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Cullin 4B (CUL4B) is the scaffold protein in the CUL4B-RING E3 ubiquitin ligase (CRL4B) complex. Loss-of-function mutations in the human CUL4B gene lead to syndromic X-linked intellectual disability (XLID). Till now, the mechanism of intellectual disability caused by CUL4B mutation still needs to be elucidated. In this study, we used single-nucleus RNA sequencing (snRNA-seq) to investigate the impact of CUL4B deficiency on the transcriptional programs of diverse cell types. The results revealed that depletion of CUL4B resulted in impaired intercellular communication and elicited cell type-specific transcriptional changes relevant to synapse dysfunction. Golgi-Cox staining of brain slices and immunostaining of in vitro cultured neurons revealed remarkable synapse loss in CUL4B-deficient mice. Ultrastructural analysis via transmission electron microscopy (TEM) showed that the width of the synaptic cleft was significantly greater in CUL4B-deficient mice. Electrophysiological experiments found a decrease in the amplitude of AMPA receptor-mediated EPSCs in the hippocampal CA1 pyramidal neurons of CUL4B-deficient mice. These results indicate that depletion of CUL4B in mice results in morphological and functional abnormalities in synapses. Furthermore, behavioral tests revealed that depletion of CUL4B in the mouse nervous system results in impaired spatial learning and memory. Taken together, the findings of this study reveal the pathogenesis of neurological disorders associated with CUL4B mutations and promote the identification of therapeutic targets that can halt synaptic abnormalities and preserve memory in individuals., Competing Interests: Declarations. Ethics approval and consent to participate: All animal care and experiments were approved by the Animal Care and Use Committee of Shandong University School of Basic Medical Sciences (No. ECSBMSSDU2023-2-27). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

In humans, a quantifiable number of cortical synapses appears early in fetal life. In this paper, we present a bridge across different scales of resolution and the distribution of synapses across the transient cytoarchitectonic compartments: marginal zone (MZ), cortical plate (CP), subplate (SP), and in vivo MR images. The tissue of somatosensory cortex (7-26 postconceptional weeks (PCW)) was prepared for electron microscopy, and classified synapses with a determined subpial depth were used for creating histograms matched to the histological sections immunoreacted for synaptic markers and aligned to in vivo MR images (1.5 T) of corresponding fetal ages (maternal indication). Two time periods and laminar patterns of synaptogenesis were identified: an early and midfetal two-compartmental distribution (MZ and SP) and a late fetal three-compartmental distribution (CP synaptogenesis). During both periods, a voluminous, synapse-rich SP was visualized on the in vivo MR. Another novel finding concerns the phase of secondary expansion of the SP (13 PCW), where a quantifiable number of synapses appears in the upper SP. This lamina shows a T2 intermediate signal intensity below the low signal CP. In conclusion, the early fetal appearance of synapses shows early differentiation of putative genetic mechanisms underlying the synthesis, transport and assembly of synaptic proteins. "Pioneering" synapses are likely to play a morphogenetic role in constructing of fundamental circuitry architecture due to interaction between neurons. They underlie spontaneous, evoked, and resting state activity prior to ex utero experience. Synapses can also mediate genetic and environmental triggers, adversely altering the development of cortical circuitry and leading to neurodevelopmental disorders., Competing Interests: Declarations. Conflict of interest: The author have no relevant financial or non-financial interests to disclose. Ethical approval: This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by Ethics Committee of School of Medicine University of Zagreb (19.11.2015./protocol number 380–59-10,106–15-168/314)., (© 2024. The Author(s).)

Human vocalization is a complex process that is still only partially understood. Previous studies have suggested the possibility of a localized neuromuscular network of the larynx. Here we investigate this structure in human dissection specimens using multiple immunofluorescence and transmission electron microscopy (TEM). In the area of the pars interna of the thyroarytenoid muscle, muscle fibers are present that are clearly differentiated from skeletal or cardiac muscle cells and show an intermediate ultrastructure. In addition, intramuscular neurons are present that are detectable by both electron and fluorescence microscopy and may have a sensory function in a local neuronal network. Also, several types of sensory and motor synapses are detectable and distributed throughout the pars interna of the thyroarytenoid muscle, with multisynaptic muscle fibers being a common feature. These findings suggest the existence of a previously unrecognized type of muscle fiber coupled to an intramuscular neuronal network, the presence of which could explain functional peculiarities at the laryngeal level.

Chemical synapses are the major sites of communication between neurons in the nervous system and mediate either excitatory or inhibitory signaling. At excitatory synapses, glutamate is the primary neurotransmitter and upon release from presynaptic vesicles, is detected by postsynaptic glutamate receptors, which include ionotropic AMPA and NMDA receptors. Here, we have developed methods to identify glutamatergic synapses in brain tissue slices, label AMPA receptors with small gold nanoparticles (AuNPs), and prepare lamella for cryo-electron tomography studies. The targeted imaging of glutamatergic synapses in the lamella is facilitated by fluorescent pre- and postsynaptic signatures, and the subsequent tomograms allow for the identification of key features of chemical synapses, including synaptic vesicles, the synaptic cleft, and AuNP-labeled AMPA receptors. These methods pave the way for imaging brain regions at high resolution, using unstained, unfixed samples preserved under near-native conditions., Competing Interests: AM, CS, JE, MS, NJ, XZ, MQ, HZ, ZY, EG No competing interests declared, (© 2024, Matsui, Spangler et al.)

