron microscope techniques. However, we have shown that the 3D reconstruction method using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) can be applied to study in detail the synaptic organization of the human brain obtained from autopsies, yielding excellent results. Using this technology, 24,752 synapses were fully reconstructed in CA1, revealing that most of them were excitatory, targeting dendritic spines and displaying a macular shape, regardless of the layer examined. However, remarkable differences were observed between layers. These data constitute the first extensive description of the synaptic organization of the neuropil of the human CA1 region.

There are billions of nerve cells or neurons in the human brain, and each one can form thousands of connections, also called synapses, with other neurons. That means there are trillions of synapses in the brain that keep information flowing. Studying the arrangement of individual neurons in the human brain, and the connections between them, is incredibly difficult because of its complexity. Scientists have tools that can image the whole brain and can measure the activity in different regions, but these tools only visualize brain structures that are large enough to be seen with human eyes. Synapses are much smaller (in the range of nanometers), and can only be seen using thin slices of preserved brain tissue through a technique called electron microscopy. The hippocampus is a part of the human brain that is critical for memory, learning and spatial orientation, and is affected in epilepsy and Alzheimer's disease. Although numerous studies of the hippocampus have been performed in laboratory animals, such as mice, the question remains as to how much of the information gained from these studies applies to humans. Thus, studying the human brain directly is a major goal in neuroscience. However, the scarcity of human brain tissue suitable for the study of synapses is one of the most important issues to overcome. Fortunately, healthy human brain tissue that can be studied using electron microscopy is sometimes donated after death. Using these donations could improve the understanding of the synapses in normal brains and possible changes associated with disease. Now, Montero-Crespo et al. have mapped synapses in the normal human hippocampus in three dimensions ­ providing the first detailed description of synaptic structure in this part of the brain. Using high-powered electron microscopes and donated brain tissue samples collected after death, Montero-Crespo et al. imaged almost 25,000 connections between neurons. The analysis showed that synapses were more densely packed in some layers of the hippocampus than in others. Most synapses were found to be connected to tiny dendritic 'spines' that sprout from dendritic branches of the neuron, and they activated (not suppressed) the next neuron. Beyond its implications for better understanding of brain health and disease, this work could also advance computer modelling attempts to mimic the structure of the brain and its activity.

Alterations of the microvascular network in sclerotic hippocampi from patients with epilepsy.

The main hallmarks of human hippocampal sclerosis are neuronal loss and gliosis; reductions in microvasculature labeling in the cornu Ammonis 1 in this condition have been detected using alkaline phosphatase histochemistry. To determine whether the reduction in alkaline phosphatase activity is coupled with a loss of blood vessels,we examined the volume fraction occupied by blood vessels in toluidine blue-stained hippocampal sections from 24 epilepsy patient resections (19 with hippocampal sclerosis, 5 without hippocampal sclerosis) and 5 normal autopsy controls. Light and electron microscopy and immunohistochemistry were used to determine the distribution of collagen Type IV in relation to the fine structure of the hippocampal microvascular network. We found a consistent and highly significant loss of microvessels in the sclerotic hippocampal cornu Ammonis 1 field; a variety of vascular alterations including spinelike protrusions, disruptions, and atrophic branching, were observed in the remaining blood vessels. We suggest that blood vessel alterations are an additional pathological hallmark of hippocampal sclerosis associated with temporal lobe epilepsy and that they may relate to the pathogenesis of this condition.

Subregional Density of Neurons, Neurofibrillary Tangles and Amyloid Plaques in the Hippocampus of Patients With Alzheimer's Disease.

A variety of anatomical alterations have been reported in the hippocampal formation of patients with Alzheimer's Disease (AD) and these alterations have been correlated with cognitive symptoms in the early stages of the disease. Major hallmarks in AD are the presence of paired helical filaments of tau protein (PHFTau) within neurons, also known as neurofibrillary tangles (NFTs), and aggregates of amyloid-ß protein (Aß) which form plaques in the extracellular space. Nevertheless, how the density of plaques and NFTs relate to the severity of cell loss and cognitive decline is not yet clear. The aim of the present study was to further examine the possible relationship of both Aß plaques and NFTs with neuronal loss in several hippocampal fields (DG, CA3, CA1, and subiculum) of 11 demented AD patients. For this purpose, using stereological techniques, we compared neuronal densities (Nissl-stained, and immunoreactive neurons for NeuN) with: (i) numbers of neurons immunostained for two isoforms of PHFTau (PHFTau-AT8 and PHFTau-pS396); and (ii) number of Aß plaques. We found that CA1 showed the highest number of NFTs and Aß plaques, whereas DG and CA3 displayed the lowest number of these markers. Furthermore, AD patients showed a variable neuronal loss in CA1 due to tangle-related cell death, which seems to correlate with the presence of extracellular tangles.

InTool Explorer: An Interactive Exploratory Analysis Tool for Versatile Visualizations of Neuroscientific Data.

