Structural changes of CA1 pyramidal neurons after stroke in the contralesional hippocampus.

Significant progress has been made with regard to understanding how the adult brain responds after a stroke. However, a large number of patients continue to suffer lifelong disabilities without adequate treatment. In the present study, we have analyzed possible microanatomical alterations in the contralesional hippocampus from the ischemic stroke mouse model tMCAo 12-14 weeks after transient middle cerebral artery occlusion. After individually injecting Lucifer yellow into pyramidal neurons from the CA1 field of the hippocampus, we performed a detailed three-dimensional analysis of the neuronal complexity, dendritic spine density, and morphology. We found that, in both apical (stratum radiatum) and basal (stratum oriens) arbors, CA1 pyramidal neurons in the contralesional hippocampus of tMCAo mice have a significantly higher neuronal complexity, as well as reduced spine density and alterations in spine volume and spine length. Our results show that when the ipsilateral hippocampus is dramatically damaged, the contralesional hippocampus exhibits several statistically significant selective alterations. However, these alterations are not as significant as expected, which may help to explain the recovery of hippocampal function after stroke. Further anatomical and physiological studies are necessary to better understand the modifications in the "intact" contralesional lesioned brain regions, which are probably fundamental to recover functions after stroke.

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.

Single-Neuron Labeling in Fixed Tissue and Targeted Volume Electron Microscopy.

The structural complexity of nervous tissue makes it very difficult to unravel the connectivity between neural elements at different scales. Numerous methods are available to trace long-range projections at the light microscopic level, and to identify the actual synaptic connections at the electron microscopic level. However, correlating mesoscopic and nanoscopic scales in the same cell, cell population or brain region is a problematic, laborious and technically demanding task. Here we present an effective method for the 3D reconstruction of labeled subcellular structures at the ultrastructural level, after single-neuron labeling in fixed tissue. The brain is fixed by intracardial perfusion of aldehydes and thick vibratome sections (250 µm) are obtained. Single cells in these vibratome sections are intracellularly injected with horseradish peroxidase (HRP), so that the cell body and its processes can be identified. The thick sections are later flat-embedded in epoxy resin and re-sectioned into a series of thinner (7 µm) sections. The sections containing the regions of interest of the labeled cells are then imaged with automated focused ion beam milling and scanning electron microscopy (FIB-SEM), acquiring long series of high-resolution images that can be reconstructed, visualized, and analyzed in 3D. With this methodology, we can accurately select any cellular segment at the light microscopic level (e.g., proximal, intermediate or distal dendrites, collateral branches, axonal segments, etc.) and analyze its synaptic connections at the electron microscopic level, along with other ultrastructural features. Thus, this method not only facilitates the mapping of the synaptic connectivity of single-labeled neurons, but also the analysis of the surrounding neuropil. Since the labeled processes can be located at different layers or subregions, this method can also be used to obtain data on the differences in local synaptic organization that may exist at different portions of the labeled neurons.

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.

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.

MultiMap: A Tool to Automatically Extract and Analyse Spatial Microscopic Data From Large Stacks of Confocal Microscopy Images.

The development of 3D visualization and reconstruction methods to analyse microscopic structures at different levels of resolutions is of great importance to define brain microorganization and connectivity. MultiMap is a new tool that allows the visualization, 3D segmentation and quantification of fluorescent structures selectively in the neuropil from large stacks of confocal microscopy images. The major contribution of this tool is the posibility to easily navigate and create regions of interest of any shape and size within a large brain area that will be automatically 3D segmented and quantified to determine the density of puncta in the neuropil. As a proof of concept, we focused on the analysis of glutamatergic and GABAergic presynaptic axon terminals in the mouse hippocampal region to demonstrate its use as a tool to provide putative excitatory and inhibitory synaptic maps. The segmentation and quantification method has been validated over expert labeled images of the mouse hippocampus and over two benchmark datasets, obtaining comparable results to the expert detections.

GSK-3ß Overexpression Alters the Dendritic Spines of Developmentally Generated Granule Neurons in the Mouse Hippocampal Dentate Gyrus.

