Biology subjects

Merchan-Perez, A.

Publications and source records attributed to Merchan-Perez, A..

5 recordsLinked to original sources

bioRxiv2025

Volume Electron Microscopy of Cortical Organoids: Methods for Region Identification, Connectome Reconstruction, and Organelle Segmentation

Volume electron microscopy (vEM) has become a powerful tool for 3D ultrastructural analysis of neural circuits, yet its application to human brain organoids remains limited, particularly for connectomic studies. Here, we established a comprehensive and scalable workflow for applying vEM to human cortical organoids, integrating correlative light and electron microscopy, large-area SEM mosaic imaging, focused ion beam-scanning electron microscopy (FIB-SEM), and transmission electron microscopy (TEM) validation. By systematically comparing two embedding protocols in use, we demonstrated that the DeFelipe and Fairen (1993)/Cano-Astorga et al. (2024) method provides optimal compatibility with toluidine blue-stained semithin sectioning and enables reliable synapse segmentation and neurite tracing. In contrast, the Deerinck et al. (2010) protocol offers enhanced membrane contrast but limits postsynaptic density visualization. Using FIB-SEM imaging of peripheral, neuropil-like regions of cortical organoids, we achieved accurate 3D reconstruction of synapses, neurites and intracellular organelles, enabling quantitative assessment of synaptic apposition surfaces, neurite trajectories, and organelle distribution across defined cellular compartments. Together, our results demonstrate for the first time the feasibility of micro-connectomic reconstruction in human cortical organoids at nanometer resolution. This methodological framework expands the applicability of vEM to organoid systems and provides a robust foundation for future studies of human brain development, disease modeling, and therapeutic evaluation at the synaptic and subcellular level.

neuroscience↗

bioRxiv2023

Nanoconnectivity: a quantitative approach to examine the structural complexity of the brain

Unraveling the highly complex connectivity at the synaptic level is critical for the understanding of brain function. However, investigating the structure of the brain is time consuming and technically demanding. We have developed and applied a tracing method that allows us to obtain a simplified or skeletonized version of all nerve fibers present within a given brain sample, without the need for full 3D reconstructions. Every tracing or "skeleton" is linked to its corresponding synaptic contacts, so the result is an intricate meshwork of axons and dendrites interconnected by a cloud of synaptic junctions. The tissue sample is obtained by volume electron microscopy (FIB-SEM) and tracing is performed by dedicated software. In addition, the software provides the length of each skeleton as well as the number and positions of its corresponding synapses. With this approach, we have identified clear as well as subtle quantitative differences between three brain regions (the stratum radiatum of the hippocampus and layers 1 and 3 of the somatosensory cortex). For example, we have found that axons and dendrites are densely packed in the neuropil of these three regions, reaching close to 8 kilometers of nerve fibers per cubic mm in layer 1. In all cases, the predominant fibers were excitatory axons, followed by spiny dendrites, inhibitory axons and smooth dendrites. Other examples include the fact that the excitatory axons in the stratum radiatum establish many more synapses per micron than the axons of the other regions, or the lack of correlation between the number of synapses established on the spines of a given dendrite and the number of synapses established on the dendritic shaft. Using this methodology, we obtain a set of quantitative parameters including the number and proportions of excitatory and inhibitory synapses; the numbers of dendrites and axons; the proportion of excitatory and inhibitory axons; the linear densities of synapses established on dendritic spines and dendritic shafts; the linear densities of synapses established by excitatory and inhibitory axons; and the lengths of the different types of dendrites and axons. Taken together, these data characterize each region, allowing us to compare them on a quantitative basis and to identify subtle traits and differences that would have escaped a qualitative analysis. With this strategy, we can achieve a better understanding of the structure and dynamics of the brain, and we are better equipped to explore how they change during both normal and pathological conditions.

neuroscience↗

bioRxiv2022

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. This study constitutes the first description of the Etruscan shrew synaptic characteristics using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM). We analyzed the synaptic density and a variety of structural characteristics of 7,239 3D reconstructed synapses, obtaining the following major results: (i) cortical synaptic density was very high, particularly in layer I; (ii) the vast majority of synapses were excitatory, with the highest proportion found in layer I; (iii) excitatory synapses were larger than inhibitory synapses in all layers except in layer VI; and (iv) synapses were either randomly distributed in space or showed a slight tendency to be organised in a regular arrangement. Some of these 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. The present work provides a quantitative dataset from the Etruscan shrew synapses -- not only contributing to the knowledge of the ultrastructure of the mammalian cortex, but also identifying common and differing principles of synaptic organization.

neuroscience↗

bioRxiv2020

Estimation of the number of synapses in the hippocampus and brain-wide by volume electron microscopy and genetic labeling

Determining the number of synapses that are present in different brain regions is crucial to understand brain connectivity as a whole. Membrane-associated guanylate kinases (MAGUKs) are a family of scaffolding proteins that are expressed in excitatory glutamatergic synapses. We used genetic labeling of two of these proteins (PSD95 and SAP102), and Spinning Disc confocal Microscopy (SDM), to estimate the number of fluorescent puncta in the CA1 area of the hippocampus. We also used FIB-SEM, a three-dimensional electron microscopy technique, to calculate the actual numbers of synapses in the same area. We then estimated the ratio between the three-dimensional densities obtained with FIB-SEM (synapses/m3) and the bi-dimensional densities obtained with SDM (puncta/100 m2). Given that it is impractical to use FIB-SEM brain-wide, we used previously available SDM data from other brain regions and we applied this ratio as a conversion factor to estimate the minimum density of synapses in those regions. We found the highest densities of synapses in the isocortex, olfactory areas, hippocampal formation and cortical subplate. Low densities were found in the pallidum, hypothalamus, brainstem and cerebellum. Finally, the striatum and thalamus showed a wide range of synapse densities.

neuroscience↗

bioRxiv2019

Area-specific synapse structure in branched axons reveals a subcellular level of complexity in thalamocortical networks

Thalamocortical Posterior nucleus (Po) axons innervating the somatosensory (S1) and motor (MC) vibrissal cortices are key links in the brain neuronal network that allows rodents to explore the environment whisking with their motile vibrissae. Here, using high-end 3D electron microscopy, we demonstrate massive differences between MC vs. S1 Po synapses in a) bouton and active zone size; b) neurotransmitter vesicle pool size; c) mitochondria distribution near synapses; and d) proportion of non-spinous dendrite contacts. These differences are as large, or bigger, than those between Po and ventroposterior thalamic nucleus synapses in S1. Moreover, using single-axon transfection labeling, we show that the structure of boutons in the MC vs. S1 branches of individual Po axons is different. These structural differences parallel striking, recently-discovered divergences in functional efficacy and plasticity between S1 and MC Po synapses, and overall reveal a new, subcellular level of thalamocortical circuit complexity, unaccounted for in current models.

neuroscience↗