Cell Body Volume Research Articles

Redox flow stacks with tubular cell design—Feasibility and performance

Redox flow batteries are a reliable option for the storage of energy from renewable resources and the all vanadium cell chemistry features the highest level of commercialization. The storage market demands further cost reductions. A tubular cell design can lower power specific costs by featuring reduced material needs and enabling the use of cost efficient extrusion processes of cell components. The feasibility of tubular all vanadium cells has already been shown in previous studies of the authors. In this study, we report on the progress of a recent cell generation and the integration of single cells into stacks. The performance of the current cell and stack generation has reached the region of planar cells with discharge current densities of idch> 300mAcm-2 at SOC≈0.5 and Ucell=0.8V as well as maximum power densities of P/Area> 300mWcm-2 with respect to the membrane area and of P/Volume> 750kWm-3 with respect to the cell body volume. The single cell performance turns out to be reproduceable with negligible losses at stack level in a 5p setup. In this way, the feasibility of tubular stacks is demonstrated. We claim the tubular approach to be transferrable to other cell chemistries.

Hydroxysafflor Yellow A and Tenuigenin Exhibit Neuroprotection Effects Against Focal Cerebral Ischemia Via Differential Regulation of JAK2/STAT3 and SOCS3 Signaling Interaction.

Numerous natural bioactive compounds extracted from Chinese medicines have been proved to be promising and potent agents in the treatment of ischemic stroke. Hydroxysafflor yellow A (HSYA), separated from Carthamus tinctorius, has increasingly attracted attention for its broad spectrum of pharmacological effects, especially of its neuroprotective action. Our previous studies revealed that HSYA plays significant beneficial roles in a dose-dependent manner in rats with focal cerebral ischemia. However, treatment with higher doses of HSYA appeared to bring about adverse reactions in the rats. In present study, we adopted tenuigenin (TEN), extracted from the Polygala tenuifolia root, in combination with HSYA to optimize the therapeutic strategy against ischemic stroke, and further explored the underlying mechanisms of action of the combination in vivo and in vitro. We firstly confirmed the pharmacological efficacies of co-treatment of HSYA and TEN in middle cerebral ischemia occlusion (MCAO) rats and observed the synergistic improvement of infarct volume, cerebral edema, and morphology of neuron cell body. Behavioral experiments indicated that combination of HSYA and TEN could synergistically improve motor and cognitive function in MCAO rats. We also observed increased viability and suppressed cell apoptosis after HSYA and TEN co-treatments in the oxygen-glucose deprivation/reperfusion (OGD/R) SH-SY5Y cells. Furthermore, JAK2/STAT3 and SOCS3 signaling interaction was demonstrated to be a critical responsor to the co-treatment of HSYA and TEN. In the subsequent experiments with silencing SOCS3 in OGD/R-exposed cells, we found that HSYA and TEN might suppress JAK2/STAT3 pathway through different regulatory mechanisms targeting SOCS3-negative feedback signaling. HSYA seemed to impose excessive activation of JAK2/STAT3 to trigger SOCS3-negative feedback signaling, while TEN appeared to provoke SOCS3 inhibitory feedback role directly to further attenuate JAK2-mediated signaling. Collectively, HSYA and TEN might modulate the crosstalk between JAK2/STAT3 and SOCS3 signaling pathways in different manners that eventually contributed to their synergistic therapeutic effects against cerebral ischemic stroke.

Volumetric Reconstruction of a Human Retinal Pigment Epithelial Cell Reveals Specialized Membranes and Polarized Distribution of Organelles.