The dentate gyrus (DG) plays a crucial role in hippocampal-related memory. The most abundant cellular type in the DG, namely granule neurons, are developmentally generated around postnatal day P6 in mice. Moreover, a unique feature of the DG is the occurrence of adult hippocampal neurogenesis, a process that gives rise to newborn granule neurons throughout life. Adult-born and developmentally generated granule neurons share some maturational aspects but differ in others, such as in their positioning within the granule cell layer. Adult hippocampal neurogenesis encompasses a series of plastic changes that modify the function of the hippocampal trisynaptic network. In this regard, it is known that glycogen synthase kinase 3ß (GSK-3ß) regulates both synaptic plasticity and memory. By using a transgenic mouse overexpressing GSK-3ß in hippocampal neurons, we previously demonstrated that the overexpression of this kinase has deleterious effects on the maturation of newborn granule neurons. In the present study, we addressed the effects of GSK-3ß overexpression on the morphology and number of dendritic spines of developmentally generated granule neurons. To this end, we performed intracellular injections of Lucifer Yellow in developmentally generated granule neurons of wild-type and GSK-3ß-overexpressing mice and analyzed the number and morphologies of dendritic spines (namely, stubby, thin and mushroom). GSK-3ß overexpression led to a general reduction in the number of dendritic spines. In addition, it caused a slight reduction in the percentage, head diameter and length of thin spines, whereas the head diameter of mushroom spines was increased.

Rat-strain dependent changes of dendritic and spine morphology in the hippocampus after cocaine self-administration.

We previously showed that cocaine self-administration increases spine density in CA1 hippocampal neurons in Lewis (LEW) but not in Fischer 344 (F344) rats. Dendritic spine morphology is intimately related to its function. Thus, we conducted a 3D morphological analysis of CA1 dendrites and dendritic spines in these two strains of rats. Strain-specific differences were observed prior to cocaine self-administration: LEW rats had significantly larger dendritic diameters but lower spine density than the F344 strain. After cocaine self-administration, proximal dendritic volume, dendritic surface area and spine density were increased in LEW rats, where a higher percentage of larger spines were also observed. In addition, we found a strong positive correlation between dendritic volume and spine morphology, and a moderate correlation between dendritic volume and spine density in cocaine self-administered LEW rats, an effect that was not evident in any other condition. By contrast, after cocaine self-administration, F334 rats showed decreased spine head volumes. Our findings suggest that genetic differences could play a key role in the structural plasticity induced by cocaine in CA1 pyramidal neurons. These cocaine-induced alterations could be related to differences in the memory processing of drug reward cues that could potentially explain differential individual vulnerability to cocaine addiction.

Specific cytoarchitectureal changes in hippocampal subareas in daDREAM mice.

BACKGROUND: Transcriptional repressor DREAM (downstream regulatory element antagonist modulator) is a Ca(2+)-binding protein that regulates Ca(2+) homeostasis through gene regulation and protein-protein interactions. It has been shown that a dominant active form (daDREAM) is implicated in learning-related synaptic plasticity such as LTP and LTD in the hippocampus. Neuronal spines are reported to play important roles in plasticity and memory. However, the possible role of DREAM in spine plasticity has not been reported. RESULTS: Here we show that potentiating DREAM activity, by overexpressing daDREAM, reduced dendritic basal arborization and spine density in CA1 pyramidal neurons and increased spine density in dendrites in dentate gyrus granule cells. These microanatomical changes are accompanied by significant modifications in the expression of specific genes encoding the cytoskeletal proteins Arc, Formin 1 and Gelsolin in daDREAM hippocampus. CONCLUSIONS: Our results strongly suggest that DREAM plays an important role in structural plasticity in the hippocampus.

Dendritic branching angles of pyramidal cells across layers of the juvenile rat somatosensory cortex.

The characterization of the structural design of cortical microcircuits is essential for understanding how they contribute to function in both health and disease. Since pyramidal neurons represent the most abundant neuronal type and their dendritic spines constitute the major postsynaptic elements of cortical excitatory synapses, our understanding of the synaptic organization of the neocortex largely depends on the available knowledge regarding the structure of pyramidal cells. Previous studies have identified several apparently common rules in dendritic geometry. We study the dendritic branching angles of pyramidal cells across layers to further shed light on the principles that determine the geometric shapes of these cells. We find that the dendritic branching angles of pyramidal cells from layers II-VI of the juvenile rat somatosensory cortex suggest common design principles, despite the particular morphological and functional features that are characteristic of pyramidal cells in each cortical layer. J. Comp. Neurol. 524:2567-2576, 2016. © 2016 Wiley Periodicals, Inc.

