Pelvic Nerve Injury Causes a Rapid Decrease in Expression of Choline Acetyltransferase and Upregulation of c-Jun and ATF-3 in a Distinct Population of Sacral Preganglionic Neurons

Autonomic regulation of the urogenital organs is impaired by injuries sustained during pelvic surgery or compression of lumbosacral spinal nerves (e.g., cauda equina syndrome). To understand the impact of injury on both sympathetic and parasympathetic components of this nerve supply, we performed an experimental surgical and immunohistochemical study on adult male rats, where the structure of this complex part of the nervous system has been well defined. We performed unilateral transection of pelvic or hypogastric nerves and analyzed relevant regions of lumbar and sacral spinal cord, up to 4 weeks after injury. Expression of c-Jun, the neuronal injury marker activating transcription factor-3 (ATF-3), and choline acetyltransferase (ChAT) were examined. We found little evidence for chemical or structural changes in substantial numbers of functionally related but uninjured spinal neurons (e.g., in sacral preganglionic neurons after hypogastric nerve injury), failing to support the concept of compensatory events. The effects of injury were greatest in sacral cord, ipsilateral to pelvic nerve transection. Here, around half of all preganglionic neurons expressed c-Jun within 1 week of injury, and substantial ATF-3 expression also occurred, especially in neurons with complete loss of ChAT-immunoreactivity. There did not appear to be any death of retrogradely labeled neurons, in contrast to axotomy studies performed on other regions of spinal cord or sacral ventral root avulsion models. Each of the effects we observed occurred in only a subpopulation of preganglionic neurons at that spinal level, raising the possibility that distinct functional subgroups have different susceptibility to trauma-induced degeneration and potentially different regenerative abilities. Identification of the cellular basis of these differences may provide insights into organ-specific strategies for attenuating degeneration or promoting regeneration of these circuits after trauma

Correlative super-resolution fluorescence and electron microscopy using conventional fluorescent proteins in vacuo

Super-resolution light microscopy, correlative light and electron microscopy, and volume electron microscopy are revolutionising the way in which biological samples are examined and understood. Here, we combine these approaches to deliver super-accurate correlation of fluorescent proteins to cellular structures. We show that YFP and GFP have enhanced blinking properties when embedded in acrylic resin and imaged under partial vacuum, enabling in vacuo single molecule localisation microscopy. In conventional section-based correlative microscopy experiments, the specimen must be moved between imaging systems and/or further manipulated for optimal viewing. These steps can introduce undesirable alterations in the specimen, and complicate correlation between imaging modalities. We avoided these issues by using a scanning electron microscope with integrated optical microscope to acquire both localisation and electron microscopy images, which could then be precisely correlated. Collecting data from ultrathin sections also improved the axial resolution and signal-to-noise ratio of the raw localisation microscopy data. Expanding data collection across an array of sections will allow 3-dimensional correlation over unprecedented volumes. The performance of this technique is demonstrated on vaccinia virus (with YFP) and diacylglycerol in cellular membranes (with GFP)

Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure

Femtosecond laser preparation of resin embedded samples for correlative microscopy workflows in life sciences

Correlative multimodal imaging is a useful approach to investigate complex structural relations in life sciences across multiple scales. For these experiments, sample preparation workflows that are compatible with multiple imaging techniques must be established. In one such implementation, a fluorescently labeled region of interest in a biological soft tissue sample can be imaged with light microscopy before staining the specimen with heavy metals, enabling follow-up higher resolution structural imaging at the targeted location, bringing context where it is required. Alternatively, or in addition to fluorescence imaging, other microscopy methods, such as synchrotron x-ray computed tomography with propagation-based phase contrast or serial blockface scanning electron microscopy, might also be applied. When combining imaging techniques across scales, it is common that a volumetric region of interest (ROI) needs to be carved from the total sample volume before high resolution imaging with a subsequent technique can be performed. In these situations, the overall success of the correlative workflow depends on the precise targeting of the ROI and the trimming of the sample down to a suitable dimension and geometry for downstream imaging. Here, we showcase the utility of a femtosecond laser (fs laser) device to prepare microscopic samples (1) of an optimized geometry for synchrotron x-ray tomography as well as (2) for volume electron microscopy applications and compatible with correlative multimodal imaging workflows that link both imaging modalities

Functional and multiscale 3D structural investigation of brain tissue through correlative in vivo physiology, synchrotron microtomography and volume electron microscopy

