Somatic and autonomic nerve density and distribution within the clitoris: an immunohistochemical study in adult female cadavers.

INTRODUCTION AND HYPOTHESIS: Knowledge of clitoral neuroanatomy is critical to vulvar surgery. We sought to characterize the density and distribution of autonomic and somatic nerves supplying the clitoris. METHODS: Pelvic tissue harvested from female cadavers was sectioned axially at three anatomic levels: the proximal aspect of the clitoral body (CB), the distal CB, and the glans. The CB, glans, and the surrounding connective tissue (dorsal, lateral, and ventral) were outlined microscopically. An area containing large nerve bundles dorsal to the CB, referred to as the dorsal nerve subregion, was analyzed separately. Double-immunofluorescent staining for beta III tubulin (ßIIIT), a global axonal marker, and myelin basic protein (MBP), a myelinated nerve marker, was performed. Threshold-based automatic image-segmentation distinguished stained areas. Autonomic and somatic density were calculated as percentage of tissue stained with ßIIIT alone, and ßIIIT and MBP respectively. Comparisons were made using nonparametric Friedman tests. RESULTS: Seven cadavers, aged 22-81, were examined. Somatic (mean 4.42%, SD ± 1.97) and autonomic (2.14% ± 2.42) nerve density was highest in the dorsal nerve subregion and dorsal region at the distal CB level. Compared with the CB, somatic density was higher in proximal (0.05% ± 0.03 vs 1.27% ± 0.69, p = 0.03) and distal (0.29% ± 0.25 vs 1.09% ± 0.41, p = 0.05) dorsal regions. Somatic density was greater in the glans than in the surrounding lateral (0.78% ± 0.47 vs 0.43% ± 0.23, p = 0.03) and ventral (0.78% ± 0.47 vs 0.52% ± 0.2, p = 0.03) regions. Autonomic density was greater than somatic in all areas, except for the dorsal nerve subregion. CONCLUSIONS: Somatic and autonomic nerve density were greatest in a well-defined region dorsal to the CB. Surgical preservation of this region is critical for maintaining nerve supply to the clitoris.

Somatic and autonomic nerve density of the urethra, periurethral tissue, and anterior vaginal wall: an immunohistochemical study in adult female cadavers.

INTRODUCTION AND HYPOTHESIS: Retropubic procedures may disrupt nerves supplying the pelvic viscera; however, knowledge of pelvic neuroanatomy is limited. We sought to characterize somatic and autonomic nerve density within the urethra, periurethral tissue, and anterior vagina. METHODS: Axial sections were obtained from pelvic tissue harvested from female cadavers ≤24 h from death at three anatomical levels: the midurethra, proximal urethra, and upper trigone. Periurethral/perivesical tissue was divided into medial and lateral sections, and the anterior vagina into middle, medial, and lateral sections. Double immunofluorescent staining for beta III tubulin (ßIIIT), a global axonal marker, and myelin basic protein (MBP), a myelinated nerve marker, was performed. Threshold-based automatic image segmentation distinguished stained areas. Autonomic and somatic density were calculated as percentage of tissue stained with ßIIIT alone, and with ßIIIT and MBP respectively. Statistical comparisons were made using nonparametric Friedman tests. RESULTS: Six cadavers, aged 22-73, were examined. Overall, autonomic nerve density was highest at the midurethral level in the lateral and middle anterior vagina. Somatic density was highest in the external urethral sphincter (midurethra mean 0.15%, SD ±0.11; proximal urethra 0.19%, SD ±0.19). Comparison of annotated sections revealed significant differences in autonomic density among the lateral, medial, and middle vagina at the midurethra level (0.71%, SD ±0.48 vs 0.60%, SD ±0.48 vs 0.70%, SD ±0.63, p=0.03). Autonomic density was greater than somatic density in all sections. CONCLUSIONS: Autonomic and somatic nerves are diffusely distributed throughout the periurethral tissue and anterior vagina, with few significant differences in nerve density among sections analyzed. Minimizing tissue disruption near urethral skeletal muscle critical for urinary continence may prevent adverse postoperative urinary symptoms.

Endogenous pathology in tauopathy mice progresses via brain networks.