Laminar Differences in Dendritic Structure of Pyramidal Neurons in the Juvenile Rat Somatosensory Cortex.

Pyramidal cell structure varies between different cortical areas and species, indicating that the cortical circuits that these cells participate in are likely to be characterized by different functional capabilities. Structural differences between cortical layers have been traditionally reported using either the Golgi method or intracellular labeling, but the structure of pyramidal cells has not previously been systematically analyzed across all cortical layers at a particular age. In the present study, we investigated the dendritic architecture of complete basal arbors of pyramidal neurons in layers II, III, IV, Va, Vb, and VI of the hindlimb somatosensory cortical region of postnatal day 14 rats. We found that the characteristics of basal dendritic morphologies are statistically different in each cortical layer. The variations in size and branching pattern that exist between pyramidal cells of different cortical layers probably reflect the particular functional properties that are characteristic of the cortical circuit in which they participate. This new set of complete basal dendritic arbors of 3D-reconstructed pyramidal cell morphologies across each cortical layer will provide new insights into interlaminar information processing in the cerebral cortex.

Structural plasticity in hippocampal cells related to the facilitative effect of intracranial self-stimulation on a spatial memory task.

Posttraining intracranial self-stimulation (SS) in the lateral hypothalamus facilitates the acquisition and retention of several implicit and explicit memory tasks. Here, intracellular injections of Lucifer yellow were used to assess morphological changes in hippocampal neurons that might be specifically related to the facilitative posttraining SS effect upon the acquisition and retention of a distributed spatial task in the Morris water maze. We examined the structure, size and branching complexity of cornus ammonis 1 (CA1) cells, and the spine density of CA1 pyramidal neurons and granular cells of the dentate gyrus (DG). Animals that received SS after each acquisition session performed faster and better than Sham ones--an improvement that was also evident in a probe trial 3 days after the last training session. The neuromorphological analysis revealed an increment in the size and branching complexity in apical CA1 dendritic arborization in SS-treated subjects as compared with Sham animals. Furthermore, increased spine density was observed in the CA1 field in SS animals, whereas no effects were observed in DG cells. Our results support the hypothesis that the facilitating effect of SS on the acquisition and retention of a spatial memory task could be related to structural plasticity in CA1 hippocampal cells.

The effects of cocaine self-administration on dendritic spine density in the rat hippocampus are dependent on genetic background.

Chronic exposure to cocaine induces modifications to neurons in the brain regions involved in addiction. Hence, we evaluated cocaine-induced changes in the hippocampal CA1 field in Fischer 344 (F344) and Lewis (LEW) rats, 2 strains that have been widely used to study genetic predisposition to drug addiction, by combining intracellular Lucifer yellow injection with confocal microscopy reconstruction of labeled neurons. Specifically, we examined the effects of cocaine self-administration on the structure, size, and branching complexity of the apical dendrites of CA1 pyramidal neurons. In addition, we quantified spine density in the collaterals of the apical dendritic arbors of these neurons. We found differences between these strains in several morphological parameters. For example, CA1 apical dendrites were more branched and complex in LEW than in F344 rats, while the spine density in the collateral dendrites of the apical dendritic arbors was greater in F344 rats. Interestingly, cocaine self-administration in LEW rats augmented the spine density, an effect that was not observed in the F344 strain. These results reveal significant structural differences in CA1 pyramidal cells between these strains and indicate that cocaine self-administration has a distinct effect on neuron morphology in the hippocampus of rats with different genetic backgrounds.

DREAM controls the on/off switch of specific activity-dependent transcription pathways.