The present study uses electron microscopy to document ultrastructural characteristics of hippocampal GABAergic inhibitory synapses under resting and stimulated conditions in three experimental systems. Synaptic profiles were sampled from stratum pyramidale and radiatum of the CA1 region from (1) perfusion fixed mouse brains, (2) immersion fixed rat organotypic slice cultures, and from (3) rat dissociated hippocampal cultures of mixed cell types. Synapses were stimulated in the brain by a 5 min delay in perfusion fixation to trigger an ischemia-like excitatory condition, and by treating the two culture systems with 90 mM high K + for 2-3 min to depolarize the neurons. Upon such stimulation conditions, the presynaptic terminals of the inhibitory synapses exhibited similar structural changes to those seen in glutamatergic excitatory synapses, with depletion of synaptic vesicles, increase of clathrin-coated vesicles and appearance of synaptic spinules. However, in contrast to excitatory synapses, no structural differences were detected in the postsynaptic compartment of the inhibitory synapses upon stimulation. There were no changes in the appearance of material associated with the postsynaptic membrane or the length and curvature of the membrane. Also no change was detected in the labeling density of gephyrin, a GABAergic synaptic marker, lining the postsynaptic membrane. Furthermore, virtually all inhibitory synaptic clefts remained rigidly apposed, unlike in the case of excitatory synapses where ~ 20-30% of cleft edges were open upon stimulation, presumably to facilitate the clearance of neurotransmitters from the cleft. The fact that no open clefts were induced in inhibitory synapses upon stimulation suggests that inhibitory input may not need to be toned down under these conditions. On the other hand, similar to excitatory synapse, EGTA (a calcium chelator) induced open clefts in ~ 18% of inhibitory synaptic cleft edges, presumably disrupting similar calcium-dependent trans-synaptic bridges in both types of synapses., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Adapting to environmental changes and formulating behavioral strategies are central to the nervous system, with the prefrontal cortex being crucial. Chronic stress impacts this region, leading to disorders including major depression. This review discusses the roles for prefrontal cortex and the effects of stress, highlighting similarities and differences between human/primates and rodent brains. Notably, the rodent medial prefrontal cortex is analogous to the human subgenual anterior cingulate cortex in terms of emotional regulation, sharing similarities in cytoarchitecture and circuitry, while also performing cognitive functions similar to the human dorsolateral prefrontal cortex. It has been shown that chronic stress induces atrophic changes in the rodent mPFC, which mirrors the atrophy observed in the subgenual anterior cingulate cortex and dorsolateral prefrontal cortex of depression patients. However, the precise alterations in neural circuitry due to chronic stress are yet to be fully unraveled. The use of advanced imaging techniques, particularly volume electron microscopy, is emphasized as critical for the detailed examination of synaptic changes, providing a deeper understanding of stress and depression at the molecular, cellular and circuit levels. This approach offers invaluable insights into the alterations in neuronal circuits within the medial prefrontal cortex caused by chronic stress, significantly enriching our understanding of stress and depression pathologies., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Odor information is modulated by centrifugal inputs from other brain regions to the olfactory bulb (OB). Neurons containing monoamines, such as serotonin, acetylcholine, and noradrenaline, are well known as centrifugal inputs; however, the role of histamine, which is also present in the OB, is not well understood. In this study, we examined the histaminergic neurons projecting from the hypothalamus to the OB. We used an antibody against histidine decarboxylase (HDC), a synthesizing enzyme of histamine, to identify histaminergic neurons and assess their localization within the OB and the ultrastructure of their fibers and synapses using multiple immunostaining laser microscopy, ultra-high voltage electron microscopy (EM), and EM to confirm their relationships with other neurons. To further identify the origin nucleus of the histaminergic neurons projecting to the OB, we injected the retrograde tracer FluoroGold and analyzed the pathway to the OB anterogradely. HDC-immunoreactive (-ir) fibers were abundant in the olfactory nerve (ON) layer compared to other monoamines. HDC-ir neurons received asymmetrical synapses from ONs and formed synapses containing pleomorphic vesicles with variable postsynaptic densities to non-ON elements, thus forming serial synapses. We also confirmed that histaminergic neurons project from the rostral ventral tuberomammillary nucleus to the granule cell layer of the OB and, for the first time, successfully visualized their axons from the hypothalamus to the OB. These findings indicate that histamine may regulate odor discrimination in the OB, suggesting a regulatory relationship between hypothalamic function and olfaction. We thus elucidate morphological mechanisms with tuberomammillary nucleus-derived histaminergic neurons involved in olfactory information., (© 2024 Wiley Periodicals LLC.)

Many animals use visual information to navigate 1-4 , but how such information is encoded and integrated by the navigation system remains incompletely understood. In Drosophila melanogaster, EPG neurons in the central complex compute the heading direction 5 by integrating visual input from ER neurons 6-12 , which are part of the anterior visual pathway (AVP) 10,13-16 . Here we densely reconstruct all neurons in the AVP using electron-microscopy data 17 . The AVP comprises four neuropils, sequentially linked by three major classes of neurons: MeTu neurons 10,14,15 , which connect the medulla in the optic lobe to the small unit of the anterior optic tubercle (AOTUsu) in the central brain; TuBu neurons 9,16 , which connect the AOTUsu to the bulb neuropil; and ER neurons 6-12 , which connect the bulb to the EPG neurons. On the basis of morphologies, connectivity between neural classes and the locations of synapses, we identify distinct information channels that originate from four types of MeTu neurons, and we further divide these into ten subtypes according to the presynaptic connections in the medulla and the postsynaptic connections in the AOTUsu. Using the connectivity of the entire AVP and the dendritic fields of the MeTu neurons in the optic lobes, we infer potential visual features and the visual area from which any ER neuron receives input. We confirm some of these predictions physiologically. These results provide a strong foundation for understanding how distinct sensory features can be extracted and transformed across multiple processing stages to construct higher-order cognitive representations., (© 2024. The Author(s).)

The basal ganglia play pivotal roles in motor control and cognitive functioning. These nuclei are embedded in an anatomical loop: cortex to basal ganglia to thalamus back to cortex. We focus here on an essential synapse for descending control, from cortical layer 5 (L5) onto the GABAergic spiny projection neurons (SPNs) of the caudoputamen (CP). We employed genetic labeling to distinguish L5 neurons from somatosensory (S1) and motor (M1) cortices in large volume serial electron microscopy and electrophysiology datasets to better detail these inputs. First, M1 and S1 synapses showed a strong preference to innervate the spines of SPNs and rarely contacted aspiny cells, which are likely to be interneurons. Second, L5 inputs commonly converge from both areas onto single SPNs. Third, compared to unlabeled terminals in CP, those labeled from M1 and S1 show ultrastructural hallmarks of strong driver synapses: They innervate larger spines that were more likely to contain a spine apparatus, more often had embedded mitochondria, and more often contacted multiple targets. Finally, these inputs also demonstrated driver-like functional properties: SPNs responded to optogenetic activation from S1 and M1 with large EPSP/Cs that depressed and were dependent on ionotropic but not metabotropic receptors. Together, our findings suggest that individual SPNs integrate driver input from multiple cortical areas with implications for how the basal ganglia relay cortical input to provide inhibitory innervation of motor thalamus., (© 2024 The Author(s). European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Most excitatory synapses in the mammalian brain are contacted or ensheathed by astrocyte processes, forming tripartite synapses. Astrocytes are thought to be critical regulators of the structural and functional dynamics of synapses. While the degree of synaptic coverage by astrocytes is known to vary across brain regions and animal species, the reason for and implications of this variability remains unknown. Further, how astrocyte coverage of synapses relates to in vivo functional properties of individual synapses has not been investigated. Here, we characterized astrocyte coverage of synapses of pyramidal neurons in the ferret visual cortex and, using correlative light and electron microscopy, examined their relationship to synaptic strength and sensory-evoked Ca 2+ activity. Nearly, all synapses were contacted by astrocytes, and most were contacted along the axon-spine interface. Structurally, we found that the degree of synaptic astrocyte coverage directly scaled with synapse size and postsynaptic density complexity. Functionally, we found that the amount of astrocyte coverage scaled with how selectively a synapse responds to a particular visual stimulus and, at least for the largest synapses, scaled with the reliability of visual stimuli to evoke postsynaptic Ca 2+ events. Our study shows astrocyte coverage is highly correlated with structural metrics of synaptic strength of excitatory synapses in the visual cortex and demonstrates a previously unknown relationship between astrocyte coverage and reliable sensory activation., (© 2024 The Author(s). GLIA published by Wiley Periodicals LLC.)

Cryo-electron tomography (cryo-ET) has the potential to reveal cell structure down to atomic resolution. Nevertheless, cellular cryo-ET data is highly complex, requiring image segmentation for visualization and quantification of subcellular structures. Due to noise and anisotropic resolution in cryo-ET data, automatic segmentation based on classical computer vision approaches usually does not perform satisfactorily. Communication between neurons relies on neurotransmitter-filled synaptic vesicle (SV) exocytosis. Cryo-ET study of the spatial organization of SVs and their interconnections allows a better understanding of the mechanisms of exocytosis regulation. Accurate SV segmentation is a prerequisite to obtaining a faithful connectivity representation. Hundreds of SVs are present in a synapse, and their manual segmentation is a bottleneck. We addressed this by designing a workflow consisting of a convolutional network followed by post-processing steps. Alongside, we provide an interactive tool for accurately segmenting spherical vesicles. Our pipeline can in principle segment spherical vesicles in any cell type as well as extracellular and in vitro spherical vesicles., (© 2024 Khosrozadeh et al.)