Neurodegenerative tauopathies are hypothesized to propagate via brain networks. This is uncertain because we have lacked precise network resolution of pathology. We therefore developed whole-brain staining methods with anti-p-tau nanobodies and imaged in 3D PS19 tauopathy mice, which have pan-neuronal expression of full-length human tau containing the P301S mutation. We analyzed patterns of p-tau deposition across established brain networks at multiple ages, testing the relationship between structural connectivity and patterns of progressive pathology. We identified core regions with early tau deposition, and used network propagation modeling to determine the link between tau pathology and connectivity strength. We discovered a bias towards retrograde network-based propagation of tau. This novel approach establishes a fundamental role for brain networks in tau propagation, with implications for human disease.

Anatomic relationships of the clitoral body, bulbs of the vestibule, and urethra.

BACKGROUND: Although recent studies have enhanced our understanding of the anatomy of the clitoris and its somatic innervation, less emphasis has been placed on the anatomic relationships of the clitoris to its surrounding structures. OBJECTIVE: This study aimed to further characterize the gross and histologic relationships of the clitoris, vestibular bulbs, and urethra. STUDY DESIGN: Detailed dissections were performed in 30 unembalmed female cadavers. In 23 specimens, gross dissections were performed, and relationships of the clitoris, vestibular bulbs, and urethra were annotated. Histologic evaluation was performed in 7 specimens, in which tissues were harvested within 24 hours from death. Descriptive statistics were used for data analyses. RESULTS: The clitoral body consisted of 2 components, the proximal body and the distal body. The distal body was oriented ≤90° from the proximal body, forming an outer and inner angle at the inflection point. A "septumlike" arrangement of fibroconnective and vascular tissues was noted between the inner angle of the clitoral body and the urethra. Neurovascular bundles coursed laterally along the clitoral body and the surfaces of the crura and vestibular bulbs. The vestibular bulbs approached each other over the ventral surface of the urethra, at the commissure of the vestibular bulbs. Each bulb was separated by fibrous tissue and did not merge along the midline. The vestibular bulbs approximated the clitoral body, but the erectile tissue of the vestibular bulbs was separated from the corpora cavernosa of the clitoral body by the tunica albuginea. The erectile tissue of the vestibular bulbs abutted the ventrolateral walls of the urethra but was separated from the urethral mucosa by an indiscrete layer of erectilelike tissue with dense stroma. CONCLUSION: This study provided gross and histological confirmation of the relationships of the clitoris, vestibular bulbs, and urethra. Detailed knowledge of the anatomy of the clitoris is crucial for reducing surgical complications associated with periclitoral and distal urethral procedures, which may adversely affect sexual arousal and sexual function.

Review: Milk Small Extracellular Vesicles for Use in the Delivery of Therapeutics.

Small extracellular vesicles (sEVs, "exosomes") in milk have attracted considerable attention for use in delivering therapeutics to diseased tissues because of the following qualities. The production of milk sEVs is scalable, e.g., more than 1021 sEVs may be obtained annually from a single cow. Milk EVs protect their cargo against degradation in the gastrointestinal tract and during industrial processing. Milk sEVs and their cargo are absorbed following oral administration and they cross barriers such as intestinal mucosa, placenta and the blood-brain barrier in humans, pigs, and mice. Milk sEVs do no alter variables of liver and kidney function, or hematology, and do not elicit immune responses in humans, rats, and mice. Protocols are available for loading milk sEVs with therapeutic cargo, and a cell line is available for assessing effects of milk sEV modifications on drug delivery. Future research will need to assess and optimize sEV shelf-life and storage and effects of milk sEV modifications on the delivery of therapeutic cargo to diseased tissues.

Small Extracellular Vesicles in Milk Cross the Blood-Brain Barrier in Murine Cerebral Cortex Endothelial Cells and Promote Dendritic Complexity in the Hippocampus and Brain Function in C57BL/6J Mice.