Despite the centrality of the retinal pigment epithelium (RPE) in vision and retinopathy our picture of RPE morphology is incomplete. With a volumetric reconstruction of human RPE ultrastructure, we aim to characterize major membranous features including apical processes and their interactions with photoreceptor outer segments, basolateral infoldings, and the distribution of intracellular organelles. A parafoveal retinal sample was acquired from a 21-year-old male organ donor. With serial block-face scanning electron microscopy, a tissue volume from the inner-outer segment junction to basal RPE was captured. Surface membranes and complete internal ultrastructure of an individual RPE cell were achieved with a combination of manual and automated segmentation methods. In one RPE cell, apical processes constitute 69% of the total cell surface area, through a dense network of over 3000 terminal branches. Single processes contact several photoreceptors. Basolateral infoldings facing the choriocapillaris resemble elongated filopodia and comprise 22% of the cell surface area. Membranous tubules and sacs of endoplasmic reticulum represent 20% of the cell body volume. A dense basal layer of mitochondria extends apically to partly overlap electron-dense pigment granules. Pores in the nuclear envelope form a distinct pattern of rows aligned with chromatin. Specialized membranes at the apical and basal side of the RPE cell body involved in intercellular uptake and transport represent over 90% of the total surface area. Together with the polarized distribution of organelles within the cell body, these findings are relevant for retinal clinical imaging, therapeutic approaches, and disease pathomechanisms.

Stromal cell-derived factor 1 (SDF-1) increases the number of telocytes in exvivo and invitro assays.

Telocytes are interstitial cells that are present in various tissues, have long cytoplasmic projections known as telopodes, and are classified as CD34+ cells. Telopodes form extensive networks that permeate the stroma, and there is evidence that these networks connect several stromal cell types, giving them an important role in intercellular communication and the maintenance of tissue organisation. Data have also shown that these networks can be impaired and the number of telocytes reduced in association with many pathological conditions such as cancer and fibrosis. Thus, techniques that promote telocyte proliferation have become an important therapeutic target. In this study, exvivo and invitro assays were conducted to evaluate the impact on prostatic telocytes of SDF-1, a factor involved in the proliferation and migration of CD34+ cells. SDF-1 caused an increase in the number of telocytes in explants, as well as morphological changes that were possibly related to the proliferation of these cells. These changes involved the fusion of telopode segments, linked to an increase in cell body volume. Invitro assays also showed that SDF-1 enriched prostate stromal cells with telocytes. Altogether, the data indicate that SDF-1 may offer promising uses in therapies that aim to increase the number of telocytes. However, further studies are needed to confirm the efficiency of this factor in different tissues/pathological conditions.

Neuropathology and morphometry of dentate nucleus neurons in DYT1 brains: Cerebellar abnormalities in isolated dystonia.

Brain lesions exclusive to dystonia, or specific forms of it, such as isolated dystonia, have been rarely described. While the identification of distinctive intra- or extraneuronal abnormalities in childhood-onset generalized dystonia (DYT1) brains remains lacking, recent stereology-based findings demonstrated hypertrophy of neurons in the substantia nigra (SN) of DYT1-carriers manifesting dystonia (DYT1-manif) versus DYT1-carriers nonmanifesting dystonia (DYT1-nonmanif), and age-matched control subjects (C). Because other brain regions including the cerebellum (CRB) have been implicated in the pathomechanisms of dystonia, we investigated neurons of the dentate nucleus (DN), the "door-out" nucleus of the CRB. We performed systematic neuropathologic assessments and stereology-based measurements of 7 DN from DYT1-carriers (DYT1-DN; 4 DYT1-manif and 3 DYT1-nonmanif), and 5 age-matched control (C-DN) subjects. Data demonstrated larger cell body (+14.1%), nuclear (+10.6%), and nucleolar (+48.3%) volumes of DYT1-DN versus C-DN neurons. No differences in intra- and extracellular pathological indicators (β-amyloid, pTau, α-synuclein, Torsin1A, Negri, Bunina, Hirano, Marinesco, Nissl bodies, Buscaino bodies, granulovacuolar degeneration, or cerebrovascular lesions) were detected in DYT1-DN versus C-DN. Astroglial reactivity (GFAP) and microglial activation (IBA1) were observed in some DYT1-DNs. These novel findings confirm involvement of the DN and CRB in the pathogenesis of DYT1 and perhaps of other forms of isolated dystonia.

Toxicity of silver nanoparticles on Achromobacter denitrificans: effect of concentration, temperature and coexisting anions.