The cerebral cortex accounts for substantial energy expenditure, primarily driven by the metabolic demands of synaptic signaling. Mitochondria, the organelles responsible for generating cellular energy, play a crucial role in this process. We investigated ultrastructural characteristics of the primary visual cortex in 18 phylogenetically diverse mammals, spanning a broad range of brain sizes from mouse to elephant. Our findings reveal remarkable uniformity in synapse density, postsynaptic density (PSD) length, and mitochondria density, indicating functional and metabolic constraints that maintain these fundamental features. Notably, we observed an average of 1.9 mitochondria per synapse across mammalian species. When considered together with the trend of decreasing neuron density with larger brain size, we find that brain enlargement in mammals is characterized by increasing proportions of synapses and mitochondria per cortical neuron. These results shed light on the adaptive mechanisms and metabolic dynamics that govern cortical ultrastructure across mammals., (© 2024 Wiley Periodicals LLC.)

Functional and structural studies investigating macroscopic connectivity in the human cerebral cortex suggest that high-order associative regions exhibit greater connectivity compared to primary ones. However, the synaptic organization of these brain regions remains unexplored. In the present work, we conducted volume electron microscopy to investigate the synaptic organization of the human brain obtained at autopsy. Specifically, we examined layer III of Brodmann areas 17, 3b, and 4, as representative areas of primary visual, somatosensorial, and motor cortex. Additionally, we conducted comparative analyses with our previous datasets of layer III from temporopolar and anterior cingulate associative cortical regions (Brodmann areas 24, 38, and 21). 9,690 synaptic junctions were 3D reconstructed, showing that certain synaptic characteristics are specific to particular regions. The number of synapses per volume, the proportion of the postsynaptic targets, and the synaptic size may distinguish one region from another, regardless of whether they are associative or primary cortex. By contrast, other synaptic characteristics were common to all analyzed regions, such as the proportion of excitatory and inhibitory synapses, their shapes, their spatial distribution, and a higher proportion of synapses located on dendritic spines. The present results provide further insights into the synaptic organization of the human cerebral cortex., (© The Author(s) 2024. Published by Oxford University Press.)

The retina comprises numerous cells forming diverse neuronal circuits, which constitute the first stage of the visual pathway. Each circuit is characterized by unique features and distinct neurotransmitters, determining its role and functional significance. Given the intricate cell types within its structure, the complexity of neuronal circuits in the retina poses challenges for exploration. To better investigate retinal circuits and cross-talk, such as the link between cone and rod pathways, and precise molecular localization (neurotransmitters or neuropeptides), such as the presence of substance P-like immunoreactivity in the mouse retina, we employed a pre-embedding immunoelectron microscopy (immuno-EM) method to explore synaptic connections and organization. This approach enables us to pinpoint specific intercellular synaptic connections and precise molecular localization and could play a guiding role in exploring its function. This article describes the protocol, reagents used, and detailed steps, including (1) retina fixation preparation, (2) pre-embedding immunostaining, and (3) post-fixation and embedding.

Synaptic ribbons are the eponymous specializations of continuously active ribbon synapses. They are primarily composed of the RIBEYE protein that consists of a unique amino-terminal A-domain and carboxy-terminal B-domain that is largely identical to the ubiquitously expressed transcriptional regulator protein CtBP2. Both RIBEYE A-domain and RIBEYE B-domain are essential for the assembly of the synaptic ribbon, as shown by previous analyses of RIBEYE knockout and knockin mice and related investigations. How exactly the synaptic ribbon is assembled from RIBEYE subunits is not yet clear. To achieve further insights into the architecture of the synaptic ribbon, we performed analytical post-embedding immunogold-electron microscopy with direct gold-labelled primary antibodies against RIBEYE A-domain and RIBEYE B-domain for improved ultrastructural resolution. With direct gold-labelled monoclonal antibodies against RIBEYE A-domain and RIBEYE B-domain, we found that both domains show a very similar localization within the synaptic ribbon of mouse photoreceptor synapses, with no obvious differential gradient between the centre and surface of the synaptic ribbon. These data favour a model of the architecture of the synaptic ribbon in which the RIBEYE A-domain and RIBEYE B-domain are located similar distances from the midline of the synaptic ribbon.

The spatial distribution of proteins and their arrangement within the cellular ultrastructure regulates the opening of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in response to glutamate release at the synapse. Fluorescence microscopy imaging revealed that the postsynaptic density (PSD) and scaffolding proteins in the presynaptic active zone (AZ) align across the synapse to form a trans-synaptic "nanocolumn," but the relation to synaptic vesicle release sites is uncertain. Here, we employ focused-ion beam (FIB) milling and cryoelectron tomography to image synapses under near-native conditions. Improved image contrast, enabled by FIB milling, allows simultaneous visualization of supramolecular nanoclusters within the AZ and PSD and synaptic vesicles. Surprisingly, membrane-proximal synaptic vesicles, which fuse to release glutamate, are not preferentially aligned with AZ or PSD nanoclusters. These synaptic vesicles are linked to the membrane by peripheral protein densities, often consistent in size and shape with Munc13, as well as globular densities bridging the synaptic vesicle and plasma membrane, consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using biorealistic models guided by our tomograms predict that clustering AMPARs within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. Together, our data support a model in which synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites., Competing Interests: Competing interests statement:The authors declare no competing interest.

The highly complex structure of the brain requires an approach that can unravel its connectivity. Using volume electron microscopy and a dedicated software we can trace and measure all nerve fibers present within different samples of brain tissue. With this software tool, individual dendrites and axons are traced, obtaining a simplified "skeleton" of each fiber, which is linked to its corresponding synaptic contacts. The result is an intricate meshwork of axons and dendrites interconnected by a cloud of synaptic junctions. To test this methodology, we apply it to the stratum radiatum of the hippocampus and layers 1 and 3 of the somatosensory cortex of the mouse. We find that nerve fibers are densely packed in the neuropil, reaching up to 9 kilometers per cubic mm. We obtain the number of synapses, the number and lengths of dendrites and axons, the linear densities of synapses established by dendrites and axons, and their location on dendritic spines and shafts. The quantitative data obtained through this method enable us to identify subtle traits and differences in the synaptic organization of the samples, which might have been overlooked in a qualitative analysis., (© 2024. The Author(s).)