Human milk contains large amounts of small extracellular vesicles (sEVs) and their microRNA cargos, whereas infant formulas contain only trace amounts of sEVs and microRNAs. We assessed the transport of sEVs across the blood-brain barrier (BBB) and sEV accumulation in distinct regions of the brain in brain endothelial cells and suckling mice. We further assessed sEV-dependent gene expression profiles and effects on the dendritic complexity of hippocampal granule cells and phenotypes of EV depletion in neonate, juvenile and adult mice. The transfer of sEVs across the BBB was assessed by using fluorophore-labeled bovine sEVs in brain endothelial bEnd.3 monolayers and dual chamber systems, and in wild-type newborn pups fostered to sEV and cargo tracking (ECT) dams that express sEVs labeled with a CD63-eGFP fusion protein for subsequent analysis by serial two-photon tomography and staining with anti-eGFP antibodies. Effects of EVs on gene expression and dendritic architecture of granule cells was analyzed in hippocampi from juvenile mice fed sEV and RNA-depleted (ERD) and sEV and RNA-sufficient (ERS) diets by using RNA-sequencing analysis and Golgi-Cox staining followed by integrated neuronal tracing and morphological analysis of neuronal dendrites, respectively. Spatial learning and severity of kainic acid-induced seizures were assessed in mice fed ERD and ERS diets. bEnd.3 cells internalized sEVs by using a saturable transport mechanism and secreted miR-34a across the basal membrane. sEVs penetrated the entire brain in fostering experiments; major regions of accumulation included the hippocampus, cortex and cerebellum. Two hundred ninety-five genes were differentially expressed in hippocampi from mice fed ERD and ERS diets; high-confidence gene networks included pathways implicated in axon guidance and calcium signaling. Juvenile pups fed the ERD diet had reduced dendritic complexity of dentate granule cells in the hippocampus, scored nine-fold lower in the Barnes maze test of spatial learning and memory, and the severity of seizures was 5-fold higher following kainic acid administration in adult mice fed the ERD diet compared to mice fed the ERS diet. We conclude that sEVs cross the BBB and contribute toward optimal neuronal development, spatial learning and memory, and resistance to kainic acid-induced seizures in mice.

Autoantigen microarrays reveal myelin basic protein autoantibodies in morphea.

BACKGROUND: Morphea is an autoimmune, sclerosing skin disorder. Despite the recent emphasis on immune dysregulation in morphea, the role of autoantibodies in morphea pathogenesis or utility as biomarkers are poorly defined. METHODS: Autoantigen microarray was used to profile autoantibodies from the serum of participants from the Morphea in Adults and Children (MAC) cohort. Clinical and demographic features of morphea patients with myelin basic protein (MBP) autoantibodies were compared to those without. MBP immunohistochemistry staining was subsequently performed in morphea skin to assess for perineural inflammation in areas of staining. Immunofluorescence staining on mouse brain tissue was also performed using patient sera and mouse anti-myelin basic protein antibody to confirm the presence of MBP antibodies in patient sera. RESULTS: Myelin basic protein autoantibodies were found in greater frequency in morphea (n = 50, 71.4%) compared to systemic sclerosis (n = 2, 6.7%) and healthy controls (n = 7, 20%). Patients with MBP antibodies reported pain at higher frequencies. Morphea skin biopsies, highlighted by immunohistochemistry, demonstrated increased perineural inflammation in areas of MBP expression. Immunofluorescence staining revealed an increased fluorescence signal in myelinated areas of mouse brain tissue (i.e. axons) when incubated with sera from MBP antibody-positive morphea patients compared to sera from MBP antibody-negative morphea patients. Epitope mapping revealed target epitopes for MBP autoantibodies in morphea are distinct from those reported in MS, and included fragments 11-30, 41-60, 51-70, and 91-110. CONCLUSIONS: A molecular classification of morphea based on distinct autoantibody biosignatures may be used to differentially classify morphea. We have identified anti-MBP as a potential antibody associated with morphea due to its increased expression in morphea compared to healthy controls and systemic sclerosis patients.

Establishment of patient-derived organoid models of lower-grade glioma.

