Distribution Of Cell Bodies Research Articles

Indications of Peripheral Pain, Dermal Hypersensitivity, and Neurogenic Inflammation in Patients with Lipedema.

Lipedema is a disease with abnormally increased adipose tissue deposition and distribution. Pain sensations have been described in the clinical evaluation of lipedema, but its etiology remains poorly understood. We hypothesized that pain sensitivity measurements and ex vivo quantitation of neuronal cell body distribution in the skin would be lipedema stage-dependent, and could, thus, serve to objectively characterize neuropathic pain in lipedema. The pain was assessed by questionnaire and peripheral cutaneous mechanical sensitization (von-Frey) in lipedema (n = 27) and control (n = 23) consenting female volunteers. Dermal biopsies from (n = 11) Stages 1–3 lipedema and control (n = 10) participants were characterized for neuronal cell body and nociceptive neuropeptide calcitonin gene-related peptide (CGRP) and nerve growth factor (NGF) distribution. Stage 2 or 3 lipedema participants responded positively to von Frey sensitization in the calf and thigh, and Stage 3 participants also responded in the arm. Lipedema abdominal skin displayed reduced Tuj-1+ neuronal cell body density, compared to healthy controls, while CGRP and NGF was significantly elevated in Stage 3 lipedema tissues. Together, dermal neuronal cell body loss is consistent with hyper-sensitization in patients with lipedema. Further study of neuropathic pain in lipedema may elucidate underlying disease mechanisms and inform lipedema clinical management and treatment impact.

The mosaic of AII amacrine cell bodies in rat retina is indistinguishable from a random distribution.

The vertebrate retina contains a large number of different types of neurons that can be distinguished by their morphological properties. Assuming that no location should be without a contribution from the circuitry and function linked to a specific type of neuron, it is expected that the dendritic trees of neurons belonging to a type will cover the retina in a regular manner. Thus, for most types of neurons, the contribution to visual processing is thought to be independent of the exact location of individual neurons across the retina. Here, we have investigated the distribution of AII amacrine cells in rat retina. The AII is a multifunctional amacrine cell found in mammals and involved in synaptic microcircuits that contribute to visual processing under both scotopic and photopic conditions. Previous investigations have suggested that AIIs are regularly distributed, with a nearest-neighbor distance regularity index of ~4. It has been argued, however, that this presumed regularity results from treating somas as points, without taking into account their actual spatial extent which constrains the location of other cells of the same type. When we simulated random distributions of cell bodies with size and density similar to real AIIs, we confirmed that the simulated distributions could not be distinguished from the distributions observed experimentally for AIIs in different regions and eccentricities of the retina. The developmental mechanisms that generate the observed distributions of AIIs remain to be investigated.

Neuroanatomical distribution of the enkephalinergic and tachykininergic systems in the alpaca brainstem: an immunohistochemical study.

A recent study has shown a close neuroanatomical relationship between the enkephalinergic (methionine-enkephalin) and tachykininergic (substance P) systems in the alpaca diencephalon. In this study, our aim is to show this relationship in the alpaca brainstem. Using an immunohistochemical technique, the distribution of immunoreactive (Ir) fibers and cell bodies containing substance P (SP) or methionine-enkephalin (MET) has been studied in the alpaca brainstem. Five adult males were used; brain tissue was fixed and processed by standard methods. SP- and MET-Ir fibers showed a widespread and similar distribution in the mesencephalon, pons and medulla oblongata. The co-localization of fibers containing SP or MET was found in most of the nuclei/tracts of the alpaca brainstem. This close neuroanatomical relationship suggests multiple physiological interactions between both neuropeptides. The distribution of the cell bodies containing SP was very restricted (cell bodies were only observed in a few nuclei located in the mesencephalon and medulla oblongata), whereas MET-Ir perikarya showed a moderately widespread distribution in the mesencephalon, pons and medulla oblongata. This study increases the knowledge on the neuroanatomical distribution/relationship of the tachykininergic (SP) and enkephalinergic (MET) systems in the alpaca central nervous system.