The synaptic basal lamina of the electrocytes was disclosed to be electron-translucent to some extent when viewed in an en-face direction in embedment-free section transmission electron microscopy (EFS-TEM), and synaptic vesicles located close to the presynaptic membrane were seen through the synaptic basal lamina together with the presynaptic and postsynaptic membranes. This feature of translucency has the potential to analyze possible spatial interrelations in situ between bioactive molecules in the synaptic basal lamina and the synaptic vesicles in further studies. The synaptic basal lamina, appearing as an electron-dense line sandwiched by two parallel lines representing the presynaptic and postsynaptic membranes in ultrathin sections cut right to the synaptic junctional plane in conventional TEM, was not fully continuous but randomly intermittent along its trajectory. Compatible with the intermittent line appearance, the en-face 3D view in embedment-free section TEM revealed for the first time partial irregular defects of the synaptic basal lamina. Considering the known functional significance of several molecules contained in the synaptic basal lamina in the maintenance and exertion of the synapse, its partial defects may not represent its rigid structural features, but its immature structure under remodeling or its dynamic changes in consistency such as the sol/gel transition, whose validity needs further examination. RESEARCH HIGHLIGHTS: In embedment-free section TEM, a 3D en-face view of synaptic basal lamina in situ is reliably possible. The basal lamina en-face is electron-translucent, which makes it possible to analyze spatial interrelation between pre- and post-synaptic components. Partial irregular defects in the basal lamina are revealed in Torpedo electrocytes, suggesting its remodeling or dynamic changes in consistency., (© 2024 Wiley Periodicals LLC.)

A deep understanding of how the brain controls behaviour requires mapping neural circuits down to the muscles that they control. Here, we apply automated tools to segment neurons and identify synapses in an electron microscopy dataset of an adult female Drosophila melanogaster ventral nerve cord (VNC) 1 , which functions like the vertebrate spinal cord to sense and control the body. We find that the fly VNC contains roughly 45 million synapses and 14,600 neuronal cell bodies. To interpret the output of the connectome, we mapped the muscle targets of leg and wing motor neurons using genetic driver lines 2 and X-ray holographic nanotomography 3 . With this motor neuron atlas, we identified neural circuits that coordinate leg and wing movements during take-off. We provide the reconstruction of VNC circuits, the motor neuron atlas and tools for programmatic and interactive access as resources to support experimental and theoretical studies of how the nervous system controls behaviour., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Single-cell microcultures (SCMs) form a monosynaptic circuit that allows stimulation and recording of postsynaptic responses using a single electrode. Here, we present a protocol to establish autaptic cultures from rat superior cervical ganglion neurons. We describe the steps for preparing SCMs, recording synaptic currents, and identifying and processing the recorded neurons for electron microscopy. We then detail procedures for visualizing synapses. This protocol is illustrated by correlating evoked and spontaneous neurotransmitter release with the ultrastructural features of synapses recorded. For complete details on the use and execution of this protocol, please refer to Velasco et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Vertebrate vision begins with light absorption by rod and cone photoreceptors, which transmit signals from their synaptic terminals to second-order neurons: bipolar and horizontal cells. In mouse rods, there is a single presynaptic ribbon-type active zone at which the release of glutamate occurs tonically in the dark. This tonic glutamatergic signaling requires continuous exo- and endocytosis of synaptic vesicles. At conventional synapses, endocytosis commonly requires dynamins: GTPases encoded by three genes ( Dnm1-3 ), which perform membrane scission. Disrupting endocytosis by dynamin deletions impairs transmission at conventional synapses, but the impact of disrupting endocytosis and the role(s) of specific dynamin isoforms at rod ribbon synapses are understood incompletely. Here, we used cell-specific knock-outs (KOs) of the neuron-specific Dnm1 and Dnm3 to investigate the functional roles of dynamin isoforms in rod photoreceptors in mice of either sex. Analysis of synaptic protein expression, synapse ultrastructure, and retinal function via electroretinograms (ERGs) showed that dynamins 1 and 3 act redundantly and are essential for supporting the structural and functional integrity of rod ribbon synapses. Single Dnm3 KO showed no phenotype, and single Dnm1 KO only modestly reduced synaptic vesicle density without affecting vesicle size and overall synapse integrity, whereas double Dnm1/Dnm3 KO impaired vesicle endocytosis profoundly, causing enlarged vesicles, reduced vesicle density, reduced ERG responses, synaptic terminal degeneration, and disassembly and degeneration of postsynaptic processes. Concurrently, cone function remained intact. These results show the fundamental redundancy of dynamins 1 and 3 in regulating the structure and function of rod ribbon synapses., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

A decade ago, in 2013, and over the course of 4 summer months, three separate observations were reported that each shed light independently on a new molecular organization that fundamentally reshaped our perception of excitatory synaptic transmission (Fukata et al., 2013; MacGillavry et al., 2013; Nair et al., 2013). This discovery unveiled an intricate arrangement of AMPA-type glutamate receptors and their principal scaffolding protein PSD-95, at synapses. This breakthrough was made possible, thanks to advanced super-resolution imaging techniques. It fundamentally changed our understanding of excitatory synaptic architecture and paved the way for a brand-new area of research. In this Progressions article, the primary investigators of the nanoscale organization of synapses have come together to chronicle the tale of their discovery. We recount the initial inquiry that prompted our research, the preceding studies that inspired our work, the technical obstacles that were encountered, and the breakthroughs that were made in the subsequent decade in the realm of nanoscale synaptic transmission. We review the new discoveries made possible by the democratization of super-resolution imaging techniques in the field of excitatory synaptic physiology and architecture, first by the extension to other glutamate receptors and to presynaptic proteins and then by the notion of trans-synaptic organization. After describing the organizational modifications occurring in various pathologies, we discuss briefly the latest technical developments made possible by super-resolution imaging and emerging concepts in synaptic physiology., Competing Interests: The authors declare no competing financial interest., (Copyright © 2024 the authors.)

A primary cilium is a membrane-bound extension from the cell surface that contains receptors for perceiving and transmitting signals that modulate cell state and activity. Primary cilia in the brain are less accessible than cilia on cultured cells or epithelial tissues because in the brain they protrude into a deep, dense network of glial and neuronal processes. Here, we investigated cilia frequency, internal structure, shape, and position in large, high-resolution transmission electron microscopy volumes of mouse primary visual cortex. Cilia extended from the cell bodies of nearly all excitatory and inhibitory neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) but were absent from oligodendrocytes and microglia. Ultrastructural comparisons revealed that the base of the cilium and the microtubule organization differed between neurons and glia. Investigating cilia-proximal features revealed that many cilia were directly adjacent to synapses, suggesting that cilia are poised to encounter locally released signaling molecules. Our analysis indicated that synapse proximity is likely due to random encounters in the neuropil, with no evidence that cilia modulate synapse activity as would be expected in tetrapartite synapses. The observed cell class differences in proximity to synapses were largely due to differences in external cilia length. Many key structural features that differed between neuronal and glial cilia influenced both cilium placement and shape and, thus, exposure to processes and synapses outside the cilium. Together, the ultrastructure both within and around neuronal and glial cilia suggest differences in cilia formation and function across cell types in the brain., Competing Interests: Declaration of interests The authors have no competing financial interests to declare., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

To fully understand how the human brain works, knowledge of its structure at high resolution is needed. Presented here is a computationally intensive reconstruction of the ultrastructure of a cubic millimeter of human temporal cortex that was surgically removed to gain access to an underlying epileptic focus. It contains about 57,000 cells, about 230 millimeters of blood vessels, and about 150 million synapses and comprises 1.4 petabytes. Our analysis showed that glia outnumber neurons 2:1, oligodendrocytes were the most common cell, deep layer excitatory neurons could be classified on the basis of dendritic orientation, and among thousands of weak connections to each neuron, there exist rare powerful axonal inputs of up to 50 synapses. Further studies using this resource may bring valuable insights into the mysteries of the human brain.