BACKGROUND: Historically, creating patient-derived models of lower-grade glioma (LGG) has been challenging, contributing to few experimental platforms that support laboratory-based investigations of this disease. Although organoid modeling approaches have recently been employed to create in vitro models of high-grade glioma (HGG), it is unknown whether this approach can be successfully applied to LGG. METHODS: In this study, we developed an optimized protocol for the establishment of organoids from LGG primary tissue samples by utilizing physiologic (5%) oxygenation conditions and employed it to produce the first known suite of these models. To assess their fidelity, we surveyed key biological features of patient-derived organoids using metabolic, genomic, histologic, and lineage marker gene expression assays. RESULTS: Organoid models were created with a success rate of 91% (n = 20/22) from primary tumor samples across glioma histological subtypes and tumor grades (WHO Grades 1-4), and a success rate of 87% (13/15) for WHO Grade 1-3 tumors. Patient-derived organoids recapitulated stemness, proliferative, and tumor-stromal composition profiles of their respective parental tumor specimens. Cytoarchitectural, mutational, and metabolic traits of parental tumors were also conserved. Importantly, LGG organoids were maintained in vitro for weeks to months and reanimated after biobanking without loss of integrity. CONCLUSIONS: We report an efficient method for producing faithful in vitro models of LGG. New experimental platforms generated through this approach are well positioned to support preclinical studies of this disease, particularly those related to tumor immunology, tumor-stroma interactions, identification of novel drug targets, and personalized assessments of treatment response profiles.

VAMP4 Maintains a Ca2+-Sensitive Pool of Spontaneously Recycling Synaptic Vesicles.

Spontaneous neurotransmitter release is a fundamental property of synapses in which neurotransmitter filled vesicles release their content independent of presynaptic action potentials (APs). Despite their seemingly random nature, these spontaneous fusion events can be regulated by Ca2+ signaling pathways. Here, we probed the mechanisms that maintain Ca2+ sensitivity of spontaneous release events in synapses formed between hippocampal neurons cultured from rats of both sexes. In this setting, we examined the potential role of vesicle-associated membrane protein 4 (VAMP4), a vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein in spontaneous neurotransmission. Our results show that VAMP4 is required for Ca2+-dependent spontaneous excitatory neurotransmission, with a limited role in spontaneous inhibitory neurotransmission. Key residues in VAMP4 that regulate its retrieval as well as functional clathrin-mediated vesicle trafficking were essential for the maintenance of VAMP4-mediated spontaneous release. Moreover, high-frequency stimulation (HFS) that typically triggers asynchronous release and retrieval of VAMP4 from the plasma membrane also augmentsCa2+-sensitive spontaneous release for up to 30 min in a VAMP4-dependent manner. This VAMP4-mediated link between asynchronous and spontaneous excitatory neurotransmission might serve as a presynaptic substrate for synaptic plasticity coupling distinct forms of release.SIGNIFICANCE STATEMENT Spontaneous neurotransmitter release that occurs independent of presynaptic action potentials (APs) shows significant sensitivity to intracellular Ca2+ levels. In this study, we identify the vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) molecule vesicle-associated membrane protein 4 (VAMP4) as a key component of the machinery that maintains these Ca2+-sensitive fraction of spontaneous release events. Following brief intense activity, VAMP4-dependent synaptic vesicle retrieval supports a pool of vesicles that fuse spontaneously in the long term. We propose that this vesicle trafficking pathway acts to shape spontaneous release and associated signaling based on previous activity history of synapses.

Visualization and Quantification of Post-stroke Neural Connectivity and Neuroinflammation Using Serial Two-Photon Tomography in the Whole Mouse Brain.

Whole-brain volumetric microscopy techniques such as serial two-photon tomography (STPT) can provide detailed information on the roles of neuroinflammation and neuroplasticity throughout the whole brain post-stroke. STPT automatically generates high-resolution images of coronal sections of the entire mouse brain that can be readily visualized in three dimensions. We developed a pipeline for whole brain image analysis that includes supervised machine learning (pixel-wise random forest models via the "ilastik" software package) followed by registration to a standardized 3-D atlas of the adult mouse brain (Common Coordinate Framework v3.0; Allen Institute for Brain Science). These procedures allow the detection of cellular fluorescent signals throughout the brain in an unbiased manner. To illustrate our imaging techniques and automated image quantification, we examined long-term post-stroke motor circuit connectivity in mice that received a motor cortex photothrombotic stroke. Two weeks post-stroke, mice received intramuscular injections of pseudorabies virus (PRV-152), a trans-synaptic retrograde herpes virus driving expression of green fluorescent protein (GFP), into the affected contralesional forelimb to label neurons in descending tracts to the forelimb musculature. Mice were sacrificed 3 weeks post-stroke. We also quantified sub-acute neuroinflammation in the post-stroke brain in a separate cohort of mice following a 60 min transient middle cerebral artery occlusion (tMCAo). Naive e450+-labeled splenic CD8+ cytotoxic T cells were intravenously injected at 7, 24, 48, and 72 h post-tMCAo. Mice were sacrificed 4 days after stroke. Detailed quantification of post-stroke neural connectivity and neuroinflammation indicates a role for remote brain regions in stroke pathology and recovery. The workflow described herein, incorporating STPT and automated quantification of fluorescently labeled features of interest, provides a framework by which one can objectively evaluate labeled neuronal or lymphocyte populations in healthy and injured brains. The results provide region-specific quantification of neural connectivity and neuroinflammation, which could be a critical tool for investigating mechanisms of not only stroke recovery, but also a wide variety of brain injuries or diseases.