CI in single-sided deafness

The cochlear implant (CI) as a treatment option for single-sided deafness (SSD) started with a clinical study looking in to the influence of cochlear implantation with a MED-EL device on incapacitating unilateral tinnitus in SSD. The study began in 2003 and was conducted by P. Van de Heyning and his team in Antwerp, Belgium. The first CI in SSD without tinnitus in Germany was implanted by J. Mueller and R. Jacob in Koblenz in 2005. Translational research activities took place since then to evaluate the CI as a treatment option for SSD not only in adults but also in children. They assessed the hearing performance of SSD patients implanted with CI, importance of long electrode arrays in SSD patients, degree of acceptance of CI by SSD children, importance of early CI implantation in SSD children in developing language skills, music enjoyment by hearing with two ears and evidence on spiral ganglion cell body distribution. In 2013, MED-EL was the first CI manufacturer to receive the CE mark for the indication of SSD and asymmetric hearing loss (AHL) in adults and children. In 2019, MED-EL was the first CI manufacturer to get its CI device approved for patients over the age of five with SSD and AHL, by the FDA in the USA. This article covers the milestones of translational research from the first concept to the widespread clinical use of CI in SSD.

A single-cell transcriptomic and anatomic atlas of mouse dorsal raphe Pet1 neurons.

Among the brainstem raphe nuclei, the dorsal raphe nucleus (DR) contains the greatest number of Pet1-lineage neurons, a predominantly serotonergic group distributed throughout DR subdomains. These neurons collectively regulate diverse physiology and behavior and are often therapeutically targeted to treat affective disorders. Characterizing Pet1 neuron molecular heterogeneity and relating it to anatomy is vital for understanding DR functional organization, with potential to inform therapeutic separability. Here we use high-throughput and DR subdomain-targeted single-cell transcriptomics and intersectional genetic tools to map molecular and anatomical diversity of DR-Pet1 neurons. We describe up to fourteen neuron subtypes, many showing biased cell body distributions across the DR. We further show that P2ry1-Pet1 DR neurons - the most molecularly distinct subtype - possess unique efferent projections and electrophysiological properties. These data complement and extend previous DR characterizations, combining intersectional genetics with multiple transcriptomic modalities to achieve fine-scale molecular and anatomic identification of Pet1 neuron subtypes.

Anatomically Defined and Functionally Distinct Dorsal Raphe Serotonin Sub-systems

The dorsal raphe (DR) constitutes a major serotonergic input to the forebrain and modulates diverse functions and brain states, including mood, anxiety, and sensory and motor functions. Most functional studies to date have treated DR serotonin neurons as a single population. Using viral-genetic methods, we found that subcortical- and cortical-projecting serotonin neurons have distinct cell-body distributions within the DR and differentially co-express a vesicular glutamate transporter. Further, amygdala- and frontal-cortex-projecting DR serotonin neurons have largely complementary whole-brain collateralization patterns, receive biased inputs from presynaptic partners, and exhibit opposite responses to aversive stimuli. Gain- and loss-of-function experiments suggest that amygdala-projecting DR serotonin neurons promote anxiety-like behavior, whereas frontal-cortex-projecting neurons promote active coping in the face of challenge. These results provide compelling evidence that the DR serotonin system contains parallel sub-systems that differ ininput and output connectivity, physiological response properties, and behavioral functions.

Neuropeptide- and serotonin- cells in the brain of Rhodnius prolixus (Hemiptera) associated with the circadian clock

The neuronal pathways of the circadian clock in the brain of R. prolixus have been described in detail previously, but there is no information concerning the cells or their pathways which relay either inputs to the clock (e.g. for light entrainment), or outputs from it to driven rhythms. Here, we employ antisera to three neuropeptides (type A allatostatin-7, crustacean cardioactive peptide and FMRFamide), and serotonin in confocal laser scanning immunohistochemistry to analyze the distribution of cell bodies and their projections in relation to the principle circadian clock cells (lateral cells, LNs) for all four neuron types. LNs are revealed following labelling with anti- pigment dispersing factor in double labelled preparations. Regions of potential communication between ramifications of the LNs and each of the four other neuron types is described (identified by close superposition of their neurites in various brain regions), as is their detailed projections within the brain. Neuromodulation is sometimes suggested by close, but not intimate, proximity of varicosities of neurites. We infer that some neuron types comprise input pathways to the LNs, some are outputs to neuroendocrine or behavioral rhythms, and others participate in both input and output pathways, sometimes by the same neuron type but in different locations. For example, one retinula cell in each ommatidium is immunoreactive for allatostatin A; its axon projects to the medulla making superpositions with LNs, as do serotonin cells in the optic lobe, indicating roles of both neuron types in light input (entrainment) to the clock. But in other brain areas, these same types appear to mediate outputs from the clock. The accessory medulla has been widely reported as the principle center of integration in other insects; but we found sparse evidence of this in R. prolixus as it contains few neurites other than those from the clock cells. Rather, the importance of neural pathways involving the medulla and the superior protocerebrum is emphasized. We conclude that there is a vast and complex web of interactions in the brain with the LNs, which potentially receive multiple pathways of inputs and outputs that could drive rhythmicity in a multitude of downstream cells, rendering a host of output pathways rhythmic, notably hormone release from neurosecretory cells and behaviors.