High-resolution electron microscopy of nervous systems has enabled the reconstruction of synaptic connectomes. However, we do not know the synaptic sign for each connection (i.e., whether a connection is excitatory or inhibitory), which is implied by the released transmitter. We demonstrate that artificial neural networks can predict transmitter types for presynapses from electron micrographs: a network trained to predict six transmitters (acetylcholine, glutamate, GABA, serotonin, dopamine, octopamine) achieves an accuracy of 87% for individual synapses, 94% for neurons, and 91% for known cell types across a D. melanogaster whole brain. We visualize the ultrastructural features used for prediction, discovering subtle but significant differences between transmitter phenotypes. We also analyze transmitter distributions across the brain and find that neurons that develop together largely express only one fast-acting transmitter (acetylcholine, glutamate, or GABA). We hope that our publicly available predictions act as an accelerant for neuroscientific hypothesis generation for the fly., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mapping neuronal networks from three-dimensional electron microscopy (3D-EM) data still poses substantial reconstruction challenges, in particular for thin axons. Currently available automated image segmentation methods require manual proofreading for many types of connectomic analysis. Here we introduce RoboEM, an artificial intelligence-based self-steering 3D 'flight' system trained to navigate along neurites using only 3D-EM data as input. Applied to 3D-EM data from mouse and human cortex, RoboEM substantially improves automated state-of-the-art segmentations and can replace manual proofreading for more complex connectomic analysis problems, yielding computational annotation cost for cortical connectomes about 400-fold lower than the cost of manual error correction., (© 2024. The Author(s).)

The structure of photoreceptors (PR) and the arrangement of neurons in the retina of red-tail shark were investigated using light and electron microscopy. The PR showed a mosaic arrangement and included double cones, single cones (SC), and single rods. Most cones occur as SC. The ratio between the number of cones and rods was 3:1.39 (±0.29). The rods were tall that reached the pigmented epithelium. The outer plexiform layer (OPL) showed a complex synaptic connection between the horizontal and photoreceptor terminals that were surrounded by Müller cell processes. Electron microscopy showed that the OPL possessed both cone pedicles and rod spherules. Each rod spherule consisted of a single synaptic ribbon within the invaginating terminal endings of the horizontal cell (hc) processes. In contrast, the cone pedicles possessed many synaptic ribbons within their junctional complexes. The inner nuclear layer consisted of bipolar, amacrine, Müller cells, and hc. Müller cells possessed intermediate filaments and cell processes that can reach the outer limiting membrane and form connections with each other by desmosomes. The ganglion cells were large multipolar cells with a spherical nucleus and Nissl' bodies in their cytoplasm. The presence of different types of cones arranged in a mosaic pattern in the retina of this species favors the spatial resolution of visual objects. RESEARCH HIGHLIGHTS: This is the first study demonstrating the structure and arrangement of retinal neurons of red-tail shark using light and electron microscopy. The current study showed the presence of different types of cones arranged in a mosaic pattern that may favor the spatial resolution of visual objects in this species. The bipolar, amacrine, Müller, and horizontal cells could be demonstrated., (© 2024 Wiley Periodicals LLC.)

The computational capabilities of neuronal networks are fundamentally constrained by their specific connectivity. Previous studies of cortical connectivity have mostly been carried out in rodents; whether the principles established therein also apply to the evolutionarily expanded human cortex is unclear. We studied network properties within the human temporal cortex using samples obtained from brain surgery. We analyzed multineuron patch-clamp recordings in layer 2-3 pyramidal neurons and identified substantial differences compared with rodents. Reciprocity showed random distribution, synaptic strength was independent from connection probability, and connectivity of the supragranular temporal cortex followed a directed and mostly acyclic graph topology. Application of these principles in neuronal models increased dimensionality of network dynamics, suggesting a critical role for cortical computation.

Synaptic vesicles can undergo several modes of exocytosis, endocytosis, and trafficking within individual synapses, and their fates may be linked to different vesicular protein compositions. Here, we mapped the intrasynaptic distribution of the synaptic vesicle proteins SV2B and SV2A in glutamatergic synapses of the hippocampus using three-dimensional electron microscopy. SV2B was almost completely absent from docked vesicles and a distinct cluster of vesicles found near the active zone. In contrast, SV2A was found in all domains of the synapse and was slightly enriched near the active zone. SV2B and SV2A were found on the membrane in the peri-active zone, suggesting the recycling from both clusters of vesicles. SV2B knockout mice displayed an increased seizure induction threshold only in a model employing high-frequency stimulation. Our data show that glutamatergic synapses generate molecularly distinct populations of synaptic vesicles and are able to maintain them at steep spatial gradients. The almost complete absence of SV2B from vesicles at the active zone of wildtype mice may explain why SV2A has been found more important for vesicle release., (© The Author(s) (2023). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

DYT1 dystonia is associated with decreased striatal dopamine release. In this study, we examined the possibility that ultrastructural changes of nigrostriatal dopamine terminals could contribute to this neurochemical imbalance using a serial block face/scanning electron microscope (SBF/SEM) and three-dimensional reconstruction to analyse striatal tyrosine hydroxylase-immunoreactive (TH-IR) terminals and their synapses in a DYT1(ΔE) knockin (DYT1-KI) mouse model of DYT1 dystonia. Furthermore, to study possible changes in vesicle packaging capacity of dopamine, we used transmission electron microscopy to assess the synaptic vesicle size in striatal dopamine terminals. Quantitative comparative analysis of 80 fully reconstructed TH-IR terminals in the WT and DYT1-KI mice indicate (1) no significant difference in the volume of TH-IR terminals; (2) no major change in the proportion of axo-spinous versus axo-dendritic synapses; (3) no significant change in the post-synaptic density (PSD) area of axo-dendritic synapses, while the PSDs of axo-spinous synapses were significantly smaller in DYT1-KI mice; (4) no significant change in the contact area between TH-IR terminals and dendritic shafts or spines, while the ratio of PSD area/contact area decreased significantly for both axo-dendritic and axo-spinous synapses in DYT1-KI mice; (5) no significant difference in the mitochondria volume; and (6) no significant difference in the synaptic vesicle area between the two groups. Altogether, these findings suggest that abnormal morphometric changes of nigrostriatal dopamine terminals and their post-synaptic targets are unlikely to be a major source of reduced striatal dopamine release in DYT1 dystonia., (© 2023 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

