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Oxytocin (OXT) is a peptide hormone and a neuropeptide that regulates various peripheral physiological processes and modulates behavioral responses in the central nervous system. While the humoral release occurs from the axons arriving at the median eminence, the neuropeptide is also released from oxytocinergic cell axons in various brain structures that contain its receptor, and from their dendrites in hypothalamic nuclei and potentially into the cerebrospinal fluid (CSF). Understanding oxytocin's complex functions requires the knowledge on patterns of oxytocinergic projections in relationship to its receptor (OXTR). This study provides the first comprehensive examination of the oxytocinergic system in the prairie vole (Microtus ochrogaster), an animal exhibiting social behaviors that mirror human social behaviors linked to oxytocinergic functioning. Using light and electron microscopy, we characterized the neuroanatomy of the oxytocinergic system in this species. OXT+ cell bodies were found primarily in the hypothalamus, and axons were densest in subcortical regions. Examination of the OXT+ fibers and their relationship to oxytocin receptor transcripts (Oxtr) revealed that except for some subcortical structures, the presence of axons was not correlated with the amount of Oxtr across the brain. Of particular interest, the cerebral cortex that had high expression of Oxtr transcripts contained little to no fibers. Electron microscopy is used to quantify dense cored vesicles (DCV) in OXT+ axons and to identify potential axonal release sites. The ependymal cells that line the ventricles were frequently permissive of DCV-containing OXT+ dendrites reaching the third ventricle. Our results highlight a mechanism in which oxytocin is released directly into the ventricles and circulates throughout the ventricular system, may serve as the primary source for oxytocin that binds to OXTR in the cerebral cortex., (© 2024. The Author(s).)

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).)

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.

Mapping single-neuron projections is essential for understanding brain-wide connectivity and diverse functions of the hippocampus (HIP). Here, we reconstructed 10,100 single-neuron projectomes of mouse HIP and classified 43 projectome subtypes with distinct projection patterns. The number of projection targets and axon-tip distribution depended on the soma location along HIP longitudinal and transverse axes. Many projectome subtypes were enriched in specific HIP subdomains defined by spatial transcriptomic profiles. Furthermore, we delineated comprehensive wiring diagrams for HIP neurons projecting exclusively within the HIP formation (HPF) and for those projecting to both intra- and extra-HPF targets. Bihemispheric projecting neurons generally projected to one pair of homologous targets with ipsilateral preference. These organization principles of single-neuron projectomes provide a structural basis for understanding the function of HIP neurons.

Due to its proximity to the axon initial segment (AIS), the paranode of the first myelin segment can influence the threshold for action potentials and how a neuron participates in a neuronal circuit. Using serial section electron microscopy, we examined its three-dimensional (3D) organization in the ventral horn of the mouse spinal cord. The myelin loops of postnatal day 18 mice resemble those at the node of Ranvier. However, in 3-month-old mice, 13 of 22 para-AIS showed 4 types of alteration: (A) A cytoplasmic foot process, with ultrastructural characteristics of an astrocyte, was interposed between the axolemma and the myelin loops. (B) A thin extension of the inner tongue was present between the foot process and axolemma. (C) The foot process was absent. The inner tongue extension was a broad lamella from which a thin extension reached beyond the loops and spiraled around axon. (D) One set of loops was adjacent to the axon, and another was further back and underlain by compact myelin. We suggest that (A)-(C) are steps in a progression toward (D). In this progression, a glial process displaces the original loops, the inner tongue reactivates and extends beneath the foot process, then wraps around the axon to form a new set of loops. This is the first study of the 3D organization of myelin at the AIS and provides evidence for glia-mediated age-dependent remodeling at this critical region., (© 2024 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

Early postnatal brain development involves complex interactions among maturing neurons and glial cells that drive tissue organization. We previously analyzed gene expression in tissue from the mouse medial nucleus of the trapezoid body (MNTB) during the first postnatal week to study changes that surround rapid growth of the large calyx of Held (CH) nerve terminal. Here, we present genes that show significant changes in gene expression level during the second postnatal week, a developmental timeframe that brackets the onset of airborne sound stimulation and the early stages of myelination. Gene Ontology analysis revealed that many of these genes are related to the myelination process. Further investigation of these genes using a previously published cell type-specific bulk RNA-Seq data set in cortex and our own single-cell RNA-Seq data set in the MNTB revealed enrichment of these genes in the oligodendrocyte lineage (OL) cells. Combining the postnatal day (P)6-P14 microarray gene expression data with the previously published P0-P6 data provided fine temporal resolution to investigate the initiation and subsequent waves of gene expression related to OL cell maturation and the process of myelination. Many genes showed increasing expression levels between P2 and P6 in patterns that reflect OL cell maturation. Correspondingly, the first myelin proteins were detected by P4. Using a complementary, developmental series of electron microscopy 3D image volumes, we analyzed the temporal progression of axon wrapping and myelination in the MNTB. By employing a combination of established ultrastructural criteria to classify reconstructed early postnatal glial cells in the 3D volumes, we demonstrated for the first time that astrocytes within the mouse MNTB extensively wrap the axons of the growing CH terminal prior to OL cell wrapping and compaction of myelin. Our data revealed significant expression of several myelin genes and enrichment of multiple genes associated with lipid metabolism in astrocytes, which may subserve axon wrapping in addition to myelin formation. The transition from axon wrapping by astrocytes to OL cells occurs rapidly between P4 and P9 and identifies a potential new role of astrocytes in priming calyceal axons for subsequent myelination., (© 2024 Wiley Periodicals LLC.)

Birds have a well-developed retinopetal system projecting from the midbrain to the contralateral retina. The signal sent to the retina through the retinopetal system facilitates visual responses of the retinal ganglion cells (RGCs), and the retinopetal signals function as attentional signals during visual search. Thus, the retinopetal signal somehow reaches and facilitates visual responses of the RGCs. However, the tertiary neuron of the retinopetal system, the isthmo-optic target cell (IOTC), is unlikely to contact most RGCs directly, because the IOTCs form axon terminals localized in the outermost sublayer (lamina 1) of the inner plexiform layer (IPL) where few RGC dendrites terminate. Therefore, some other intrinsic retinal neurons must be involved in the centrifugal attentional enhancement of visual responses of the RGCs. We investigated connections of the target cells of the IOTCs in chicken and quail, using light and electron microscopic immunohistochemistry. We show that axon terminals of the IOTC make synaptic contacts with protein kinase Cα (PKCα)-immunoreactive (ir) bipolar cells (PKCα-BCs) in lamina 1 of the IPL. Furthermore, with prolonged electrical stimulation of the isthmo-optic nucleus (ION) on one side, whose neurons send their axons to the contralateral retina and make synaptic contacts there with IOTCs, phosphorylation of cAMP response element-binding protein was observed in the PKCα-BCs in the contralateral retina, but not in the ipsilateral retina. This suggests that electrical stimulation of ION activated PKCα-BCs through synapses from IOTCs to PKCα-BCs, thus stimulating transcription in PKCα-BCs. Thus, centrifugal attentional signals may facilitate visual responses of RGCs via the PKCα-BCs., (© 2023 Wiley Periodicals LLC.)