Anatomical, Physiological, and Functional Heterogeneity of the Dorsal Raphe Serotonin System

The dorsal raphe (DR) constitutes a major serotonergic input to the forebrain, and modulates diverse functions and brain states including mood, anxiety, and sensory and motor functions. Most functional studies to date have treated DR serotonin neurons as a single, homogeneous population. Using viral-genetic methods, we found that subcortical - vs. cortical - projecting serotonin neurons have distinct cell body distributions within the DR and different degrees of co-expressing a vesicular glutamate transporter. Further, the amygdala- and frontal cortex-projecting DR serotonin neurons have largely complementary whole-brain collateralization patterns, receive biased inputs from presynaptic partners, and exhibit opposite responses to aversive stimuli. Gain- and loss-of-function experiments suggest that amygdala-projecting DR serotonin neurons promote anxiety-like behavior, whereas frontal cortex-projecting neurons promote active coping in face of challenge. These results provide compelling evidence that the DR serotonin system contains parallel subsystems that differ in input and output connectivity, physiological response properties, and behavioral functions.

Distribution of glutamatergic, GABAergic, and glycinergic neurons in the auditory pathways of macaque monkeys

Macaque monkeys use complex communication calls and are regarded as a model for studying the coding and decoding of complex sound in the auditory system. However, little is known about the distribution of excitatory and inhibitory neurons in the auditory system of macaque monkeys. In this study, we examined the overall distribution of cell bodies that expressed mRNAs for VGLUT1, and VGLUT2 (markers for glutamatergic neurons), GAD67 (a marker for GABAergic neurons), and GLYT2 (a marker for glycinergic neurons) in the auditory system of the Japanese macaque. In addition, we performed immunohistochemistry for VGLUT1, VGLUT2, and GAD67 in order to compare the distribution of proteins and mRNAs. We found that most of the excitatory neurons in the auditory brainstem expressed VGLUT2. In contrast, the expression of VGLUT1 mRNA was restricted to the auditory cortex (AC), periolivary nuclei, and cochlear nuclei (CN). The co-expression of GAD67 and GLYT2 mRNAs was common in the ventral nucleus of the lateral lemniscus (VNLL), CN, and superior olivary complex except for the medial nucleus of the trapezoid body, which expressed GLYT2 alone. In contrast, the dorsal nucleus of the lateral lemniscus, inferior colliculus, thalamus, and AC expressed GAD67 alone. The absence of co-expression of VGLUT1 and VGLUT2 in the medial geniculate, medial superior olive, and VNLL suggests that synaptic responses in the target neurons of these nuclei may be different between rodents and macaque monkeys.

Distribution of Neurotensin and Somatostatin-28 (1-12) in the Minipig Brainstem.

Using an indirect immunoperoxidase technique, an in depth study has been carried out for the first time on the distribution of fibres and cell bodies containing neurotensin and somatostatin-28 (1-12) (SOM) in the minipig brainstem. The animals used were not treated with colchicine. The distribution of neurotensin- and SOM-immunoreactive fibres was seen to be quite similar and was moderate in the minipig brainstem: a close anatomical relationship between both neuropeptides was observed. The distribution of cell bodies containing neurotensin or SOM was quite different and restricted. Cell bodies containing neurotensin were found in four brainstem nuclei: nucleus centralis raphae, nucleus dorsalis raphae, in the pars centralis of the nucleus tractus spinalis nervi trigemini and in the nucleus ventralis raphae. Cell bodies containing SOM were found in six nuclei/regions of the brainstem: nucleus ambiguus, nucleus dorsalis motorius nervi vagus, formatio reticularis, nucleus parabrachialis medialis, nucleus reticularis lateralis and nucleus ventralis raphae. According to the observed anatomical distribution of the immunoreactive structures containing neurotensin or SOM, the peptides could be involved in sleep-waking, nociceptive, gustatory, motor, respiratory and autonomic mechanisms.