The posterior parietal cortex exhibits choice-selective activity during perceptual decision-making tasks 1-10 . However, it is not known how this selective activity arises from the underlying synaptic connectivity. Here we combined virtual-reality behaviour, two-photon calcium imaging, high-throughput electron microscopy and circuit modelling to analyse how synaptic connectivity between neurons in the posterior parietal cortex relates to their selective activity. We found that excitatory pyramidal neurons preferentially target inhibitory interneurons with the same selectivity. In turn, inhibitory interneurons preferentially target pyramidal neurons with opposite selectivity, forming an opponent inhibition motif. This motif was present even between neurons with activity peaks in different task epochs. We developed neural-circuit models of the computations performed by these motifs, and found that opponent inhibition between neural populations with opposite selectivity amplifies selective inputs, thereby improving the encoding of trial-type information. The models also predict that opponent inhibition between neurons with activity peaks in different task epochs contributes to creating choice-specific sequential activity. These results provide evidence for how synaptic connectivity in cortical circuits supports a learned decision-making task., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The digenean complex life cycle includes various morphological forms with different locomotory and behavioral activities, and the functional specialization of their nervous system is of importance for the transmission of these parasites. Adult digeneans acquire many adaptive features associated with the final settlement in a vertebrate host. Our study describes the general morphology and ultrastructure of the nervous system of the adult renicolid digenean Renicola parvicaudatus parasitizing the renal tubules of herring gulls. Using immunocytochemical and electron microscopic methods, we identified the distinctive characteristics of ganglia and synapses in the studied species. A comparative analysis of the organization of the nervous system of adult individuals and their continuously-swimming stylet cercariae revealed a number of stage-related differences in the composition of ganglia, the distribution of serotonin- and FMRFamide-immunoreactive neurons, the cytomorphology of neuron somata and free sensory endings. Thus, in adults, the presence of FMRFamide-positive neuron somata, accessory muscle bundles in the ganglionic cortex, and eight types of neuronal vesicles was detected, but no glia-like elements were identified. Their neurons are characterized by a larger volume of cytoplasm and also show greater ultrastructural diversity. Although the sensory papillae of adults do not vary in their external morphology as much as those of larvae, their sensory bulbs are more diverse in cytomorphology. Following our previous data on the "support" cell processes related to various tissues of the larvae and considered as glia-like structures, we also briefly present the identified features of the parenchyma, attachment organs and excretory system of adult individuals. The excretory system of adult R. parvicaudatus is characterized by the presence of unique terminal cells with several flame tufts, which are not typical either for the larvae of this species or for other digeneans studied so far. We also used molecular phylogenetic analysis to clarify species identification., (© 2024 Wiley Periodicals LLC.)

Array tomography (AT) allows one to localize sub-cellular components within the structural context of cells in 3D through the imaging of serial sections. Using this technique, the z-resolution can be improved physically by cutting ultra-thin sections. Nevertheless, conventional immunofluorescence staining of those sections is time consuming and requires relatively large amounts of costly antibody solutions. Moreover, epitopes are only readily accessible at the section's surface, leaving the volume of the serial sections unlabeled. Localization of receptors at neuronal synapses in 3D in their native cellular ultrastructural context is important for understanding signaling processes. Here, we present in vivo labeling of receptors via fluorophore-coupled tags in combination with super-resolution AT. We present two workflows where we label receptors at the plasma membrane: first, in vivo labeling via microinjection with a setup consisting of readily available components and self-manufactured microscope table equipment and second, live receptor labeling by using a cell-permeable tag. To take advantage of a near-to-native preservation of tissues for subsequent scanning electron microscopy (SEM), we also apply high-pressure freezing and freeze substitution. The advantages and disadvantages of our workflows are discussed., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Taste receptor cells are morphologically classified as types II and III. Type II cells form a unique type of synapses referred to as channel synapses where calcium homeostasis modulator 1 (CALHM1) together with CALHM3 forms voltage-gated channels that release the neurotransmitter, adenosine triphosphate (ATP). To validate the proposed structural model of channel synapses, the ultrastructural localization of CALHM1 in type II cells of both fungiform and circumvallate taste buds was examined. A monoclonal antibody against CALHM1 was developed and its localization was evaluated via immunofluorescence and immunoelectron microscopy using the immunogold-silver labeling technique. CALHM1 was detected as puncta using immunofluorescence and along the presynaptic membrane of channel synapses facing atypical mitochondria, which provide ATP, by immunoelectron microscopy. In addition, it was detected along the plasma membrane lined by subsurface cisternae at sites apposed to afferent nerve fibers. Our results support the validity of a previously proposed structural model for channel synapses and provide insights into the function of subsurface cisternae whose function in taste receptor cells is unknown. We also examined the localization of CALHM1 in hybrid synapses of type III cells, which are conventional chemical synapses accompanied by mitochondria similar to atypical mitochondria of channel synapses. CALHM1 was not detected in the six hybrid synapses examined using immunoelectron microscopy. We further performed double immunolabeling for CALHM1 and Bassoon, which is detected as puncta corresponding to conventional vesicular synapses in type III cells. Our observations suggest that at least some, and probably most, hybrid synapses are not accompanied by CALHM1., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Correlative light and electron microscopy (CLEM) can provide valuable information about a biological sample by giving information on the specific localization of a molecule of interest within an ultrastructural context. In this work, we describe a simple CLEM method to obtain high-resolution images of neurotransmitter receptor distribution in synapses by electron microscopy (EM). We use hippocampal organotypic slices from a previously reported mouse model expressing a modified AMPA receptor (AMPAR) subunit that binds biotin at the surface (Getz et al., 2022). This tag can be recognized by StreptAvidin-Fluoronanogold™ conjugates (SA-FNG), which reach receptors at synapses (synaptic cleft is 50-100nm thick). By using pre-embedding labeling, we found that SA-FNG reliably bind synaptic receptors and penetrate around 10-15μm in depth in live tissue. However, the silver enhancement was only reaching the surface of the slices. We show that permeabilization with triton is highly effective at increasing the in depth-gold amplification and that the membrane integrity is well preserved. Finally, we also apply high-resolution electron tomography, thus providing important information about the 3D organization of surface AMPA receptors in synapses at the nanoscale., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Age-related decline in physical and cognitive functions are facts of life that do not affect everyone to the same extent. We had reported earlier that such cognitive decline is both sex- and context-dependent. Moreover, age-associated ultrastructural changes were observed in the hippocampus of male rats. In this study, we sought to determine potential differences in ultrastructural changes between male and female rats at various stages of life. We performed quantitative electron microscopic evaluation of hippocampal CA1 region, an area intimately involved in cognitive behavior, in both male and female adolescent, adult and old Wistar rats. Specifically, we measured the number of docking synaptic vesicles in axo-dendritic synapses, the length of active zone as well as the total number of synaptic vesicles. Distinct age- and sex-dependent effects were observed in several parameters. Thus, adult female rats had the lowest synaptic active zone compared to both adolescent and old female rats. Moreover, the same parameter was significantly lower in adult and old female rats compared to their male counterparts. On the other hand, old male rats had significantly lower number of total synaptic vesicles compared to both adolescent and adult male rats as well as compared to their female counterparts. Taken together, it may be suggested that age- and sex-dependent ultrastructural changes in the hippocampus may underlie at least some of the differences in cognitive functions among these groups., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Zebrafish (Danio rerio) is a well-established model for studying the nervous system. Findings in zebrafish often inform studies on human diseases of the nervous system and provide crucial insight into disease mechanisms. The functions of the nervous system often rely on communication between neurons. Signal transduction is achieved via release of signaling molecules in the form of neuropeptides or neurotransmitters at synapses. Snapshots of membrane dynamics of these processes are imaged by electron microscopy. Electron microscopy can reveal ultrastructure and thus synaptic processes. This is crucial both for mapping synaptic connections and for investigating synaptic functions. In addition, via volumetric electron microscopy, the overall architecture of the nervous system becomes accessible, where structure can inform function. Electron microscopy is thus of particular value for studying the nervous system. However, today a plethora of electron microscopy techniques and protocols exist. Which technique is most suitable highly depends on the research question and scope as well as on the type of tissue that is examined. This review gives an overview of the electron microcopy techniques used on the zebrafish nervous system. It aims to give researchers a guide on which techniques are suitable for their specific questions and capabilities as well as an overview of the capabilities of electron microscopy in neurobiological research in the zebrafish model., (© 2023 The Authors. Development, Growth & Differentiation published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Developmental Biologists.)