To enable rapid propagation of action potentials, axons are ensheathed by myelin, a multilayered insulating membrane formed by oligodendrocytes. Most of the myelin is generated early in development, resulting in the generation of long-lasting stable membrane structures. Here, we explored structural and dynamic changes in central nervous system myelin during development. To achieve this, we performed an ultrastructural analysis of mouse optic nerves by serial block face scanning electron microscopy (SBF-SEM) and confocal time-lapse imaging in the zebrafish spinal cord. We found that myelin undergoes extensive ultrastructural changes during early postnatal development. Myelin degeneration profiles were engulfed and phagocytosed by microglia using exposed phosphatidylserine as one "eat me" signal. In contrast, retractions of entire myelin sheaths occurred independently of microglia and involved uptake of myelin by the oligodendrocyte itself. Our findings show that the generation of myelin early in development is an inaccurate process associated with aberrant ultrastructural features that require substantial refinement., (© 2023 Djannatian et al.)

Adolescence is a critical period of postnatal development characterized by social, emotional, and cognitive changes. These changes are increasingly understood to depend on white matter development. White matter is highly vulnerable to the effects of injury, including secondary degeneration in regions adjacent to the primary injury site which alters the myelin ultrastructure. However, the impact of such alterations on adolescent white matter maturation is yet to be investigated. To address this, female piebald-virol-glaxo rats underwent partial transection of the optic nerve during early adolescence (postnatal day (PND) 56) with tissue collection two weeks (PND 70) or three months later (PND 140). Axons and myelin in the transmission electron micrographs of tissue adjacent to the injury were classified and measured based on the appearance of the myelin laminae. Injury in adolescence impaired the myelin structure in adulthood, resulting in a lower percentage of axons with compact myelin and a higher percentage of axons with severe myelin decompaction. Myelin thickness did not increase as expected into adulthood after injury and the relationship between the axon diameter and myelin thickness in adulthood was altered. Notably, dysmyelination was not observed 2 weeks postinjury. In conclusion, injury in adolescence altered the developmental trajectory, resulting in impaired myelin maturation when assessed at the ultrastructural level in adulthood.

There is an increased need and focus to understand how local brain microstructure affects the transport of drug molecules directly administered to the brain tissue, for example in convection-enhanced delivery procedures. This study reports a systematic attempt to characterize the cytoarchitecture of commissural, long association and projection fibres, namely the corpus callosum, the fornix and the corona radiata, with the specific aim to map different regions of the tissue and provide essential information for the development of accurate models of brain biomechanics. Ovine samples are imaged using scanning electron microscopy combined with focused ion beam milling to generate 3D volume reconstructions of the tissue at subcellular spatial resolution. Focus is placed on the characteristic cytological feature of the white matter: the axons and their alignment in the tissue. For each tract, a 3D reconstruction of relatively large volumes, including a significant number of axons, is performed and outer axonal ellipticity, outer axonal cross-sectional area and their relative perimeter are measured. The study of well-resolved microstructural features provides useful insight into the fibrous organization of the tissue, whose micromechanical behaviour is that of a composite material presenting elliptical tortuous tubular axonal structures embedded in the extra-cellular matrix. Drug flow can be captured through microstructurally-based models using 3D volumes, either reconstructed directly from images or generated in silico using parameters extracted from the database of images, leading to a workflow to enable physically-accurate simulations of drug delivery to the targeted tissue., (© 2022. The Author(s).)

Traditional histopathologic evaluation of peripheral nerve employs brightfield microscopy with diffraction limited resolution of ~ 250 nm. Though electron microscopy yields nanoscale resolution of the nervous system, sample preparation is costly and the technique is incompatible with living samples. Super-resolution microscopy (SRM) comprises a set of imaging techniques that permit nanoscale resolution of fluorescent objects using visible light. The advent of SRM has transformed biomedical science in establishing non-toxic means for investigation of nanoscale cellular structures. Herein, sciatic nerve sections from GFP-variant expressing mice, and regenerating human nerve from cross-facial autografts labelled with a myelin-specific fluorescent dye were imaged by super-resolution radial fluctuation microscopy, stimulated emission depletion microscopy, and structured illumination microscopy. Super-resolution imaging of axial cryosections of murine sciatic nerves yielded robust visualization myelinated and unmyelinated axons. Super-resolution imaging of axial cryosections of human cross-facial nerve grafts demonstrated enhanced resolution of small-caliber thinly-myelinated regenerating motor axons. Resolution and contrast enhancement afforded by super-resolution imaging techniques enables visualization of unmyelinated axons, regenerating axons, cytoskeleton ultrastructure, and neuronal appendages of mammalian peripheral nerves using light microscopes., (© 2022. The Author(s).)

Background and Objective: Advances in electron microscopy (EM) now allow three-dimensional (3D) imaging of hundreds of micrometers of tissue with nanometer-scale resolution, providing new opportunities to study the ultrastructure of the brain. In this work, we introduce a freely available Matlab-based gACSON software for visualization, segmentation, assessment, and morphology analysis of myelinated axons in 3D-EM volumes of brain tissue samples., Methods: The software is equipped with a graphical user interface (GUI). It automatically segments the intra-axonal space of myelinated axons and their corresponding myelin sheaths and allows manual segmentation, proofreading, and interactive correction of the segmented components. gACSON analyzes the morphology of myelinated axons, such as axonal diameter, axonal eccentricity, myelin thickness, or g-ratio., Results: We illustrate the use of the software by segmenting and analyzing myelinated axons in six 3D-EM volumes of rat somatosensory cortex after sham surgery or traumatic brain injury (TBI). Our results suggest that the equivalent diameter of myelinated axons in somatosensory cortex was decreased in TBI animals five months after the injury., Conclusion: Our results indicate that gACSON is a valuable tool for visualization, segmentation, assessment, and morphology analysis of myelinated axons in 3D-EM volumes. It is freely available at https://github.com/AndreaBehan/g-ACSON under the MIT license., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Non-invasive assessment of axon radii via MRI bears great potential for clinical and neuroscience research as it is a main determinant of the neuronal conduction velocity. However, there is a lack of representative histological reference data at the scale of the cross-section of MRI voxels for validating the MRI-visible, effective radius (r eff ). Because the current gold standard stems from neuroanatomical studies designed to estimate the bulk-determined arithmetic mean radius (r arith ) on small ensembles of axons, it is unsuited to estimate the tail-weighted r eff . We propose CNN-based segmentation on high-resolution, large-scale light microscopy (lsLM) data to generate a representative reference for r eff . In a human corpus callosum, we assessed estimation accuracy and bias of r arith and r eff . Furthermore, we investigated whether mapping anatomy-related variation of r arith and r eff is confounded by low-frequency variation of the image intensity, e.g., due to staining heterogeneity. Finally, we analyzed the error due to outstandingly large axons in r eff . Compared to r arith , r eff was estimated with higher accuracy (maximum normalized-root-mean-square-error of r eff : 8.5 %; r arith : 19.5 %) and lower bias (maximum absolute normalized-mean-bias-error of r eff : 4.8 %; r arith : 13.4 %). While r arith was confounded by variation of the image intensity, variation of r eff seemed anatomy-related. The largest axons contributed between 0.8 % and 2.9 % to r eff . In conclusion, the proposed method is a step towards representatively estimating r eff at MRI voxel resolution. Further investigations are required to assess generalization to other brains and brain areas with different axon radii distributions., Competing Interests: Declaration of Competing Interest The Max Planck Institute for Human Cognitive and Brain Sciences has an institutional research agreement with Siemens Healthcare. NW was a speaker at an event organized by Siemens Healthcare and was reimbursed for the travel expenses., (Copyright © 2022. Published by Elsevier Inc.)