Anatomy, histology, and nerve density of clitoris and associated structures: clinical applications to vulvar surgery.

BACKGROUND: A precise understanding of structures comprising the female external genitalia is essential in obstetric and gynecologic practice. OBJECTIVE: To further characterize the anatomy, histology, and nerve density of the clitoris and associated structures, and to provide clinical correlations to vulvar surgery. MATERIALS AND METHODS: Unembalmed female cadavers were examined. The length and width of the body, glans, and crura of the clitoris were measured. Distances from the glans to the urethra and from the dorsal surface of the clitoral body to the mid pubic arch were recorded. The path of the dorsal nerve of the clitoris was examined, and the nerve width was measured as it emerged from the lateral surface of crura and at the distal clitoral body. Distances from where the dorsal nerve emerged from the perineal membrane to the posterior surface of the membrane and to mid pubic arch were measured. Connective tissue layers associated with the clitoris were examined. Tissue was harvested from additional unembalmed cadavers, and nerve density of the labia minora, glans, and clitoral body were analyzed. Histological examination was performed on vulvar structures to clarify tissue composition. Descriptive statistics were used for data analyses. RESULTS: A total of 27 cadavers (aged 48-96 years) were examined, 22 grossly and 5 histologically. The median length and width of clitoral body were 29 mm (range, 13-59 mm) and 9 mm (range, 5-14 mm), respectively. The glans was 8 mm (range, 5-12 mm) long and 4 mm (range, 3-10 mm) wide. The length of the crura was 50 mm (range, 25-68 mm), and the width at the anterior portion was 9 mm (range, 2-13 mm). The closest distance from the glans to the urethra was 25 mm (range, 14-37 mm) and from the clitoral body to the mid pubic arch was 29 mm (range, 14-46 mm). The widths of the dorsal nerve at the lateral crura and at the distal clitoral body were 3 mm (range, 2-4 mm) and 1 mm (range, 1-2 mm), respectively. The distance from the dorsal nerve as it emerged from the perineal membrane to the mid pubic arch was 34 mm (range, 20-48 mm) and to the posterior surface of the membrane was 20 mm (range, 8-31 mm). The dorsal nerve and artery of the clitoris coursed adjacent to the medial surface of the inferior pubic ramus surrounded by a dense fibrous capsule adherent to the periosteum. The nerve and artery then coursed deep to dense connective tissue layers, which were contiguous with the suspensory ligament and fascia of the clitoris. Histologic examination revealed the presence of erectile tissue in the clitoral body, crura, and vestibular bulbs, but such tissue was absent in the glans and labia minora. Nerve density analysis revealed statistically significant greater density in the dorsal compared with ventral half of the clitoral body. Although not statistically significant, there was increased nerve density in the distal compared to the proximal half of the labia minora. CONCLUSION: Precise knowledge of clitoral anatomy and associated neurovascular structures is essential to safely complete partial vulvectomies, clitoral and vulvar reconstructive procedures, anti-incontinence surgeries, and repair of obstetric lacerations. Understanding the range of anatomic variations and awareness of the areas of increased nerve density is important during counseling and surgical planning. Although the dorsal nerve of the clitoris courses deep to dense connective tissue layers, inadvertent injury may occur in the setting of deep dissection or suture placement. The dorsal nerve seems most vulnerable with surgical entry or lacerations that extend from the midline of the prepuce to the inferior pubic rami.