Changes in nuclear Ca(2+) homeostasis activate specific gene expression programs and are central to the acquisition and storage of information in the brain. DREAM (downstream regulatory element antagonist modulator), also known as calsenilin/KChIP-3 (K(+) channel interacting protein 3), is a Ca(2+)-binding protein that binds DNA and represses transcription in a Ca(2+)-dependent manner. To study the function of DREAM in the brain, we used transgenic mice expressing a Ca(2+)-insensitive/CREB-independent dominant active mutant DREAM (daDREAM). Using genome-wide analysis, we show that DREAM regulates the expression of specific activity-dependent transcription factors in the hippocampus, including Npas4, Nr4a1, Mef2c, JunB, and c-Fos. Furthermore, DREAM regulates its own expression, establishing an autoinhibitory feedback loop to terminate activity-dependent transcription. Ablation of DREAM does not modify activity-dependent transcription because of gene compensation by the other KChIP family members. The expression of daDREAM in the forebrain resulted in a complex phenotype characterized by loss of recurrent inhibition and enhanced long-term potentiation (LTP) in the dentate gyrus and impaired learning and memory. Our results indicate that DREAM is a major master switch transcription factor that regulates the on/off status of specific activity-dependent gene expression programs that control synaptic plasticity, learning, and memory.

Machine learning approach for the outcome prediction of temporal lobe epilepsy surgery.

Epilepsy surgery is effective in reducing both the number and frequency of seizures, particularly in temporal lobe epilepsy (TLE). Nevertheless, a significant proportion of these patients continue suffering seizures after surgery. Here we used a machine learning approach to predict the outcome of epilepsy surgery based on supervised classification data mining taking into account not only the common clinical variables, but also pathological and neuropsychological evaluations. We have generated models capable of predicting whether a patient with TLE secondary to hippocampal sclerosis will fully recover from epilepsy or not. The machine learning analysis revealed that outcome could be predicted with an estimated accuracy of almost 90% using some clinical and neuropsychological features. Importantly, not all the features were needed to perform the prediction; some of them proved to be irrelevant to the prognosis. Personality style was found to be one of the key features to predict the outcome. Although we examined relatively few cases, findings were verified across all data, showing that the machine learning approach described in the present study may be a powerful method. Since neuropsychological assessment of epileptic patients is a standard protocol in the pre-surgical evaluation, we propose to include these specific psychological tests and machine learning tools to improve the selection of candidates for epilepsy surgery.

The influence of phospho-τ on dendritic spines of cortical pyramidal neurons in patients with Alzheimer's disease.

The dendritic spines on pyramidal cells represent the main postsynaptic elements of cortical excitatory synapses and they are fundamental structures in memory, learning and cognition. In the present study, we used intracellular injections of Lucifer yellow in fixed tissue to analyse over 19 500 dendritic spines that were completely reconstructed in three dimensions along the length of the basal dendrites of pyramidal neurons in the parahippocampal cortex and CA1 of patients with Alzheimer's disease. Following intracellular injection, sections were immunostained for anti-Lucifer yellow and with tau monoclonal antibodies AT8 and PHF-1, which recognize tau phosphorylated at Ser202/Thr205 and at Ser396/404, respectively. We observed that the diffuse accumulation of phospho-tau in a putative pre-tangle state did not induce changes in the dendrites of pyramidal neurons, whereas the presence of tau aggregates forming intraneuronal neurofibrillary tangles was associated with progressive alteration of dendritic spines (loss of dendritic spines and changes in their morphology) and dendrite atrophy, depending on the degree of tangle development. Thus, the presence of phospho-tau in neurons does not necessarily mean that they suffer severe and irreversible effects as thought previously but rather, the characteristic cognitive impairment in Alzheimer's disease is likely to depend on the relative number of neurons that have well developed tangles.

A stereological study of synapse number in the epileptic human hippocampus.

Hippocampal sclerosis is the most frequent pathology encountered in resected mesial temporal structures from patients with intractable temporal lobe epilepsy (TLE). Here, we have used stereological methods to compare the overall density of synapses and neurons between non-sclerotic and sclerotic hippocampal tissue obtained by surgical resection from patients with TLE. Specifically, we examined the possible changes in the subiculum and CA1, regions that seem to be critical for the development and/or maintenance of seizures in these patients. We found a remarkable decrease in synaptic and neuronal density in the sclerotic CA1, and while the subiculum from the sclerotic hippocampus did not display changes in synaptic density, the neuronal density was higher. Since the subiculum from the sclerotic hippocampus displays a significant increase in neuronal density, as well as a various other neurochemical changes, we propose that the apparently normal subiculum from the sclerotic hippocampus suffers profound alterations in neuronal circuits at both the molecular and synaptic level that are likely to be critical for the development or maintenance of seizure 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.