Noninvasive estimation of axon diameter with diffusion MRI holds the potential to investigate the dynamic properties of the brain network and pathology of neurodegenerative diseases. Recent studies use powder averaging to account for complex white matter architectures, but these have not been validated for real axonal geometries from regions that contain fibre crossings. Here, we present 120-304μm long segmented axons from X-ray nano-holotomography volumes of a splenium and crossing fibre region of a vervet monkey brain. We show that the axons in the complex crossing fibre region, which contains callosal, association, and corticospinal connections, exhibit a wider diameter distribution than those of the splenium region. To accurately estimate the axon diameter in these regions, therefore, sensitivity to a wide range of diameters is required. We demonstrate how the q-value, b-value, signal-to-noise ratio and the assumed intra-axonal parallel diffusivity influence the range of measurable diameters with powder average approaches. Furthermore, we show how Gaussian distributed noise results in a wider range of measurable diameter at high b-values than Rician distributed noise, even at high signal-to-noise ratios of 100. The number of gradient directions is also shown to impose a lower bound on measurable diameter. Our results indicate that axon diameter estimation can be performed with only few b-shells, and that additional shells do not improve the accuracy of the estimate. For strong gradients available on human Connectom and preclinical scanners, Monte Carlo simulations of diffusion confirm that powder averaging techniques succeed in providing accurate estimates of axon diameter across a range of diameters, sequence parameters and diffusion times, even in complex white matter architectures. At relatively low b-values, the diameter estimate becomes sensitive to axonal microdispersion and the intra-axonal parallel diffusivity shows time dependency at both in vivo and ex vivo intrinsic diffusivities., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021. Published by Elsevier Inc.)

The reuniens nucleus (RE) is situated at the most ventral position of the midline thalamus. In rats and mice RE is distinguished by bidirectional connections with the hippocampus and medial prefrontal cortex (mPFC) and a role in memory and cognition. In primates, many foundational questions pertaining to RE remain unresolved. We addressed these issues by investigating the composition of the rhesus monkey RE in both sexes by labeling for GABA, a marker of inhibitory neurons, and for the calcium-binding proteins parvalbumin (PV), calbindin (CB), and calretinin (CR), which label thalamic excitatory neurons that project to cortex. As in rats and mice, the macaque RE was mostly populated by CB and CR neurons, characteristic of matrix-dominant nuclei, and had bidirectional connections with hippocampus and mPFC area 25 (A25). Unlike rodents, we found GABAergic neurons in the monkey RE and a sparser but consistent population of core-associated thalamocortical PV neurons. RE had stronger connections with the basal amygdalar complex than in rats or mice. Amygdalar terminations were enriched with mitochondria and frequently formed successive synapses with the same postsynaptic structures, suggesting an active and robust pathway to RE. Significantly, hippocampal pathways formed multisynaptic complexes that uniquely involved excitatory projection neurons and dendrites of local inhibitory neurons in RE, extending this synaptic principle beyond sensory to high-order thalamic nuclei. Convergent pathways from hippocampus, A25, and amygdala in RE position it to flexibly coordinate activity for memory, cognition, and emotional context, which are disrupted in several psychiatric and neurologic diseases in humans. SIGNIFICANCE STATEMENT The primate RE is a central node for memory and cognition through connections with the hippocampus and mPFC. As in rats or mice, the primate RE is a matrix-dominant thalamic nucleus, suggesting signal traffic to the upper cortical layers. Unlike rats or mice, the primate RE contains inhibitory neurons, synaptic specializations with the hippocampal pathway, and robust connections with the amygdala, suggesting unique adaptations. Convergence of hippocampal, mPFC, and amygdalar pathways in RE may help unravel a circuit basis for binding diverse signals for conscious flexible behaviors and the synthesis of memory with affective significance in primates, whereas disruption of distinct circuit nodes may occur in psychiatric disorders in humans., (Copyright © 2022 the authors.)

The neuronal axon is packed with cytoskeletal filaments, membranes, and organelles, many of which move between the cell body and axon tip. Here, we used cryo-electron tomography to survey the internal components of mammalian sensory axons. We determined the polarity of the axonal microtubules (MTs) by combining subtomogram classification and visual inspection, finding MT plus and minus ends are structurally similar. Subtomogram averaging of globular densities in the MT lumen suggests they have a defined structure, which is surprising given they likely contain the disordered protein MAP6. We found the endoplasmic reticulum in axons is tethered to MTs through multiple short linkers. We surveyed membrane-bound cargos and describe unexpected internal features such as granules and broken membranes. In addition, we detected proteinaceous compartments, including numerous virus-like capsid particles. Our observations outline novel features of axonal cargos and MTs, providing a platform for identification of their constituents., (© 2021 MRC Laboratory of Molecular Biology.)

Dopaminergic (DA) neurons exert profound influences on behavior including addiction. However, how DA axons communicate with target neurons and how those communications change with drug exposure remains poorly understood. We leverage cell type-specific labeling with large volume serial electron microscopy to detail DA connections in the nucleus accumbens (NAc) of the mouse ( Mus musculus ) before and after exposure to cocaine. We find that individual DA axons contain different varicosity types based on their vesicle contents. Spatially ordering along individual axons further suggests that varicosity types are non-randomly organized. DA axon varicosities rarely make specific synapses (<2%, 6/410), but instead are more likely to form spinule-like structures (15%, 61/410) with neighboring neurons. Days after a brief exposure to cocaine, DA axons were extensively branched relative to controls, formed blind-ended 'bulbs' filled with mitochondria, and were surrounded by elaborated glia. Finally, mitochondrial lengths increased by ~2.2 times relative to control only in DA axons and NAc spiny dendrites after cocaine exposure. We conclude that DA axonal transmission is unlikely to be mediated via classical synapses in the NAc and that the major locus of anatomical plasticity of DA circuits after exposure to cocaine are large-scale axonal re-arrangements with correlated changes in mitochondria., Competing Interests: GW, AS, JK, AM, CH, MS, XZ, DM, BK No competing interests declared, (© 2021, Wildenberg et al.)

Parvalbumin-containing (PV + ) basket cells are specialized cortical interneurons that regulate the activity of local neuronal circuits with high temporal precision and reliability. To understand how the PV + interneuron connectivity underlying these functional properties is established during development, we used array tomography to map pairs of synaptically connected PV + interneurons and postsynaptic neurons from the neocortex of mice of both sexes. We focused on the axon-myelin unit of the PV + interneuron and quantified the number of synapses onto the postsynaptic neuron, length of connecting axonal paths, and their myelination at different time points between 2 weeks and 7 months of age. We find that myelination of the proximal axon occurs very rapidly during the third and, to a lesser extent, fourth postnatal weeks. The number of synaptic contacts made by the PV + interneuron on its postsynaptic partner meanwhile is significantly reduced to about one-third by the end of the first postnatal month. The number of autapses, the synapses that PV + interneurons form on themselves, however, remains constant throughout the examined period. Axon reorganizations continue beyond postnatal month 2, with the postsynaptic targets of PV + interneurons gradually shifting to more proximal locations, and the length of axonal paths and their myelin becoming conspicuously uniform per connection. These continued microcircuit refinements likely provide the structural substrate for the robust inhibitory effects and fine temporal precision of adult PV + basket cells. SIGNIFICANCE STATEMENT The axon of adult parvalbumin-containing (PV + ) interneurons is highly specialized for fast and reliable neurotransmission. It is myelinated and forms synapses mostly onto the cell bodies and proximal dendrites of postsynaptic neurons for maximal impact. In this study, we follow the development of the PV + interneuron axon, its myelination and synapse formation, revealing a rapid sequence of axonal reorganization, myelination of the PV + interneuron proximal axon, and pruning of almost two-thirds of the synapses in an individual connection. This is followed by a prolonged period of axon refinement and additional myelination leading to a remarkable precision of connections in the adult mouse cortex, consistent with the temporal precision and fidelity of PV + interneuron action., (Copyright © 2021 the authors.)