Mapping of somatostatin-28 (1-12) in the alpaca (Lama pacos) brainstem.

Using an indirect immunoperoxidase technique, we studied the distribution of cell bodies and fibers containing somatostatin-28 (1-12) in the alpaca brainstem. Immunoreactive fibers were widely distributed throughout the whole brainstem: 34 brainstem nuclei/regions showed a high or a moderate density of these fibers. Perikarya containing the peptide were widely distributed throughout the mesencephalon, pons and medulla oblongata. Cell bodies containing somatostatin-28 (1-12) were observed in the lateral and medial divisions of the marginal nucleus of the brachium conjunctivum, reticular formation (mesencephalon, pons and medulla oblongata), inferior colliculus, periaqueductal gray, superior colliculus, pericentral division of the dorsal tegmental nucleus, interpeduncular nucleus, nucleus of the trapezoid body, vestibular nucleus, motor dorsal nucleus of the vagus, nucleus of the solitary tract, nucleus praepositus hypoglossi, and in the substantia nigra. This widespread distribution indicates that somatostatin-28 (1-12) is involved in multiple physiological actions in the alpaca brainstem.

Current focussing in cochlear implants: An analysis of neural recruitment in a computational model

Several multipolar current focussing strategies are examined in a computational model of the implanted human cochlea. The model includes a realistic spatial distribution of cell bodies of the auditory neurons throughout Rosenthal's canal. Simulations are performed of monopolar, (partial) tripolar and phased array stimulation. Excitation patterns, estimated thresholds, electrical dynamic range, excitation density and neural recruitment curves are determined and compared. The main findings are: (I) Current focussing requires electrical field interaction to induce spatially restricted excitation patterns. For perimodiolar electrodes the distance to the neurons is too small to have sufficient electrical field interaction, which results in neural excitation near non-centre contacts. (II) Current focussing only produces spatially restricted excitation patterns when there is little or no excitation occurring in the peripheral processes, either because of geometrical factors or due to neural degeneration. (III) The model predicts that neural recruitment with electrical stimulation is a three-dimensional process; regions of excitation not only expand in apical and basal directions, but also by penetrating deeper into the spiral ganglion. (IV) At equal loudness certain differences between the spatial excitation patterns of various multipoles cannot be simulated in a model containing linearly aligned neurons of identical morphology. Introducing a form of variability in the neurons, such as the spatial distribution of cell bodies in the spiral ganglion used in this study, is therefore essential in the modelling of spread of excitation.This article is part of a Special Issue entitled <Lasker Award>.

Functional architecture of the retina: development and disease.

Structure and function are highly correlated in the vertebrate retina, a sensory tissue that is organized into cell layers with microcircuits working in parallel and together to encode visual information. All vertebrate retinas share a fundamental plan, comprising five major neuronal cell classes with cell body distributions and connectivity arranged in stereotypic patterns. Conserved features in retinal design have enabled detailed analysis and comparisons of structure, connectivity and function across species. Each species, however, can adopt structural and/or functional retinal specializations, implementing variations to the basic design in order to satisfy unique requirements in visual function. Recent advances in molecular tools, imaging and electrophysiological approaches have greatly facilitated identification of the cellular and molecular mechanisms that establish the fundamental organization of the retina and the specializations of its microcircuits during development. Here, we review advances in our understanding of how these mechanisms act to shape structure and function at the single cell level, to coordinate the assembly of cell populations, and to define their specific circuitry. We also highlight how structure is rearranged and function is disrupted in disease, and discuss current approaches to re-establish the intricate functional architecture of the retina.