Understanding the function of biological tissues requires a coordinated study of physiology and structure, exploring volumes that contain complete functional units at a detail that resolves the relevant features. Here, we introduce an approach to address this challenge: Mouse brain tissue sections containing a region where function was recorded using in vivo 2-photon calcium imaging were stained, dehydrated, resin-embedded and imaged with synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT). SXRT provided context at subcellular detail, and could be followed by targeted acquisition of multiple volumes using serial block-face electron microscopy (SBEM). In the olfactory bulb, combining SXRT and SBEM enabled disambiguation of in vivo-assigned regions of interest. In the hippocampus, we found that superficial pyramidal neurons in CA1a displayed a larger density of spine apparati than deeper ones. Altogether, this approach can enable a functional and structural investigation of subcellular features in the context of cells and tissues

The zebrafish as a novel model for the in vivo study of Toxoplasma gondii replication and interaction with macrophages.

Toxoplasma gondii is an obligate intracellular parasite capable of invading any nucleated cell. Three main clonal lineages (type I, II, III) exist and murine models have driven the understanding of general and strain-specific immune mechanisms underlying Toxoplasma infection. However, murine models are limited for studying parasite-leukocyte interactions in vivo, and discrepancies exist between cellular immune responses observed in mouse versus human cells. Here, we developed a zebrafish infection model to study the innate immune response to Toxoplasma in vivo By infecting the zebrafish hindbrain ventricle, and using high-resolution microscopy techniques coupled with computer vision-driven automated image analysis, we reveal that Toxoplasma invades brain cells and replicates inside a parasitophorous vacuole to which type I and III parasites recruit host cell mitochondria. We also show that type II and III strains maintain a higher infectious burden than type I strains. To understand how parasites are cleared in vivo, we further analyzed Toxoplasma-macrophage interactions using time-lapse microscopy and three-dimensional correlative light and electron microscopy (3D CLEM). Time-lapse microscopy revealed that macrophages are recruited to the infection site and play a key role in Toxoplasma control. High-resolution 3D CLEM revealed parasitophorous vacuole breakage in brain cells and macrophages in vivo, suggesting that cell-intrinsic mechanisms may be used to destroy the intracellular niche of tachyzoites. Together, our results demonstrate in vivo control of Toxoplasma by macrophages, and highlight the possibility that zebrafish may be further exploited as a novel model system for discoveries within the field of parasite immunity.This article has an associated First Person interview with the first author of the paper

Validation of a novel device to measure and provide feedback on sedentary behavior

Purpose. Pedometers, which enable self-monitoring of step counts, are effective in facilitating increases in physical activity. Similar devices which provide real-time feedback on sedentary (sitting) behavior are limited. This study aimed to develop and validate a novel device – the SitFIT – which could accurately measure and provide feedback on sedentary behavior and physical activity. Methods. The SitFIT is a tri-axial accelerometer, developed by PAL Technologies, which is worn in the front trouser pocket. This enables tracking of thigh inclination and therefore differentiation between sitting and upright postures, as well as tracking of step count. It has a display to provide user feedback. To determine the validity of the SitFIT for measuring sedentary behavior and step counts, 21 men, aged 30-65 years, with body mass index 26.6±3.9 kg.m-2 wore a SitFIT in a front trouser pocket and an activPAL accelerometer attached to their thigh for up to seven days. Outputs from the SitFIT were compared with the activPAL, which was assumed to provide gold-standard measurements of sitting and step counts. Results. Mean step counts were ~4% lower with the SitFIT than activPAL, with correlation between the two methods being very high (r=0.98) and no obvious bias from the line of equality (regression line: y=1.0035x+418.35). Mean sedentary time was ~5% higher with the SitFIT than activPAL, correlation between methods was high (r=0.84) and the equation of the regression line was close to the line of equality (y=0.8728x+38.445). Conclusions. The SitFIT has excellent validity for measurement of free-living step counts and sedentary time and therefore addresses a clear need for a device that can be used as a tool to provide feedback on sedentary behavior to facilitate behavior change

ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy

<p>Data for submission to Wellcome Open Research entitled "ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy".</p> <p>Data_ultraLM.tif is an image stack from the fluorescence microscope mounted on the ultramicrotome.</p> <p>Data_miniLM.tif is an image stack from the fluorescence microscope mounted in the SBF-SEM.</p> <p>Data_miniLM_EM.tif is an image stack from the SBF-SEM while the miniLM was in-situ.</p