Uncovering the architecture of white matter axons is fundamental to the study of brain networks. We developed a method for quantifying axonal orientations at a resolution of ~15 micrometers. This method is based on the common Nissl staining technique for postmortem histological slices. Nissl staining reveals the spatial organization of glial cells along axons. Using structure tensor analysis, we leveraged this patterned organization to uncover local axonal orientation. We used Nissl-based structure tensor analysis to extract fine details of axonal architecture and demonstrated its applicability in multiple datasets of humans and nonhuman primates. Nissl-based structure tensor analysis can be used to compare fine-grained features of axonal architecture across species and is widely applicable to existing datasets.

Background: Dysfunction of glia contributes to the deterioration of the central nervous system in a wide array of neurological disorders, thus global replacement of glia is very attractive. Human glial-restricted precursors (hGRPs) transplanted intraventricularly into neonatal mice extensively migrated and rescued the lifespan in half of the studied mice, whereas mouse GRPs (mGRPs) presented no therapeutic benefit. We studied in the same experimental setting canine GRPs (cGRP) to determine whether their therapeutic potential falls between hGRPs and mGRPs. Additional motivation for the selection of cGRPs was a potential for use in veterinary medicine., Methods: cGRPs were extracted from the brain of dog fetuses. The cells were transplanted into the anterior or posterior aspect of the lateral ventricle (LV) of neonatal, immunodeficient, dysmyelinated mice ( Mbp shi , Rag2 KO; shiv/rag2). Outcome measures included early cell biodistribution, animal survival and myelination assessed with MRI, immunohistochemistry and electron microscopy., Results: Grafting of cGRP into posterior LV significantly extended animal survival, whereas no benefit was observed after anterior LV transplantation. In contrast, myelination of the corpus callosum was more prominent in anteriorly transplanted animals., Conclusions: The extended survival of animals after transplantation of cGRPs could be explained by the vicinity of the transplant near the brain stem.

The axon initial segment (AIS) is structurally and functionally distinct from other regions of the axon, yet alterations in the milieu of the AIS after brain injury have not been well characterized. In this study, we have examined extracellular and intracellular changes in the AIS after hypoglossal nerve injury. Microglial adhesions to the AIS were rarely observed in healthy controls, whereas microglial adhesions to the AIS became apparent in the axonal injury model. Regarding intra-AIS morphology, we focused on mitochondria because mitochondrial flow into the injured axon appears critical for axonal regeneration. To visualize mitochondria specifically in injured axons, we used Atf3:BAC transgenic mice whose mitochondria were labeled with GFP in response to nerve injury. These mice clearly showed mitochondrial localization in the AIS after nerve injury. To precisely confirm the light microscopic observations, we performed three-dimensional ultrastructural analysis using focused ion beam/scanning electron microscopy (FIB/SEM). Although the healthy AIS was not surrounded by microglia, tight microglial adhesions with thick processes adhering to the AIS were observed after injury. FIB/SEM simultaneously allowed the observation of mitochondrial localization in the AIS. In the AIS of non-injured neurons, few mitochondria were observed, whereas mitochondria were abundantly localized in the cell body, axon hillock, and axon. Intriguingly, in the injured AIS, numerous mitochondria were observed throughout the AIS. Taken together, axonal injury changes the extracellular glial environment surrounding the AIS and intracellular mitochondrial localization in the AIS. These changes would be crucial responses, perhaps for injured neurons to regenerate after axonal injury., (© 2021 Wiley Periodicals LLC.)

Signal processing within the retina is generally mediated by graded potentials, whereas output is conveyed by action potentials transmitted along optic nerve axons. Among retinal neurons, amacrine cells seem to be an exception to this general rule, as several types generate voltage-gated Na + (Na v ) channel-dependent action potentials. The AII, a narrow-field, bistratified axon-less amacrine cell found in mammalian retinas, displays a unique process that resembles an axon initial segment (AIS), with expression of Na v channels colocalized with the cytoskeletal protein ankyrin-G, and generates action potentials. As the role of spiking in AIIs is uncertain, we hypothesized that the morphological properties of the AIS-like process could provide information relevant for its functional importance, including potential pre- and/or postsynaptic connectivity. For morphological analysis, we injected AII amacrine cells in slices with fluorescent dye and immunolabeled the slices for ankyrin-G. Subsequently, this enabled us to reliably identify AII-type processes among ankyrin-G-labeled processes in wholemount retina. We systematically analyzed the laminar localization, spatial orientation, and distribution of the AIS-like processes as a function of retinal eccentricity. In the horizontal plane, the processes displayed no preferred orientation and terminal endings were randomly distributed. In the vertical plane, the processes displayed a horizontal preference, but also ascended and descended into the inner nuclear layer and proximal inner plexiform layer, respectively. These results suggest that the AII amacrine AIS-like process is unlikely to take part in conventional synaptic connections, but may instead be adapted to respond to volume neurotransmission by means of extrasynaptic receptors., (© 2021 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

During brain development, axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly poorly accessible to imaging at high resolution in a near-native context. Here, we present a method that combines cryo-correlative light microscopy and electron tomography with human cerebral organoid technology to visualize growing axon tracts. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These findings establish our approach as a powerful resource for investigating the ultrastructure of defined neuronal compartments., Competing Interests: PH, SG, IG, MW, MS, WK No competing interests declared, ML MAL is an inventor on several patents related to cerebral organoids, is co-founder and scientific advisory board member of a:head bio, and on the scientific advisory board of the Roche Institute for Translational Bioengineering, (© 2021, Hoffmann et al.)

Axon diameter mapping using diffusion MRI in the living human brain has attracted growing interests with the increasing availability of high gradient strength MRI systems. A systematic assessment of the consistency of axon diameter estimates within and between individuals is needed to gain a comprehensive understanding of how such methods extend to quantifying differences in axon diameter index between groups and facilitate the design of neurobiological studies using such measures. We examined the scan-rescan repeatability of axon diameter index estimation based on the spherical mean technique (SMT) approach using diffusion MRI data acquired with gradient strengths up to 300 mT/m on a 3T Connectom system in 7 healthy volunteers. We performed statistical power analyses using data acquired with the same protocol in a larger cohort consisting of 15 healthy adults to investigate the implications for study design. Results revealed a high degree of repeatability in voxel-wise restricted volume fraction estimates and tract-wise estimates of axon diameter index derived from high-gradient diffusion MRI data. On the region of interest (ROI) level, across white matter tracts in the whole brain, the Pearson's correlation coefficient of the axon diameter index estimated between scan and rescan experiments was r = 0.72 with an absolute deviation of 0.18 μm. For an anticipated 10% effect size in studies of axon diameter index, most white matter regions required a sample size of less than 15 people to observe a measurable difference between groups using an ROI-based approach. To facilitate the use of high-gradient strength diffusion MRI data for neuroscientific studies of axonal microstructure, the comprehensive multi-gradient strength, multi-diffusion time data used in this work will be made publicly available, in support of open science and increasing the accessibility of such data to the greater scientific community., (Copyright © 2021. Published by Elsevier Inc.)

