3D SYNAPTIC ORGANIZATION OF THE RAT CA1 AND ALTERATIONS INDUCED BY COCAINE SELF-ADMINISTRATION

ABSTRACTThe hippocampus plays a key role in contextual conditioning and has been proposed as an important component of the cocaine addiction brain circuit. To gain knowledge about cocaine-induced alterations in this circuit, we used Focused Ion Beam milling/Scanning Electron Microscopy (FIB/SEM) to reveal and quantify the 3D synaptic organization of the stratum radiatum of rat CA1, under normal circumstances and after cocaine-self administration (SA). Most synapses are asymmetric (excitatory), macular-shaped, and in contact with spine heads. After cocaine-SA, the size and complexity of both asymmetric and symmetric (inhibitory) synapses increased but no changes were observed in the synaptic density.This work constitutes the first detailed report on the 3D synaptic organization in the stratum radiatum of the CA1 field of cocaine-SA rats. Our data contribute to the elucidation of the normal and altered synaptic organization of the hippocampus, which is crucial for better understanding the neurobiological mechanisms underlying cocaine addiction.

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3D Synaptic Organization of the Rat CA1 and Alterations Induced by Cocaine Self-Administration

Cerebral Cortex ◽

10.1093/cercor/bhaa331 ◽

2020 ◽

Author(s):

L Blazquez-Llorca ◽

M Miguéns ◽

M Montero-Crespo ◽

A Selvas ◽

J Gonzalez-Soriano ◽

...

Keyword(s):

Focused Ion Beam ◽

Ion Beam ◽

Three Dimensional ◽

Cocaine Addiction ◽

Stratum Radiatum ◽

Inhibitory Synapses ◽

Synaptic Organization ◽

Self Administration ◽

Synaptic Density ◽

Brain Circuit

Abstract The hippocampus plays a key role in contextual conditioning and has been proposed as an important component of the cocaine addiction brain circuit. To gain knowledge about cocaine-induced alterations in this circuit, we used focused ion beam milling/scanning electron microscopy to reveal and quantify the three-dimensional synaptic organization of the neuropil of the stratum radiatum of the rat CA1, under normal circumstances and after cocaine-self administration (SA). Most synapses are asymmetric (excitatory), macular-shaped, and in contact with dendritic spine heads. After cocaine-SA, the size and the complexity of the shape of both asymmetric and symmetric (inhibitory) synapses increased but no changes were observed in the synaptic density. This work constitutes the first detailed report on the 3D synaptic organization in the stratum radiatum of the CA1 field of cocaine-SA rats. Our data contribute to the elucidation of the normal and altered synaptic organization of the hippocampus, which is crucial for better understanding the neurobiological mechanisms underlying cocaine addiction.

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Three-dimensional synaptic organization of layer III of the human temporal neocortex

10.1101/2021.01.18.427131 ◽

2021 ◽

Author(s):

Nicolás Cano-Astorga ◽

Javier DeFelipe ◽

Lidia Alonso-Nanclares

Keyword(s):

Electron Microscopy ◽

Focused Ion Beam ◽

Functional Organization ◽

Ion Beam ◽

Three Dimensional ◽

Cortical Layer ◽

Cortical Region ◽

Synaptic Organization ◽

Synaptic Density ◽

Temporal Neocortex

AbstractIn 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. We analyzed the synaptic density, 3D spatial distribution, and type (excitatory/inhibitory), as well as the shape and size of each synaptic junction of 4945 synapses that were fully reconstructed in 3D. Moreover, the postsynaptic targets of 1888 synapses were determined. 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.

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3D Ultrastructural Study of Synapses in the Human Entorhinal Cortex

Cerebral Cortex ◽

10.1093/cercor/bhaa233 ◽

2020 ◽

Vol 31 (1) ◽

pp. 410-425 ◽

Cited By ~ 1

Author(s):

M Domínguez-Álvaro ◽

M Montero-Crespo ◽

L Blazquez-Llorca ◽

J DeFelipe ◽

L Alonso-Nanclares

Keyword(s):

Entorhinal Cortex ◽

Focused Ion Beam ◽

Functional Organization ◽

Structural Parameters ◽

Ion Beam ◽

Three Dimensional ◽

Ultrastructural Level ◽

3D Analysis ◽

Synaptic Organization ◽

Health And Disease

Abstract The entorhinal cortex (EC) is a brain region that has been shown to be essential for memory functions and spatial navigation. However, detailed three-dimensional (3D) synaptic morphology analysis and identification of postsynaptic targets at the ultrastructural level have not been performed before in the human EC. In the present study, we used Focused Ion Beam/Scanning Electron Microscopy to perform a 3D analysis of the synapses in the neuropil of medial EC in layers II and III from human brain autopsies. Specifically, we studied synaptic structural parameters of 3561 synapses, which were fully reconstructed in 3D. We analyzed the synaptic density, 3D spatial distribution, and type (excitatory and inhibitory), as well as the shape and size of each synaptic junction. Moreover, the postsynaptic targets of synapses could be clearly determined. The present work constitutes a detailed description of the synaptic organization of the human EC, which is a necessary step to better understand the functional organization of this region in both health and disease.