Gross and Histologic Anatomy of the Pelvic Ureter: Clinical Applications to Pelvic Surgery.

OBJECTIVE: To further evaluate relationships of the pelvic ureter to clinically relevant structures and to characterize the anatomy, histology, and nerve density of the distal ureter. METHODS: In this observational cadaveric study, 35 female cadavers were examined, 30 by gross dissections and five microscopically. Ureter length and segments of pelvic ureter were measured. Closest distances between the ureter and clinically relevant points were recorded. The distal pelvic ureter and surrounding parametrium were evaluated microscopically. Nerve density was analyzed using automated quantification of peripheral nerve immunostaining. Average measurements of nerve density in the anterior and posterior quadrants surrounding the ureter were statistically compared using a two-tailed t test. Descriptive statistics were used for analyses with distances reported as mean±SD (range). RESULTS: Gross dissections revealed ureter length of 26.3±1.4 (range 24-29) cm (right), 27.6±1.6 (25-30.5) cm (left). Lengths of ureter from pelvic brim to uterine artery crossover were 8.2±1.9 (4.4-11.5) cm (right), 8.5±1.5 (4.5-11.5) cm (left) and from crossover to bladder wall 3.3±0.7 (2.4-5.8) cm (right), 3.2±0.4 (2.6-4.1) cm (left). Intramural ureter length was 1.5±0.3 (1-2.2) cm (right) and 1.7±1.2 (0.8-2.5) cm (left). Distances from the ureter to uterine isthmus: median 1.7 (range 1-3.0) cm (right) and 1.7 (1.0-2.9) cm (left); lateral anterior vaginal fornix 1.5 (1.0-3.1) cm (right) and 1.7 (0.8-3.2) cm (left); lateral vaginal apex 1.3 (1.0-2.6) cm (right) and 1.2 (1.1-2.2) cm (left) were recorded. Microscopy demonstrated denser fibrovascularity posteromedial to the ureter. Peripheral nerve immunostaining revealed greater nerve density posterior to the distal ureter. CONCLUSION: Proximity of the ureter to the uterine isthmus and lateral anterior vagina mandates careful surgical technique and identification. The intricacy of tissue surrounding the distal ureter within the parametrium and the increased nerve density along the posterior distal ureter emphasizes the importance of avoiding extensive ureterolysis in this region.

Cell-Specific Loss of SNAP25 from Cortical Projection Neurons Allows Normal Development but Causes Subsequent Neurodegeneration.

Synaptosomal associated protein 25 kDa (SNAP25) is an essential component of the SNARE complex regulating synaptic vesicle fusion. SNAP25 deficiency has been implicated in a variety of cognitive disorders. We ablated SNAP25 from selected neuronal populations by generating a transgenic mouse (B6-Snap25tm3mcw (Snap25-flox)) with LoxP sites flanking exon5a/5b. In the presence of Cre-recombinase, Snap25-flox is recombined to a truncated transcript. Evoked synaptic vesicle release is severely reduced in Snap25 conditional knockout (cKO) neurons as shown by live cell imaging of synaptic vesicle fusion and whole cell patch clamp recordings in cultured hippocampal neurons. We studied Snap25 cKO in subsets of cortical projection neurons in vivo (L5-Rbp4-Cre; L6-Ntsr1-Cre; L6b-Drd1a-Cre). cKO neurons develop normal axonal projections, but axons are not maintained appropriately, showing signs of swelling, fragmentation and eventually complete absence. Onset and progression of degeneration are dependent on the neuron type, with L5 cells showing the earliest and most severe axonal loss. Ultrastructural examination revealed that cKO neurites contain autophagosome/lysosome-like structures. Markers of inflammation such as Iba1 and lipofuscin are increased only in adult cKO cortex. Snap25 cKO can provide a model to study genetic interactions with environmental influences in several disorders.

Gross and histologic relationships of the retropubic urethra to lateral pelvic sidewall and anterior vaginal wall in female cadavers: clinical applications to retropubic surgery.