Dorsal root ganglion (DRG) neurons are the predominant cell type that innervates the vertebrate skin. They are typically described as pseudounipolar cells that have central and peripheral axons branching from a single root exiting the cell body. The peripheral axon travels within a nerve to the skin, where free sensory endings can emerge and branch into an arbor that receives and integrates information. In some immature vertebrates, DRG neurons are preceded by Rohon-Beard (RB) neurons. While the sensory endings of RB and DRG neurons function like dendrites, we use live imaging in zebrafish to show that they have axonal plus-end-out microtubule polarity at all stages of maturity. Moreover, we show both cell types have central and peripheral axons with plus-end-out polarity. Surprisingly, in DRG neurons these emerge separately from the cell body, and most cells never acquire the signature pseudounipolar morphology. Like another recently characterized cell type that has multiple plus-end-out neurites, ganglion cells in Nematostella, RB and DRG neurons maintain a somatic microtubule organizing center even when mature. In summary, we characterize key cellular and subcellular features of vertebrate sensory neurons as a foundation for understanding their function and maintenance., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localization and precise role in the synaptic vesicle cycle remain the subject of debate. Here, we used iGluSnFR to optically interrogate glutamate release, at the single-bouton level, in SYT7KO-dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired-pulse facilitation, and synaptic vesicle replenishment and found that SYT7 contributes to each of these processes to different degrees. 'Zap-and-freeze' electron microscopy revealed that a loss of SYT7 diminishes docking of synaptic vesicles after a stimulus and inhibits the recovery of depleted synaptic vesicles after a stimulus train. SYT7 supports these functions from the axonal plasma membrane, where its localization and stability require both γ-secretase-mediated cleavage and palmitoylation. In summary, SYT7 is a peripheral membrane protein that controls multiple modes of synaptic vesicle (SV) exocytosis and plasticity, in part, through enhancing activity-dependent docking of SVs., Competing Interests: JV, GK, KC, EC, SW, EC No competing interests declared, (© 2021, Vevea et al.)

Since the pioneering work of Ramón y Cajal, scientists have sought to unravel the complexities of axon development underlying neural circuit formation. Micrometer-scale axonal growth cones navigate to targets that are often centimeters away. To reach their targets, growth cones react to dynamic environmental cues that change in the order of seconds to days. Proper axon growth and guidance are essential to circuit formation, and progress in imaging has been integral to studying these processes. In particular, advances in high- and super-resolution microscopy provide the spatial and temporal resolution required for studying developing axons. In this Review, we describe how improved microscopy has revolutionized our understanding of axonal development. We discuss how novel technologies, specifically light-sheet and super-resolution microscopy, led to new discoveries at the cellular scale by imaging axon outgrowth and circuit wiring with extreme precision. We next examine how advanced microscopy broadened our understanding of the subcellular dynamics driving axon growth and guidance. We finally assess the current challenges that the field of axonal biology still faces for imaging axons, and examine how future technology could meet these needs., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Neuronal polarization is facilitated by the formation of axons with parallel arrays of plus-end-out and dendrites with the nonuniform orientation of microtubules. In C. elegans, the posterior lateral microtubule (PLM) neuron is bipolar with its two processes growing along the anterior-posterior axis under the guidance of Wnt signaling. Here we found that loss of the Kinesin-13 family microtubule-depolymerizing enzyme KLP-7 led to the ectopic extension of axon-like processes from the PLM cell body. Live imaging of the microtubules and axonal transport revealed mixed polarity of the microtubules in the short posterior process, which is dependent on both KLP-7 and the minus-end binding protein PTRN-1. KLP-7 is positively regulated in the posterior process by planar cell polarity components of Wnt involving rho-1/rock to induce mixed polarity of microtubules, whereas it is negatively regulated in the anterior process by the unc-73/ced-10 cascade to establish a uniform microtubule polarity. Our work elucidates how evolutionarily conserved Wnt signaling establishes the microtubule polarity in neurons through Kinesin-13.

The structural plasticity of dendritic spines is considered to be an important basis of synaptic plasticity, learning, and memory. Here, we induced input-specific structural LTP (sLTP) in single dendritic spines in organotypic hippocampal slices from mice of either sex and performed ultrastructural analyses of the spines using efficient correlative light and electron microscopy. We observed reorganization of the PSD nanostructure, such as perforation and segmentation, at 2-3, 20, and 120 min after sLTP induction. In addition, PSD and nonsynaptic axon-spine interface (nsASI) membrane expanded unevenly during sLTP. Specifically, the PSD area showed a transient increase at 2-3 min after sLTP induction. The PSD growth was to a degree less than spine volume growth at 2-3 min and 20 min after sLTP induction but became similar at 120 min. On the other hand, the nsASI area showed a profound and lasting expansion, to a degree similar to spine volume growth throughout the process. These rapid ultrastructural changes in PSD and surrounding membrane may contribute to rapid electrophysiological plasticity during sLTP. SIGNIFICANCE STATEMENT To understand the ultrastructural changes during synaptic plasticity, it is desired to efficiently image single dendritic spines that underwent structural plasticity in electron microscopy. We induced structural long-term potentiation (sLTP) in single dendritic spines by two-photon glutamate uncaging. We then identified the same spines at different phases of sLTP and performed ultrastructural analysis by using an efficient correlative light and electron microscopy method. We found that postsynaptic density undergoes dramatic modification in its structural complexity immediately after sLTP induction. Meanwhile, the nonsynaptic axon-spine interface area shows a rapid and sustained increase throughout sLTP. Our results indicate that the uneven modification of synaptic and nonsynaptic postsynaptic membrane might contribute to rapid electrophysiological plasticity during sLTP., (Copyright © 2021 the authors.)

Hereditary sensory neuropathy type 1 (HSN1) is caused by mutations in the SPTLC1 or SPTLC2 sub-units of the enzyme serine palmitoyltransferase, resulting in the production of toxic 1-deoxysphingolipid bases (DSBs). We used induced pluripotent stem cells (iPSCs) from patients with HSN1 to determine whether endogenous DSBs are neurotoxic, patho-mechanisms of toxicity and response to therapy. HSN1 iPSC-derived sensory neurons (iPSCdSNs) endogenously produce neurotoxic DSBs. Complex gangliosides, which are essential for membrane micro-domains and signaling, are reduced, and neurotrophin signaling is impaired, resulting in reduced neurite outgrowth. In HSN1 myelinating cocultures, we find a major disruption of nodal complex proteins after 8 weeks, which leads to complete myelin breakdown after 6 months. HSN1 iPSC models have, therefore, revealed that SPTLC1 mutation alters lipid metabolism, impairs the formation of complex gangliosides, and reduces axon and myelin stability. Many of these changes are prevented by l-serine supplementation, supporting its use as a rational therapy., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Neuroinflammation has been put forward as a mechanism triggering axonal regrowth in the mammalian central nervous system (CNS), yet little is known about the underlying cellular and molecular players connecting these two processes. In this study, we provide evidence that MMP2 is an essential factor linking inflammation to axonal regeneration by using an in vivo mouse model of inflammation-induced axonal regeneration in the optic nerve. We show that infiltrating myeloid cells abundantly express MMP2 and that MMP2 deficiency results in reduced long-distance axonal regeneration. However, this phenotype can be rescued by restoring MMP2 expression in myeloid cells via a heterologous bone marrow transplantation. Furthermore, while MMP2 deficiency does not affect the number of infiltrating myeloid cells, it does determine the coordinated expression of pro- and anti-inflammatory molecules. Altogether, in addition to its role in axonal regeneration via resolution of the glial scar, here, we reveal a new mechanism via which MMP2 facilitates axonal regeneration, namely orchestrating the expression of pro- and anti-inflammatory molecules by infiltrating innate immune cells.