Acoustic overexposure can eliminate synapses between inner hair cells (IHCs) and auditory nerve fibers (ANFs), even if hair-cell function recovers. This synaptopathy has been extensively studied by confocal microscopy, however, understanding the nature and sequence of damage requires ultrastructural analysis. Here, we used focused ion-beam scanning electron microscopy to mill, image, segment and reconstruct ANF terminals in mice, 1 day and 1 week after synaptopathic exposure (8-16 kHz, 98 dB SPL). At both survivals, ANF terminals were normal in number, but 62% and 53%, respectively, lacked normal synaptic specializations. Most non-synapsing fibers (57% and 48% at 1 day and 1 week) remained in contact with an IHC and contained healthy-looking organelles. ANFs showed a transient increase in mitochondrial content (51%) and efferent innervation (34%) at 1 day. Fibers maintaining synaptic connections showed hypertrophy of pre-synaptic ribbons at both 1 day and 1 week. Non-synaptic fibers were lower in mitochondrial content and typically on the modiolar side of the IHC, where ANFs with high-thresholds and low spontaneous rates are normally found. Even 1 week post-exposure, many ANF terminals remained in IHC contact despite loss of synaptic specializations, thus, regeneration efforts at early post-exposure times should concentrate on synaptogenesis rather than neurite extension., (© 2023. The Author(s).)

Using constitutive GRF1/2 knockout mice, we showed previously that GRF2 is a key regulator of nuclear migration in retinal cone photoreceptors. To evaluate the functional relevance of that cellular process for two putative targets of the GEF activity of GRF2 (RAC1 and CDC42), here we compared the structural and functional retinal phenotypes resulting from conditional targeting of RAC1 or CDC42 in the cone photoreceptors of constitutive GRF2 KO and GRF2 WT mice. We observed that single RAC1 disruption did not cause any obvious morphological or physiological changes in the retinas of GRF2 WT mice, and did not modify either the phenotypic alterations previously described in the retinal photoreceptor layer of GRF2 KO mice. In contrast, the single ablation of CDC42 in the cone photoreceptors of GRF2 WT mice resulted in clear alterations of nuclear movement that, unlike those of the GRF2 KO retinas, were not accompanied by electrophysiological defects or slow, progressive cone cell degeneration. On the other hand, the concomitant disruption of GRF2 and CDC42 in the cone photoreceptors resulted, somewhat surprisingly, in a normalized pattern of nuclear positioning/movement, similar to that physiologically observed in GRF2 WT mice, along with worsened patterns of electrophysiological responses and faster rates of cell death/disappearance than those previously recorded in single GRF2 KO cone cells. Interestingly, the increased rates of cone cell apoptosis/death observed in single GRF2 KO and double-knockout GRF2 KO /CDC42 KO retinas correlated with the electron microscopic detection of significant ultrastructural alterations (flattening) of their retinal ribbon synapses that were not otherwise observed at all in single-knockout CDC42 KO retinas. Our observations identify GRF2 and CDC42 (but not RAC1) as key regulators of retinal processes controlling cone photoreceptor nuclear positioning and survival, and support the notion of GRF2 loss-of-function mutations as potential drivers of cone retinal dystrophies.

The retinas of the vast majority of vertebrate species are termed "duplex," that is, they contain both rod and cone photoreceptor neurons in different ratios. The retina of little skate ( Leucoraja erinacea ) is a rarity among vertebrates because it contains only a single photoreceptor cell type and is thus "simplex." This unique retina provides us with an important comparative model and an exciting opportunity to study retinal circuitry within the context of a visual system with a single photoreceptor cell type. What is perhaps even more intriguing is the fact that the Leucoraja retina is able use that single photoreceptor cell type to function under both scotopic and photopic ranges of illumination. Although some ultrastructural characteristics of skate photoreceptors have been examined previously, leading to a general description of them as "rods" largely based on outer segment (OS) morphology and rhodopsin expression, a detailed study of the fine anatomy of the entire cell and its synaptic connectivity is still lacking. To address this gap in knowledge, we performed serial block-face electron microscopy imaging and examined the structure of skate photoreceptors and their postsynaptic partners. We find that skate photoreceptors exhibit unusual ultrastructural characteristics that are either common to rods or cones in other vertebrates (e.g., outer segment architecture, synaptic ribbon number, terminal extensions), or are somewhere in between those of a typical vertebrate rod or cone (e.g., number of invaginating contacts, clustering of multiple ribbons over a single synaptic invagination). We suggest that some of the ultrastructural characteristics we observe may play a role in the ability of the skate retina to function across scotopic and photopic ranges of illumination. Our findings have the potential to reveal as yet undescribed principles of vertebrate retinal design., Competing Interests: The authors declare no competing financial interests., (Copyright © 2023 Magaña-Hernández et al.)

Sex differences in the brain, encompassing variations in specific brain structures, size, cognitive function, and synaptic connections, have been identified across numerous species. While previous research has explored sex differences in postsynaptic structures, synaptic plasticity, and hippocampus-dependent functions, the hippocampal presynaptic terminals remain largely uninvestigated. The hippocampus is a critical structure responsible for multiple brain functions. This study examined presynaptic differences in cultured hippocampal neurons derived from male and female mice using a combination of biochemical assays, functional analyses measuring exocytosis and endocytosis of synaptic vesicle proteins, ultrastructural analyses via electron microscopy, and presynaptic Ca 2+ -specific optical probes. Our findings revealed that female neurons exhibited a higher number of synaptic vesicles at presynaptic terminals compared to male neurons. However, no significant differences were observed in presynaptic protein expression, presynaptic terminal ultrastructure, synaptic vesicle exocytosis and endocytosis, or presynaptic Ca 2+ alterations between male and female neurons., Competing Interests: Declaration of competing interest The authors have no relevant financial or nonfinancial interests to disclose., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The light pathways are segregated into rod and cone pathways in which rods synapse with rod bipolar cells (RBCs), while cones contact cone bipolar cells (CBCs). However, previous studies found that cones can make synapse with RBCs (cone-RBC synapses) and rods can contact OFF CBC in primate and rabbit retinas. Recently, such cone-RBC synapses have been reported physiologically and morphologically in the mouse retina. Nevertheless, the precise subcellular evidence to determine whether it is the invaginating synapse or the flat contact remains absent. This is due to a lack of immunochemically verified ultrastructural data. Here, we investigated the precise expression of protein kinase C alpha (PKCα) using pre-embedding immunoelectron microscopy (immuno-EM) with a monoclonal antibody against PKCα, a biomarker for the RBCs. We determined the nanoscale localization of PKCα in the outer plexiform layer of the mouse and guinea pig retinas. Our results demonstrate the existence of both the direct invaginating synapse and the basal/flat contact of the cone-RBCs, providing for the first time immunochemically verified ultrastructural evidence for the cone-RBC synapse in the mouse and guinea pig retinas. These results suggest that the cross talk between cone and rod pathways is much more extensive than previously assumed., (© 2023 Wiley Periodicals LLC.)