BACKGROUND: Knowledge of the retropubic space anatomy is essential for safe entry and surgical applications within this space. OBJECTIVE: The objectives of this study were to examine the gross and histologic anatomy of the retropubic urethra, paraurethral tissue, and urethrovaginal space and to correlate findings to retropubic procedures. STUDY DESIGN: Anatomic relationships of the retropubic urethra were examined grossly in unembalmed female cadavers. Measured distances included: lateral urethral wall to arcus tendineus fascia pelvis at the level of urethrovesical junction and at 1 cm distal. Other measurements included retropubic urethral length and distances from internal urethral opening to each ureteric orifice. Microscopic examination was performed at the same levels examined grossly in separate nulliparous specimens. Descriptive statistics were used for data analyses. RESULTS: In all, 25 cadavers were examined grossly. Median distance from lateral urethral wall to arcus tendineus fascia pelvis at the level of urethrovesical junction was 25 mm (range, 13-38 mm). At 1 cm distal, the median distance from aforementioned structures was 14 mm (10-26 mm). Median length of the retropubic urethra was 23 mm (range 15-30 mm). Four nulliparous specimens, ages 12 weeks, and 34, 47, and 52 years, were examined histologically. No histologic evidence of a discrete fascial layer between bladder/urethra and anterior vagina was noted at any level examined. Tissue between the urethra and the pelvic sidewall skeletal muscle was composed of dense fibrous tissue, smooth muscle bundles, scant adipose tissue, blood vessels, and nerves. The smooth muscle fibers of the vaginal muscularis interdigitated with skeletal muscle fibers in the pelvic sidewall at both levels examined. No histologic evidence of "pubourethral ligaments" within the paraurethral tissue was noticed. CONCLUSION: A 2-cm "zone of safety" exists between the urethra and arcus tendineus fascia pelvis at the urethrovesical junction level. Suture or graft placement within this region should minimize injury to the urethra, pelvic sidewall muscles, and bladder. Knowledge that the shortest length of retropubic urethra was 1.5 cm and shortest urethra to arcus tendineus fascia pelvis distance was 1 cm highlights the importance of maintaining dissection and trocar entry site close to pubic bone to avoid bladder and/or urethral injury. Histologic analysis of paraurethral tissue supports the nonexistence of pubourethral ligaments.

Loss of Doc2-Dependent Spontaneous Neurotransmission Augments Glutamatergic Synaptic Strength.

Action potential-evoked vesicle fusion comprises the majority of neurotransmission within chemical synapses, but action potential-independent spontaneous neurotransmission also contributes to the collection of signals sent to the postsynaptic cell. Previous work has implicated spontaneous neurotransmission in homeostatic synaptic scaling, but few studies have selectively manipulated spontaneous neurotransmission without substantial changes in evoked neurotransmission to study this function in detail. Here we used a quadruple knockdown strategy to reduce levels of proteins within the soluble calcium-binding double C2 domain (Doc2)-like protein family to selectively reduce spontaneous neurotransmission in cultured mouse and rat neurons. Activity-evoked responses appear normal while both excitatory and inhibitory spontaneous events exhibit reduced frequency. Excitatory miniature postsynaptic currents (mEPSCs), but not miniature inhibitory postsynaptic currents (mIPSCs), increase in amplitude after quadruple knockdown. This increase in synaptic efficacy correlates with reduced phosphorylation levels of eukaryotic elongation factor 2 and also requires the presence of elongation factor 2 kinase. Together, these data suggest that spontaneous neurotransmission independently contributes to the regulation of synaptic efficacy, and action potential-evoked and spontaneous neurotransmission can be segregated at least partially on a molecular level.SIGNIFICANCE STATEMENT Action potential-evoked and spontaneous neurotransmission have been observed in nervous system circuits as long as methods have existed to measure them. Despite being well studied, controversy still remains about whether these forms of neurotransmission are regulated independently on a molecular level or whether they are simply a continuum of neurotransmission modes. In this study, members of the Doc2 family of presynaptic proteins were eliminated, which caused a reduction in spontaneous neurotransmission, whereas action potential-evoked neurotransmission remained relatively normal. This protein loss also caused an increase in synaptic strength, suggesting that spontaneous neurotransmission is able to communicate independently with the postsynaptic neuron and trigger downstream signaling cascades that regulate the synaptic state.

Selective molecular impairment of spontaneous neurotransmission modulates synaptic efficacy.