Dendritic spines are membranous protrusions that receive essentially all excitatory inputs in most mammalian neurons. Spines, with a bulbous head connected to the dendrite by a thin neck, have a variety of morphologies that likely impact their functional properties. Nevertheless, the question of whether spines belong to distinct morphological subtypes is still open. Addressing this quantitatively requires clear identification and measurements of spine necks. Recent advances in electron microscopy enable large-scale systematic reconstructions of spines with nanometer precision in 3D. Analyzing ultrastructural reconstructions from mouse neocortical neurons with computer vision algorithms, we demonstrate that the vast majority of spine structures can be rigorously separated into heads and necks, enabling morphological measurements of spine necks. We then used a database of spine morphological parameters to explore the potential existence of different spine classes. Without exception, our analysis revealed unimodal distributions of individual morphological parameters of spine heads and necks, without evidence for subtypes of spines. The postsynaptic density size was strongly correlated with the spine head volume. The spine neck diameter, but not the neck length, was also correlated with the head volume. Spines with larger head volumes often had a spine apparatus and pairs of spines in a post-synaptic cell contacted by the same axon had similar head volumes. Our data reveal a lack of morphological subtypes of spines and indicate that the spine neck length and head volume must be independently regulated. These results have repercussions for our understanding of the function of dendritic spines in neuronal circuits., (© 2021 Wiley Periodicals, LLC.)

Functions of the neocortex depend on its bidirectional communication with the thalamus, via cortico-thalamo-cortical (CTC) loops. Recent work dissecting the synaptic connectivity in these loops is generating a clearer picture of their cellular organization. Here, we review findings across sensory, motor and cognitive areas, focusing on patterns of cell type-specific synaptic connections between the major types of cortical and thalamic neurons. We outline simple and complex CTC loops, and note features of these loops that appear to be general versus specialized. CTC loops are tightly interlinked with local cortical and corticocortical (CC) circuits, forming extended chains of loops that are probably critical for communication across hierarchically organized cerebral networks. Such CTC-CC loop chains appear to constitute a modular unit of organization, serving as scaffolding for area-specific structural and functional modifications. Inhibitory neurons and circuits are embedded throughout CTC loops, shaping the flow of excitation. We consider recent findings in the context of established CTC and CC circuit models, and highlight current efforts to pinpoint cell type-specific mechanisms in CTC loops involved in consciousness and perception. As pieces of the connectivity puzzle fall increasingly into place, this knowledge can guide further efforts to understand structure-function relationships in CTC loops.

Adenosine triphosphate (ATP) is the most vital energy source produced mainly in the mitochondria. Age-related mitochondrial dysfunction is associated with brain diseases. Nicotinamide adenine dinucleotide (NAD + ) is an essential cofactor for energy production in mitochondria. Here, we examined how the novel NAD + -assisting substance, 10-ethyl-3-methylpyrimido[4,5-b]quinoline-2,4(3H,10H)-dione (TND1128), modulates the morphological growth of cultured mouse hippocampal neurons. The morphological growth effect of TND1128 was also compared with that of β-nicotinamide mononucleotide (β-NMN). TND1128 induced the branching of axons and dendrites, and increased the number of excitatory synapses. This study provides new insight into TND1128 as a mitochondria-stimulating drug for improving brain function., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Taste neurons are functionally and molecularly diverse, but their morphologic diversity remains completely unexplored. Using sparse cell genetic labeling, we provide the first reconstructions of peripheral taste neurons. The branching characteristics across 96 taste neurons show surprising diversity in their complexities. Individual neurons had 1-17 separate arbors entering between one and seven taste buds, 18 of these neurons also innervated non-taste epithelia. Axon branching characteristics are similar in gustatory neurons from male and female mice. Cluster analysis separated the neurons into four groups according to branch complexity. The primary difference between clusters was the amount of the nerve fiber within the taste bud available to contact taste-transducing cells. Consistently, we found that the maximum number of taste-transducing cells capable of providing convergent input onto individual gustatory neurons varied with a range of 1-22 taste-transducing cells. Differences in branching characteristics across neurons indicate that some neurons likely receive input from a larger number of taste-transducing cells than other neurons (differential convergence). By dividing neurons into two groups based on the type of taste-transducing cell most contacted, we found that neurons contacting primarily sour transducing cells were more heavily branched than those contacting primarily sweet/bitter/umami transducing cells. This suggests that neuron morphologies may differ across functional taste quality. However, the considerable remaining variability within each group also suggests differential convergence within each functional taste quality. Each possibility has functional implications for the system. SIGNIFICANCE STATEMENT Taste neurons are considered relay cells, communicating information from taste-transducing cells to the brain, without variation in morphology. By reconstructing peripheral taste neuron morphologies for the first time, we found that some peripheral gustatory neurons are simply branched, and can receive input from only a few taste-transducing cells. Other taste neurons are heavily branched, contacting many more taste-transducing cells than simply branched neurons. Based on the type of taste-transducing cell contacted, branching characteristics are predicted to differ across (and within) quality types (sweet/bitter/umami vs sour). Therefore, functional differences between neurons likely depends on the number of taste-transducing cells providing input and not just the type of cell providing input., (Copyright © 2021 the authors.)

In peripheral nerves, Schwann cells form myelin and provide trophic support to axons. We previously showed that the mitochondrial protein prohibitin 2 can localize to the axon-Schwann-cell interface and is required for developmental myelination. Whether the homologous protein prohibitin 1 has a similar role, and whether prohibitins also play important roles in Schwann cell mitochondria is unknown. Here, we show that deletion of prohibitin 1 in Schwann cells minimally perturbs development, but later triggers a severe demyelinating peripheral neuropathy. Moreover, mitochondria are heavily affected by ablation of prohibitin 1 and demyelination occurs preferentially in cells with apparent mitochondrial loss. Furthermore, in response to mitochondrial damage, Schwann cells trigger the integrated stress response, but, contrary to what was previously suggested, this response is not detrimental in this context. These results identify a role for prohibitin 1 in myelin integrity and advance our understanding about the Schwann cell response to mitochondrial damage.

The chemical synapse is the principal form of contact between neurons of the central nervous system. These synapses are typically configured as presynaptic axon terminations onto postsynaptic dendrites or somata, giving rise to axo-dendritic and axo-somatic synapses, respectively. Beyond these common synapse configurations are less-studied, non-canonical synapse types that are prevalent throughout the brain and significantly contribute to neural circuit function. Among these are the axo-axonic synapses, which consist of an axon terminating on another axon or axon terminal. Here, we review evidence for axo-axonic synapse contributions to neural signaling in the mammalian nervous system and survey functional neural circuit motifs enabled by these synapses. We also detail how recent advances in microscopy, transgenics, and biological sensors may be used to identify and functionally assay axo-axonic synapses., (© 2020 Wiley Periodicals LLC.)