This paper reports on glycogen store in the retinal horizontal cells (HC) of the African mud catfish Clarias gariepinus, as seen by histochemical reaction with periodic acid Schiff (PAS) and transmission electron microscopy in light- as well as dark-adapted state. Glycogen is abundant in the large somata and less in their axons, characterised ultrastructurally by many microtubules and extensive gap junctions interconnecting them. There was no apparent difference in glycogen content in HC somata between light- and dark adaptation, but the axons clearly showed absence of glycogen in dark condition. The HC somata (presynaptic) make synapses with dendrites in the outer plexiform layer. Müller cell inner processes, which contain more densely packed glycogen, invest the HC. Other cells of the inner nuclear layer do not show any appreciable content of glycogen. Rods, but not cones, contain abundant glycogen in their inner segments and synaptic terminals. It is likely that glycogen is used as energy substrate in hypoxia for this species that dwell muddy aquatic environment with low oxygen content. They appear to have high energy demand, and a high glycogen content in HC could act as a ready source to fulfil physiological processes, like microtubule-based transport of cargo from the large somata to axons and the maintenance of electrical activities across the gap junctions between the axonal processes. It is also likely that they can supplement glucose to the neighbouring inner nuclear layer neurons, which are clearly devoid of glycogen., Competing Interests: Competing interests The author declares no known interests that influenced the work reported here., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

α-Synuclein (α-Syn) is enriched in presynaptic terminals of the central nervous system including the retina and plays a role in the synaptic vesicle cycle and synaptic transmission. Abnormal aggregation of α-Syn is considered to be the main component of the Lewy bodies that are the pathological hallmarks of Parkinson's disease. Although expression pattern of α-Syn has been described in the retinas, its precise cellular and subcellular locations are poorly understood. We investigated the precise expression of α-Syn using light microscopy (LM) and electron microscopy (EM) with antibodies against α-Syn in the mouse retina. We found that the majority of α-Syn immunoreactivity (IR) is located in GABAergic, glycinergic, and dopaminergic amacrine cells, and their processes often make a direct synapse to other labeled or unlabeled amacrine profiles, bipolar cell terminals, or ganglion cell dendrites. Further, our LM and immuno-EM results confirm the absence of α-Syn in excitatory photoreceptors, bipolar cell bodies, and their ribbon synapses, providing evidence, for the first time, that ribbon synapses do not express α-Syn. Additionally, α-Syn IR is located in the ganglion cells, some of which are intrinsically photosensitive retinal ganglion cells. These results reveal a previously unappreciated inhibitory synapse-specific expression pattern of α-Syn in the retina, suggesting that α-Syn may play a distinct role in the modulation and integration of inhibitory synaptic transmission in the retina., (© 2023 Wiley Periodicals LLC.)

Our understanding of inner ear hair cell ultrastructure has heretofore relied upon two-dimensional imaging; however, serial block-face scanning electron microscopy (SBFSEM) changes this paradigm allowing for three-dimensional evaluation. We compared inner ear hair cells of the apical cristae in myo7aa -/- null zebrafish, a model of human Usher Syndrome type 1B, to hair cells in wild-type zebrafish by SBFSEM to investigate possible ribbon synapse ultrastructural differences. Previously, it has been shown that compared to wild type, myo7aa -/- zebrafish neuromast hair cells have fewer ribbon synapses yet similar ribbon areas. We expect the recapitulation of these results within the inner ear apical crista hair cells furthering the knowledge of three-dimensional ribbon synapse structure while resolving the feasibility of therapeutically targeting myo7aa -/- mutant ribbons. In this report, we evaluated ribbon synapse number, volume, surface area, and sphericity. Localization of ribbons and their distance from the nearest innervation were also evaluated. We determined that myo7aa -/- mutant ribbon synapses are smaller in volume and surface area; however, all other measurements were not significantly different from wild-type zebrafish. Because the ribbon synapses are nearly indistinguishable between the myo7aa -/- mutant and wild type, it suggests that the ribbons are structurally receptive, supporting that therapeutic intervention may be feasible.

Brains contain networks of interconnected neurons and so knowing the network architecture is essential for understanding brain function. We therefore mapped the synaptic-resolution connectome of an entire insect brain ( Drosophila larva) with rich behavior, including learning, value computation, and action selection, comprising 3016 neurons and 548,000 synapses. We characterized neuron types, hubs, feedforward and feedback pathways, as well as cross-hemisphere and brain-nerve cord interactions. We found pervasive multisensory and interhemispheric integration, highly recurrent architecture, abundant feedback from descending neurons, and multiple novel circuit motifs. The brain's most recurrent circuits comprised the input and output neurons of the learning center. Some structural features, including multilayer shortcuts and nested recurrent loops, resembled state-of-the-art deep learning architectures. The identified brain architecture provides a basis for future experimental and theoretical studies of neural circuits.

Dense reconstruction of synaptic connectivity requires high-resolution electron microscopy images of entire brains and tools to efficiently trace neuronal wires across the volume. To generate such a resource, we sectioned and imaged a larval zebrafish brain by serial block-face electron microscopy at a voxel size of 14 × 14 × 25 nm 3 . We segmented the resulting dataset with the flood-filling network algorithm, automated the detection of chemical synapses and validated the results by comparisons to transmission electron microscopic images and light-microscopic reconstructions. Neurons and their connections are stored in the form of a queryable and expandable digital address book. We reconstructed a network of 208 neurons involved in visual motion processing, most of them located in the pretectum, which had been functionally characterized in the same specimen by two-photon calcium imaging. Moreover, we mapped all 407 presynaptic and postsynaptic partners of two superficial interneurons in the tectum. The resource developed here serves as a foundation for synaptic-resolution circuit analyses in the zebrafish nervous system., (© 2022. The Author(s).)

Through synaptic connections, long-range circuits transmit information among neurons and connect different brain regions to form functional motifs and execute specific functions. Tracing the synaptic distribution of specific neurons requires submicron-level resolution information. However, it is a great challenge to map the synaptic terminals completely because these fine structures span multiple regions, even in the whole brain. Here, we develop a pipeline including viral tracing, sample embedding, fluorescent micro-optical sectional tomography, and big data processing. We mapped the whole-brain distribution and architecture of long projections of the parvalbumin neurons in the basal forebrain at the synaptic level. These neurons send massive projections to multiple downstream regions with subregional preference. With three-dimensional reconstruction in the targeted areas, we found that synaptic degeneration was inconsistent with the accumulation of amyloid-β plaques but was preferred in memory-related circuits, such as hippocampal formation and thalamus, but not in most hypothalamic nuclei in 8-month-old mice with five familial Alzheimer's disease mutations. Our pipeline provides a platform for generating a whole-brain atlas of cell-type-specific synaptic terminals in the physiological and pathological brain, which can provide an important resource for the study of the organizational logic of specific neural circuits and the circuitry changes in pathological conditions.

Electron microscopy (EM) provided fundamental insights about the ultrastructure of neuronal synapses. The large amount of information present in the contemporary EM datasets precludes a thorough assessment by visual inspection alone, thus requiring computational methods for the analysis of the data. Here, I review image processing software methods ranging from membrane tracing in large volume datasets to high resolution structures of synaptic complexes. Particular attention is payed to molecular level analysis provided by recent cryo-electron microscopy and tomography methods., (Copyright © 2022 The Author. Published by Elsevier Ltd.. All rights reserved.)