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3D analysis of the synaptic organization in the Entorhinal cortex in Alzheimer’s disease

10.1101/2020.10.19.345025 ◽

2020 ◽

Cited By ~ 1

Author(s):

Marta Domínguez-Álvaro ◽

Marta Montero-Crespo ◽

Lidia Blazquez-Llorca ◽

Javier DeFelipe ◽

Lidia Alonso-Nanclares

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Entorhinal Cortex ◽

Focused Ion Beam ◽

Present Report ◽

Ion Beam ◽

3D Analysis ◽

Synaptic Organization ◽

Light Microscope Level ◽

Anatomical Basis

AbstractThe entorhinal cortex (EC) is especially vulnerable in the early stages of Alzheimer’s disease (AD). In particular, cognitive deficits have been linked to alterations in the upper layers of EC. In the present report, we performed light microscopy analysis and 3D ultrastructural analyses of synapses in the EC using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) to examine possible alterations related to AD. We analyzed 5000 synaptic junctions that were 3D reconstructed, representing the largest 3D ultrastructural study of synapses in the EC of the human brain from cases with AD performed to date. Structural differences were found in the AD tissue at the light microscope level and at the ultrastructural level. These differences may play a role in the anatomical basis for the impairment of cognitive functions in AD.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21155558 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5558

Author(s):

Astrid Rollenhagen ◽

Bernd Walkenfort ◽

Rachida Yakoubi ◽

Sarah A. Klauke ◽

Sandra F. Schmuhl-Giesen ◽

...

Keyword(s):

Synaptic Vesicles ◽

Focused Ion Beam ◽

Ion Beam ◽

3D Models ◽

Synaptic Organization ◽

Experimental Animals ◽

Synaptic Boutons ◽

Active Zones ◽

Em Tomography ◽

Human Temporal Lobe

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.

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Three-dimensional synaptic organization of the human hippocampal CA1 field

10.1101/2020.02.25.964080 ◽

2020 ◽

Cited By ~ 2

Author(s):

Marta Montero-Crespo ◽

Marta Domínguez-Álvaro ◽

Patricia Rondón-Carrillo ◽

Lidia Alonso-Nanclares ◽

Javier DeFelipe ◽

...

Keyword(s):

Human Brain ◽

Focused Ion Beam ◽

Ion Beam ◽

Three Dimensional ◽

Reconstruction Method ◽

Synaptic Organization ◽

Ca1 Region ◽

Hippocampal Ca1 ◽

Hippocampal Ca1 Field ◽

Cortical Inputs

AbstractThe 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.

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Neuron Class and Target Variability in the Three-Dimensional Localization of SK2 Channels in Hippocampal Neurons as Detected by Immunogold FIB-SEM

Frontiers in Neuroanatomy ◽

10.3389/fnana.2021.781314 ◽

2021 ◽

Vol 15 ◽

Author(s):

Rafael Luján ◽

Angel Merchán-Pérez ◽

Joaquim Soriano ◽

Alejandro Martín-Belmonte ◽

Carolina Aguado ◽

...

Keyword(s):

Hippocampal Neurons ◽

Spatial Organization ◽

Ion Beam ◽

Pyramidal Cells ◽

Inhibitory Neurons ◽

Stratum Radiatum ◽

Inhibitory Synapses ◽

Sk Channels ◽

Large Variability ◽

Channel Density

Small-conductance calcium-activated potassium (SK) channels are crucial for learning and memory. However, many aspects of their spatial organization in neurons are still unknown. In this study, we have taken a novel approach to answering these questions combining a pre-embedding immunogold labeling with an automated dual-beam electron microscope that integrates focused ion beam milling and scanning electron microscopy (FIB/SEM) to gather 3D map ultrastructural and biomolecular information simultaneously. Using this new approach, we evaluated the number and variability in the density of extrasynaptic SK2 channels in 3D reconstructions from six dendritic segments of excitatory neurons and six inhibitory neurons present in the stratum radiatum of the CA1 region of the mouse. SK2 immunoparticles were observed throughout the surface of hippocampal neurons, either scattered or clustered, as well as at intracellular sites. Quantitative volumetric evaluations revealed that the extrasynaptic SK2 channel density in spines was seven times higher than in dendritic shafts and thirty-five times higher than in interneurons. Spines showed a heterogeneous population of SK2 expression, some spines having a high SK2 content, others having a low content and others lacking SK2 channels. SK2 immunonegative spines were significantly smaller than those immunopositive. These results show that SK2 channel density differs between excitatory and inhibitory neurons and demonstrates a large variability in the density of SK2 channels in spines. Furthermore, we demonstrated that SK2 expression was associated with excitatory synapses, but not with inhibitory synapses in CA1 pyramidal cells. Consequently, regulation of excitability and synaptic plasticity by SK2 channels is expected to be neuron class- and target-specific. These data show that immunogold FIB/SEM represent a new powerful EM tool to correlate structure and function of ion channels with nanoscale resolution.

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Three-dimensional synaptic organization of the human hippocampal CA1 field

eLife ◽

10.7554/elife.57013 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 2

Author(s):

Marta Montero-Crespo ◽

Marta Dominguez-Alvaro ◽

Patricia Rondon-Carrillo ◽

Lidia Alonso-Nanclares ◽

Javier DeFelipe ◽

...

Keyword(s):

Human Brain ◽

Focused Ion Beam ◽

Ion Beam ◽

Three Dimensional ◽

Reconstruction Method ◽

Synaptic Organization ◽

Ca1 Region ◽

Hippocampal Ca1 ◽

Hippocampal Ca1 Field ◽

Cortical Inputs

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.

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