The bottleneck for progress in many research areas within neuroscience has shifted from the data acquisition to the data analysis stages. In the present article, we propose a method named InTool Explorer that we have developed to perform interactive exploratory data analysis, focusing on neuroanatomy as an example of its utility. This tool is freely-available software that has been designed to facilitate the study of complex neuroscience data. InTool Explorer requires no more than an internet connection and a web browser. The main contribution of this tool is to provide a user-designed canvas for data visualization and interaction, to perform specific exploratory tasks according to the user needs. Moreover, InTool Explorer permits visualization of the datasets in a very dynamic and versatile way using a linked-card approach. For this purpose, the tool allows the user to select among different predefined card types. Each card type offers an abstract data representation, a filtering tool or a set of statistical analysis methods. Additionally, InTool Explorer makes it possible linking raw images to the data. These images can be used by InTool Explorer to define new customized filtering cards. Another significant contribution of this tool is that it allows fast visualization of the data, error finding, and re-evaluation to establish new hypotheses or new lines of research. Thus, regarding its practical application in the laboratory, InTool Explorer provides a new opportunity to study and analyze neuroscience data prior to any statistical analysis being carried out.

Hippocampal sclerosis: histopathology substrate and magnetic resonance imaging.

The term hippocampal sclerosis was originally used to describe a shrunken and hardened hippocampus, which histologically displayed neuronal loss and glial proliferation. These alterations are mainly located in the hilus of the dentate gyrus and in the CA1 and CA3 pyramidal cell layers but all hippocampal regions may show neuronal cell loss to varying degrees. A number of morphologic and cytochemical findings are associated with mesial temporal sclerosis, especially within the dentate gyrus. These changes include selective loss of inhibitory interneurons, abnormal sprouting of axons, reorganization of neural transmitter receptors, alterations in second messenger systems, and hyperexcitability of the granule cells. Extrahippocampal pathology is also found at other temporal lobe structures. Frequent extrahippocampal pathology affects the amygdala, first seen with neuronal cell loss and gliosis in the laterobasal complex. Surgical removal of this epileptogenic area can be curative or provide significant reduction in seizure frequency in the majority of individuals. Magnetic resonance imaging (MRI) is highly sensitive in detecting and locating mesial temporal sclerosis when a correct MRI temporal lobe protocol is used. The most important MRI findings, atrophy and abnormal T2 signal, allow us to detect mesial temporal sclerosis in the majority of the cases. Secondary MRI findings help in the diagnosis and lateralization of mesial temporal sclerosis in patients with subtle primary findings and in cases of bilateral hippocampal abnormalities. The development of advanced magnetic resonance (MR) techniques, such as functional MR, diffusion, or transference of magnetization, will lead to greater understanding of this pathology and will improve our diagnostic capacity.

A Study of Amyloid-ß and Phosphotau in Plaques and Neurons in the Hippocampus of Alzheimer's Disease Patients.

The main pathological hallmarks in Alzheimer's disease (AD) are the presence of extracellular amyloid plaques, primarily consisting of amyloid-ß (Aß) peptide, and the accumulation of paired helical filaments of hyperphosphorylated tau protein (PHF-Tau) within neurons. Since CA1 is one of the most affected regions in AD, mainly at early stages, we have performed a detailed analysis of the CA1 region from 11 AD patients (demented and clinically similar; Braak stages IV-VI) to better understand the possible relationship between the presence and distribution of different neurochemical types of Aß plaques and PHF-Tau immunoreactive  (- ir)  neurons. Hence, we have examined hippocampal sections in confocal microscopy images from double and triple-immunostained sections, to study labeled plaques and PHF-Tau-ir neurons using specific software tools. There are four main findings in the present study. First, the pyramidal layer of proximal CA1 (close to CA2) contains the smallest number of both plaques and PHF-Tau-ir neurons. Second, a large proportion of Aß-ir plaques were also characterized by the presence of PHF-Tau-ir. Third, all plaques containing one of the two PHF-Tau isoforms also express the other isoform, that is, if a plaque contains PHFpS396, it also contains PHFAT8, and vice versa. Fourth, the coexpression study of both PHF-Tau isoforms in CA1 neurons revealed that most of the labeled neurons express only PHFpS396. Our findings further support the idea that AD is not a unique entity even within the same neuropathological stage, since the microanatomical/neurochemical changes that occur in the hippocampus greatly vary from one patient to another.

Voxel-Based Diagnosis of Alzheimer's Disease Using Classifier Ensembles.

Functional magnetic resonance imaging (fMRI) is one of the most promising noninvasive techniques for early Alzheimer's disease (AD) diagnosis. In this paper, we explore the application of different machine learning techniques to the classification of fMRI data for this purpose. The functional images were first preprocessed using the statistical parametric mapping toolbox to output individual maps of statistically activated voxels. A fast filter was applied afterwards to select voxels commonly activated across demented and nondemented groups. Four feature ranking selection techniques were embedded into a wrapper scheme using an inner-outer loop for the selection of relevant voxels. The wrapper approach was guided by the performance of six pattern recognition models, three of which were ensemble classifiers based on stochastic searches. Final classification performance was assessed from the nested internal and external cross-validation loops taking several voxel sets ordered by importance. Numerical performance was evaluated using statistical tests, and the best combination of voxel selection and classification reached a 97.14% average accuracy. Results repeatedly pointed out Brodmann regions with distinct activation patterns between demented and nondemented profiles, indicating that the machine learning analysis described is a powerful method to detect differences in several brain regions between both groups.