Myelin insulates neuronal axons and enables fast signal transmission, constituting a key component of brain development, aging and disease. Yet, myelin-specific imaging of macroscopic samples remains a challenge. Here, we exploit myelin's nanostructural periodicity, and use small-angle X-ray scattering tensor tomography (SAXS-TT) to simultaneously quantify myelin levels, nanostructural integrity and axon orientations in nervous tissue. Proof-of-principle is demonstrated in whole mouse brain, mouse spinal cord and human white and gray matter samples. Outcomes are validated by 2D/3D histology and compared to MRI measurements sensitive to myelin and axon orientations. Specificity to nanostructure is exemplified by concomitantly imaging different myelin types with distinct periodicities. Finally, we illustrate the method's sensitivity towards myelin-related diseases by quantifying myelin alterations in dysmyelinated mouse brain. This non-destructive, stain-free molecular imaging approach enables quantitative studies of myelination within and across samples during development, aging, disease and treatment, and is applicable to other ordered biomolecules or nanostructures.

Myelination is essential for central nervous system (CNS) formation, health, and function. Emerging evidence of oligodendrocyte heterogeneity in health and disease and divergent CNS gene expression profiles between mice and humans supports the development of experimentally tractable human myelination systems. Here, we developed human iPSC-derived myelinating organoids ("myelinoids") and quantitative tools to study myelination from oligodendrogenesis through to compact myelin formation and myelinated axon organization. Using patient-derived cells, we modeled a monogenetic disease of myelinated axons (Nfasc155 deficiency), recapitulating impaired paranodal axo-glial junction formation. We also validated the use of myelinoids for pharmacological assessment of myelination-both at the level of individual oligodendrocytes and globally across whole myelinoids-and demonstrated reduced myelination in response to suppressed synaptic vesicle release. Our study provides a platform to investigate human myelin development, disease, and adaptive myelination., Competing Interests: Declaration of interests S.C. is a co-founder of Pheno Therapeutics. All other authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Progranulin (PGRN) is a secreted glycoprotein with multiple biological functions in early embryogenesis, anti-inflammation, and neurodegeneration. A good model for the functional study of PGRN is the zebrafish with knockdown or knockout of grn, the gene encoding PGRN. Morpholino oligonucleotides (MOs) and zinc finger nucleases have been used to generate zebrafish grn models, yet they have shown inconsistent phenotypes due to either the neurotoxicity of the MOs or possible genetic compensation responses during gene editing. In this study, we generated stable grna (one of the major grn homologues of zebrafish) knockout zebrafish by using CRISPR/Cas9-mediated genome editing. A grna sgRNA was designed to target the similar repeated sequence shared by exon 13, exon 15, and exon 19 in zebrafish. The F1 generation with the frameshift mutation of + 4 bp (the addition of 4 bp to exon15), which causes a premature termination, was obtained and subjected to morphological and behavioral evaluation. The grna knockout zebrafish showed neurodevelopmental defects, including spinal motor neurons with shorter axons, decreased sensory hair cells, thinning of the outer nuclear layer and thickening of the inner nuclear layer of the retina, decreased expression of rhodopsin in the cone cells, and motor behavior changes. Moreover, the phenotypes of grna knockout zebrafish could be rescued with the Tol2 system carrying the grna gene. The grna knockout zebrafish model generated in this study provides a useful tool to study PGRN function and has potential for high-throughput drug screening for disease therapy., (© 2021 International Society for Neurochemistry.)

Magnetosomes are membrane-enclosed iron oxide crystals biosynthesized by magnetotactic bacteria. As the biomineralization of bacterial magnetosomes can be genetically controlled, they have become promising nanomaterials for bionanotechnological applications. In the present paper, we explore a novel application of magnetosomes as nanotool for manipulating axonal outgrowth via stretch-growth (SG). SG refers to the process of stimulation of axonal outgrowth through the application of mechanical forces. Thanks to their superior magnetic properties, magnetosomes have been used to magnetize mouse hippocampal neurons in order to stretch axons under the application of magnetic fields. We found that magnetosomes are avidly internalized by cells. They adhere to the cell membrane, are quickly internalized, and slowly degrade after a few days from the internalization process. Our data show that bacterial magnetosomes are more efficient than synthetic iron oxide nanoparticles in stimulating axonal outgrowth via SG.

One class of molecules that are now coming to be recognized as essential for our understanding of the nervous system are the lysophospholipids. One of the major signaling lysophospholipids is lysophosphatidic acid, also known as LPA. LPA activates a variety of G protein-coupled receptors (GPCRs) leading to a multitude of physiological responses. In this review, I describe our current understanding of the role of LPA and LPA receptor signaling in the development and function of the nervous system, especially the central nervous system (CNS). In addition, I highlight how aberrant LPA receptor signaling may underlie neuropathological conditions, with important clinical application.

Aging is characterized by a decline in neuronal function in all animal species investigated so far. Functional changes are accompanied by and may be in part caused by, structurally visible degenerative changes in neurons. In the mammalian brain, normal aging shows abnormalities in dendrites and axons, as well as ultrastructural changes in synapses, rather than global neuron loss. The analysis of the structural features of aging neurons, as well as their causal link to molecular mechanisms on the one hand, and the functional decline on the other hand is crucial in order to understand the aging process in the brain. Invertebrate model organisms like Drosophila and C. elegans offer the opportunity to apply a forward genetic approach to the analysis of aging. In the present review, we aim to summarize findings concerning abnormalities in morphology and ultrastructure in invertebrate brains during normal aging and compare them to what is known for the mammalian brain. It becomes clear that despite of their considerably shorter life span, invertebrates display several age-related changes very similar to the mammalian condition, including the retraction of dendritic and axonal branches at specific locations, changes in synaptic density and increased accumulation of presynaptic protein complexes. We anticipate that continued research efforts in invertebrate systems will significantly contribute to reveal (and possibly manipulate) the molecular/cellular pathways leading to neuronal aging in the mammalian brain.