Immunohistochemical mapping of pro-opiomelanocortin- and pro-dynorphin-derived peptides in the alpaca (Lamapacos) diencephalon

Using an indirect immunoperoxidase technique, we studied the distribution of cell bodies and fibres containing non-opioid peptides (adrenocorticotropin hormone (ACTH), alpha-melanocyte-stimulating hormone) and opioid peptides (beta-endorphin (1–27), alpha-neo-endorphin, leucine-enkephalin) in the alpaca diencephalon. No immunoreactive cell bodies containing ACTH were found. Perikarya containing the other four peptides were observed exclusively in the hypothalamus and their distribution was restricted. Perikarya containing alpha-melanocyte-stimulating hormone or alpha-neo-endorphin showed a more widespread distribution than those containing leucine-enkephalin or beta-endorphin (1–27). Cell bodies containing pro-opiomelanocortin-derived peptides were observed in the arcuate nucleus, anterior and lateral hypothalamic areas and in the ventromedial and supraoptic hypothalamic nuclei, whereas perikarya containing alpha-neo-endorphin (a pro-dynorphin-derived peptide) were found in the arcuate nucleus, dorsal and lateral hypothalamic areas, and in the paraventricular, ventromedial and supraoptic hypothalamic nuclei. Immunoreactive cell bodies containing leucine-enkephalin were found in the lateral hypothalamic area and in the paraventricular hypothalamic nucleus. Immunoreactive fibres expressing pro-opiomelanocortin-derived peptides were more numerous than those expressing pro-dynorphin-derived peptides. A close anatomical relationship was observed: in all the diencephalic nuclei in which beta-endorphin (1–27)-immunoreactive fibres were found, fibres containing alpha-melanocyte-stimulating hormone or alpha-neo-endorphin were also observed. Fibres containing beta-endorphin (1–27), alpha-melanocyte-stimulating hormone or alpha-neo-endorphin were widely distributed throughout the diencephalon, but fibres containing ACTH or leucine-enkephalin showed a moderate distribution. The distribution of the five peptides studied here is also compared with that reported previously in other mammalian species. The widespread distribution observed indicates that both the pro-dynorphin and the pro-opiomelanocortin systems are involved in multiple physiological actions (e.g., food intake, thermoregulation, neuroendocrine and reproductive mechanisms) in the alpaca diencephalon.

Distribution of CGRP in the minipig brainstem

For the first time, an in-depth study has been made of the distribution of fibers and cell bodies containing calcitonin gene-related peptide (CGRP) in the minipig brainstem using an indirect immunoperoxidase technique. The animals studied were not treated with colchicine. Cell bodies containing CGRP were found in 20 nuclei/regions of the brainstem. These perikarya were located in somatomotor, brachiomotor and raphae nuclei, nucleus ambiguus, substantia nigra, nucleus reticularis tegmenti pontis, nucleus prepositus hypoglossi, nuclei olivaris inferior and superior, nuclei pontis, formatio reticularis, nucleus dorsalis tegmenti of Gudden, and in the nucleus reticularis lateralis. Fourteen of the 20 brainstem nuclei showed a high density of immunoreactive cell bodies. In comparison with other species, the minipig, together with the rat, show the most widespread distribution of cell bodies containing CGRP in the mammalian brainstem. Immunoreactive fibers were also observed in the brainstem. However, in the minipig brainstem the density of these fibers is low, as in many brainstem nuclei only single immunoreactive fibers were observed. A high density of immunoreactive fibers was only observed in the pars caudalis of the nucleus tractus spinalis nervi trigemini and in the nucleus ventralis tegmenti of Gudden. According to the observed anatomical distribution of the immunoreactive structures containing CGRP, the peptide could be involved in motor, somatosensory, gustative, and autonomic mechanisms.