Recent studies suggest that stimulus-evoked and spontaneous neurotransmitter release processes are mechanistically distinct. Here we targeted the non-canonical synaptic vesicle SNAREs Vps10p-tail-interactor-1a (vti1a) and vesicle-associated membrane protein 7 (VAMP7) to specifically inhibit spontaneous release events and probe whether these events signal independently of evoked release to the postsynaptic neuron. We found that loss of vti1a and VAMP7 impairs spontaneous high-frequency glutamate release and augments unitary event amplitudes by reducing postsynaptic eukaryotic elongation factor 2 kinase (eEF2K) activity subsequent to the reduction in N-methyl-D-aspartate receptor (NMDAR) activity. Presynaptic, but not postsynaptic, loss of vti1a and VAMP7 occludes NMDAR antagonist-induced synaptic potentiation in an intact circuit, confirming the role of these vesicular SNAREs in setting synaptic strength. Collectively, these results demonstrate that spontaneous neurotransmission signals independently of stimulus-evoked release and highlight its role as a key regulator of postsynaptic efficacy.

Peripheral VH4+ plasmablasts demonstrate autoreactive B cell expansion toward brain antigens in early multiple sclerosis patients.

Plasmablasts are a highly differentiated, antibody secreting B cell subset whose prevalence correlates with disease activity in Multiple Sclerosis (MS). For most patients experiencing partial transverse myelitis (PTM), plasmablasts are elevated in the blood at the first clinical presentation of disease (known as a clinically isolated syndrome or CIS). In this study we found that many of these peripheral plasmablasts are autoreactive and recognize primarily gray matter targets in brain tissue. These plasmablasts express antibodies that over-utilize immunoglobulin heavy chain V-region subgroup 4 (VH4) genes, and the highly mutated VH4+ plasmablast antibodies recognize intracellular antigens of neurons and astrocytes. Most of the autoreactive, highly mutated VH4+ plasmablast antibodies recognize only a portion of cortical neurons, indicating that the response may be specific to neuronal subgroups or layers. Furthermore, CIS-PTM patients with this plasmablast response also exhibit modest reactivity toward neuroantigens in the plasma IgG antibody pool. Taken together, these data indicate that expanded VH4+ peripheral plasmablasts in early MS patients recognize brain gray matter antigens. Peripheral plasmablasts may be participating in the autoimmune response associated with MS, and provide an interesting avenue for investigating the expansion of autoreactive B cells at the time of the first documented clinical event.

Reelin mobilizes a VAMP7-dependent synaptic vesicle pool and selectively augments spontaneous neurotransmission.

Reelin is a glycoprotein that is critical for proper layering of neocortex during development as well as dynamic regulation of glutamatergic postsynaptic signaling in mature synapses. Here, we show that Reelin also acts presynaptically, resulting in robust rapid enhancement of spontaneous neurotransmitter release without affecting properties of evoked neurotransmission. This effect of Reelin requires a modest but significant increase in presynaptic Ca(2+) initiated via ApoER2 signaling. The specificity of Reelin action on spontaneous neurotransmitter release is encoded at the level of vesicular SNARE machinery as it requires VAMP7 and SNAP-25 but not synaptobrevin2, VAMP4, or vti1a. These results uncover a presynaptic regulatory pathway that utilizes the heterogeneity of synaptic vesicle-associated SNAREs and selectively augments action potential-independent neurotransmission.

The role of non-canonical SNAREs in synaptic vesicle recycling.

An increasing number of studies suggest that distinct pools of synaptic vesicles drive specific forms of neurotransmission. Interspersed with these functional studies are analyses of the synaptic vesicle proteome which have consistently detected the presence of so-called "non-canonical" SNAREs that typically function in fusion and trafficking of other subcellular structures within the neuron. The recent identification of certain non-canonical vesicular SNAREs driving spontaneous (e.g., VAMP7 and vti1a) or evoked asynchronous (e.g., VAMP4) release integrates and corroborates existing data from functional and proteomic studies and implies that at least some complement of non-canonical SNAREs resident on synaptic vesicles function in neurotransmission. Here, we discuss the specific roles in neurotransmission of proteins homologous to each member of the classical neuronal SNARE complex consisting of synaptobrevin2, syntaxin-1, and SNAP-25.