In neuroanatomy textbooks on humans, the posterior median septum is commonly depicted along the midline of the posterior column of the spinal cord. For intramedullary spinal cord tumors, the standard surgical treatment is posterior midline myelotomy. However, its anatomical basis is still unclear. Therefore, in this study we focused on the ultrastructural characterization of the median structure of the posterior column in an adult rat. In the median part of the fasciculi gracilis, a fine lineal tissue continued from the posterior median sulcus to the 3/4th depth of the fasciculi. At higher magnification, this fine lineal tissue consisted of bundles of astrocytes, which are often disrupted and eventually disappeared. At the junction of the ventral part of the fasciculi cuneatus and the gray commissure, short lineal figures of glial tissues extended dorsally. These lineal figures of glial tissues were morphologically similar to other lineal figures of glial tissues found in the posterior column; bundles of astrocytes extending along the axons that entered the gray commissure and the perivascular lineal figures of glial tissues. In conclusion, this study revealed that the posterior median septum is composed of very fine lineal figures of glial tissues that are often disrupted and eventually disappear. We consider these basic structures to be similar in humans. Therefore, during posterior midline myelotomy, accurately separating along the posterior median septum in the posterior column is extremely difficult., (© 2020 The Authors. The Anatomical Record published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Introduction: We recently developed an inducible model of dysphagia using intralingual injection of cholera toxin B conjugated to saporin (CTB-SAP) to cause death of hypoglossal neurons. In this study we aimed to evaluate tongue morphology and ultrastructural changes in hypoglossal neurons and nerve fibers in this model., Methods: Tissues were collected from 20 rats (10 control and 10 CTB-SAP animals) on day 9 post-injection. Tongues were weighed, measured, and analyzed for microscopic changes using laminin immunohistochemistry. Hypoglossal neurons and axons were examined using transmission electron microscopy., Results: The cross-sectional area of myofibers in the posterior genioglossus was decreased in CTB-SAP-injected rats. Degenerative changes were observed in both the cell bodies and distal axons of hypoglossal neurons., Discussion: Preliminary results indicate this model may have translational application to a variety of neurodegenerative diseases resulting in tongue dysfunction and associated dysphagia., (© 2020 Wiley Periodicals LLC.)

A quantitative description of the hippocampal formation synaptic architecture is essential for understanding the neural mechanisms of episodic memory. Yet the existing knowledge of connectivity statistics between different neuron types in the rodent hippocampus only captures a mere 5% of this circuitry. We present a systematic pipeline to produce first-approximation estimates for most of the missing information. Leveraging the www.Hippocampome.org knowledge base, we derive local connection parameters between distinct pairs of morphologically identified neuron types based on their axonal-dendritic overlap within every layer and subregion of the hippocampal formation. Specifically, we adapt modern image analysis technology to determine the parcel-specific neurite lengths of every neuron type from representative morphologic reconstructions obtained from either sex. We then compute the average number of synapses per neuron pair using relevant anatomic volumes from the mouse brain atlas and ultrastructurally established interaction distances. Hence, we estimate connection probabilities and number of contacts for >1900 neuron type pairs, increasing the available quantitative assessments more than 11-fold. Connectivity statistics thus remain unknown for only a minority of potential synapses in the hippocampal formation, including those involving long-range (23%) or perisomatic (6%) connections and neuron types without morphologic tracings (7%). The described approach also yields approximate measurements of synaptic distances from the soma along the dendritic and axonal paths, which may affect signal attenuation and delay. Overall, this dataset fills a substantial gap in quantitatively describing hippocampal circuits and provides useful model specifications for biologically realistic neural network simulations, until further direct experimental data become available. SIGNIFICANCE STATEMENT The hippocampal formation is a crucial functional substrate for episodic memory and spatial representation. Characterizing the complex neuron type circuit of this brain region is thus important to understand the cellular mechanisms of learning and navigation. Here we present the first numerical estimates of connection probabilities, numbers of contacts per connected pair, and synaptic distances from the soma along the axonal and dendritic paths, for more than 1900 distinct neuron type pairs throughout the dentate gyrus, CA3, CA2, CA1, subiculum, and entorhinal cortex. This comprehensive dataset, publicly released online at www.Hippocampome.org, constitutes an unprecedented quantification of the majority of the local synaptic circuit for a prominent mammalian neural system and provides an essential foundation for data-driven, anatomically realistic neural network models., (Copyright © 2021 the authors.)

Segmentation of axons in light and electron micrographs allows for quantitative high-resolution analysis of nervous tissues, but varied axonal dispersion angles result in over-estimates of fiber sizes. To overcome this technical challenge, we developed a novel shape-adjusted ellipse (SAE) determination of axonal size and myelination as an all-inclusive and non-biased tool to correct for oblique nerve fiber presentations. Our new resource was validated by light and electron microscopy against traditional methods of determining nerve fiber size and myelination in rhesus macaques as a model system. We performed detailed segmental mapping and characterized the morphological signatures of autonomic and motor fibers in primate lumbosacral ventral roots (VRs). An en bloc inter-subject variability for the preganglionic parasympathetic fibers within the L7-S2 VRs was determined. The SAE approach allows for morphological ground truth data collection and assignment of individual axons to functional phenotypes with direct implications for fiber mapping and neuromodulation studies.

Brain circuits in the neocortex develop from diverse types of neurons that migrate and form synapses. Here we quantify the circuit patterns of synaptogenesis for inhibitory interneurons in the developing mouse somatosensory cortex. We studied synaptic innervation of cell bodies, apical dendrites, and axon initial segments using three-dimensional electron microscopy focusing on the first 4 weeks postnatally (postnatal days P5 to P28). We found that innervation of apical dendrites occurs early and specifically: Target preference is already almost at adult levels at P5. Axons innervating cell bodies, on the other hand, gradually acquire specificity from P5 to P9, likely via synaptic overabundance followed by antispecific synapse removal. Chandelier axons show first target preference by P14 but develop full target specificity almost completely by P28, which is consistent with a combination of axon outgrowth and off-target synapse removal. This connectomic developmental profile reveals how inhibitory axons in the mouse cortex establish brain circuitry during development., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Dopamine transporter (DAT) controls dopamine neurotransmission by clearing synaptically released dopamine. However, trafficking itineraries of DAT, which determine its cell-surface concentration near synapses, are poorly characterized. It is especially unknown how DAT is transported between spatially distant midbrain somatodendritic and striatal axonal compartments. To examine this "long-range" trafficking, the localization and membrane diffusion of HA-epitope tagged DAT in the medial forebrain bundle (MFB) of a knock-in mouse (both sexes) were analyzed using confocal, super-resolution and EM in intact brain and acute brain slices. HA-DAT was abundant in the plasma membrane of MFB axons, similar to the striatum, although the intracellular fraction of HA-DAT in MFB was more substantial. Intracellular HA-DAT colocalized with VPS35, a subunit of the retromer complex mediating recycling from endosomes, in a subset of axons. Late endosomes, lysosomes, and endoplasmic reticulum were abundant in the soma but minimally present in MFB axons, suggesting that biosynthesis and lysosomal degradation of DAT are confined to soma. Together, the data suggest that membrane diffusion is the main mode of long-range DAT transport through MFB, although the contribution of vesicular traffic can be significant in a population of MFB axons. Based on HA-DAT diffusion rates, plasma membrane DAT in MFB axons turns over with a halftime of ∼20 d, which explains the extremely slow turnover of DAT protein in the brain. Unexpectedly, the mean diameter of DAT-labeled MFB axons was observed to be twice larger than reported for striatum. The implications of this finding for dopamine neuron physiology are discussed. SIGNIFICANCE STATEMENT The dopamine transporter (DAT) is a key regulator of dopamine neurotransmission and a target of abused psychostimulants. In the present study, we examined, for the first time, mechanisms of the long-range traffic of DAT in intact brain and acute brain slices from the knock-in mouse expressing epitope-tagged DAT. Using a combination of confocal, super-resolution and EM, we defined DAT localization and its membrane diffusion parameters in medial forebrain bundle axonal tracts connecting midbrain somatodendritic and striatal axonal compartments of dopaminergic neurons. In contrast to the widely accepted model of long-range axonal transport, our studies suggest that DAT traffics between midbrain and striatum, mainly by lateral diffusion in the plasma membrane with only a limited contribution of vesicular transport in recycling endosomes., (Copyright © 2021 the authors.)