Seasonal changes in RFamide‐related peptide‐3 neurons in the hypothalamus of a seasonally breeding marsupial species, the brushtail possum (Trichosurus vulpecula)

RFamide-related peptide-3 (RFRP-3) neurons have been shown to inhibit gonadotropin-releasing hormone (GnRH) neuronal activity and hence reproduction in birds and eutherian mammals. They have also been proposed to have a direct hypophysiotropic effect on pituitary gonadotropin release. We used a new RFRP-3 antibody to characterize the cell body distribution and fiber projections of RFRP-3 neurons in the adult female brushtail possum brain. RFRP-3-immunoreactive cell bodies were found scattered within the dorsomedial hypothalamus and the dorsomedial half of the ventromedial hypothalamus, while GnRH neurons were observed scattered rostrocaudally along the lateral septum, rostral to the medial septum. There was a significant 2-fold increase in the RFRP-3 cell body number during the nonbreeding season (summer) compared to the breeding season (winter). Immunoreactive RFRP-3 fibers were distributed throughout the thalamus, preoptic area, and hypothalamus. Very few fibers were observed in the median eminence, especially in the external zone. Intraperitoneal injection of the retrograde tracer Fluoro-Gold resulted in the labeling of 40% of hypophysiotropic tuberoinfundibular dopaminergic (tyrosine hydroxylase-positive) neurons; however, <10% of zona incerta dopaminergic neurons (which are not hypophysiotropic) or RFRP-3 neurons were labeled with this tracer. These observations suggest that RFRP-3 exhibits a seasonal fluctuation in cell numbers, as seen in sheep and birds, which is consistent with an increased inhibitory tone during the nonbreeding season. The lack of RFRP-3 fibers in the median eminence and of Fluoro-Gold uptake from the periphery imply that the actions of this peptide occur primarily centrally rather than at the anterior pituitary gland.

Mapping of Neurotensin in the Alpaca (Lama pacos) Brainstem

We studied the distribution of cell bodies and fibres containing neurotensin (NT) in the brainstem of the alpaca using an indirect immunoperoxidase technique. Immunoreactive fibres were widely distributed throughout the brainstem, whereas the distribution of cell bodies was less widespread. Immunoreactive perikarya were only found in the mesencephalic and bulbar reticular formation, periaqueductal grey, nucleus of the solitary tract, laminar spinal trigeminal nucleus and in the inferior colliculus. A high density of fibres containing NT was found in the dorsal nucleus of the raphe, marginal nucleus of the brachium conjunctivum, locus coeruleus, inferior colliculus, inter-peduncular nucleus, substantia nigra, periaqueductal grey, reticular formation of the mesencephalon, pons and medulla oblongata, nucleus of the solitary tract, laminar spinal trigeminal nucleus, hypoglossal nucleus, inferior central nucleus and in the tegmental reticular nucleus. The widespread distribution indicates that NT might be involved in multiple physiological actions in the alpaca brainstem; this must be investigated in the future as alpacas lives from 0 m above sea level to altitudes of up 5000 m and hence the involvement of this neuropeptide in special and unique regulatory physiological mechanisms could be suggested.

The early mouse 3D osteocyte network in the presence and absence of mechanical loading

Osteocytes are considered to act as mechanosensory cells in bone. They form a functional synctia in which their processes become interconnected to constitute a three-dimensional (3D) network. Previous studies reported that in mice, the two-dimensional osteocyte network becomes progressively more regular as they grow, although the key factors governing the arrangement of the osteocyte network during bone growth remain unknown. In this study, we characterized the 3D formation of the osteocyte network during bone growth.Morphological skeletal changes have been reported to occur in response to mechanical loading and unloading. In order to evaluate the effect of mechanical unloading on osteocyte network formation, we subjected newborn mice to sciatic neurectomy in order to immobilize their left hind limb as an unloading model. The osteocyte network was visualized by staining osteocyte cell bodies and processes with fluorescently labeled phalloidin. First, we compared the osteocyte network in the femora of embryonic and 6-week-old mice in order to understand the morphological changes that occur with normal growth and mechanical loading. In embryonic mice, the osteocyte network in the femur cortical bone displayed a random cell body distribution, non-directional orientation of cell processes, and irregularly shaped cells. In 6-week-old mice, the 3D network contained spindle-shaped osteocytes, which were arranged parallel to the longitudinal axis of the femur. In addition, more and longer cell processes radiated from each osteocyte. Second, we compared the cortical osteocyte networks of 6-week-old mice that had or had not undergone sciatic neurectomy in order to evaluate the effect of unloading on osteocyte network formation. The osteocyte network formation in both cortical bone and cancellous bone was affected by mechanical loading. However, there were differences in the extent of network formation between cortical bone and cancellous bone in response to mechanical loading with regard to the orientation, nuclear shape and branch formation.

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.