The indoor culture method was carried outto study the toxic effect of silver nanoparticles (AgNPs) on Achromobacter denitrificans.Specifically, the effects of AgNPsconcentration, temperature and coexisting anions were analyzed.The results showed that AgNPs exerted significant inhibition on the bacteria, which was closelycorrelated with its concentrationand temperature. Both the ammoniaoxidationand generationcapacityof Achromobacter denitrificansdecreased significantlywith an increase in AgNPsconcentration. Compared with the inhibition performance at 30 °C, NH4+-N generation rates decreased by 45.31% at 20 °C and 17.58% at 40 °C, respectively, revealing that too low or too high temperature induced toreduce the nitrogen conversion ability of Achromobacter denitrificans.While compared with temperature, the effect of coexistingions (Cl- and SO42-) was not significant(P > 0.05). Electron microscopy observations found that AgNPs non-specifically boundto the cells (content ranging from 0.04% to 0.10%) and actedon the cell surface structure, causingwrinkles,depressions, and ruptures on the surface of cell membranes, and leakage of substances in the membranes. AgNPs increasedthe rate of cell apoptosis and decreasedthe cell body volumemainly with short-term acute effects.

The repositioned drugs disulfiram/diethyldithiocarbamate combined to benznidazole: Searching for Chagas disease selective therapy, preventing toxicity and drug resistance.

Chagas disease (CD) affects at least 6 million people in 21 South American countries besides several thousand in other nations all over the world. It is estimated that at least 14,000 people die every year of CD. Since vaccines are not available, chemotherapy remains of pivotal relevance. About 30% of the treated patients cannot complete the therapy because of severe adverse reactions. Thus, the search for novel drugs is required. Here we tested the benznidazole (BZ) combination with the repositioned drug disulfiram (DSF) and its derivative diethyldithiocarbamate (DETC) upon Trypanosoma cruzi in vitro and in vivo. DETC-BZ combination was synergistic diminishing epimastigote proliferation and enhancing selective indexes up to over 10-fold. DETC was effective upon amastigotes of the BZ- partially resistant Y and the BZ-resistant Colombiana strains. The combination reduced proliferation even using low concentrations (e.g., 2.5 µM). Scanning electron microscopy revealed membrane discontinuities and cell body volume reduction. Transmission electron microscopy revealed remarkable enlargement of endoplasmic reticulum cisternae besides, dilated mitochondria with decreased electron density and disorganized kinetoplast DNA. At advanced stages, the cytoplasm vacuolation apparently impaired compartmentation. The fluorescent probe H2-DCFDA indicates the increased production of reactive oxygen species associated with enhanced lipid peroxidation in parasites incubated with DETC. The biochemical measurement indicates the downmodulation of thiol expression. DETC inhibited superoxide dismutase activity on parasites was more pronounced than in infected mice. In order to approach the DETC effects on intracellular infection, peritoneal macrophages were infected with Colombiana trypomastigotes. DETC addition diminished parasite numbers and the DETC-BZ combination was effective, despite the low concentrations used. In the murine infection, the combination significantly enhanced animal survival, decreasing parasitemia over BZ. Histopathology revealed that low doses of BZ-treated animals presented myocardial amastigote, not observed in combination-treated animals. The picrosirius collagen staining showed reduced myocardial fibrosis. Aminotransferase de aspartate, Aminotransferase de alanine, Creatine kinase, and urea plasma levels demonstrated that the combination was non-toxic. As DSF and DETC can reduce the toxicity of other drugs and resistance phenotypes, such a combination may be safe and effective.

Three-Dimensional Spatial Analyses of Cholinergic Neuronal Distributions Across The Mouse Septum, Nucleus Basalis, Globus Pallidus, Nucleus Accumbens, and Caudate-Putamen

Neuronal networks are regulated by three-dimensional spatial and structural properties. Despite robust evidence of functional implications in the modulation of cognition, little is known about the three-dimensional internal organization of cholinergic networks in the forebrain. Cholinergic networks in the forebrain primarily occur in subcortical nuclei, specifically the septum, nucleus basalis, globus pallidus, nucleus accumbens, and the caudate-putamen. Therefore, the present investigation analyzed the three-dimensional spatial organization of 14,000 cholinergic neurons that expressed choline acetyltransferase (ChAT) in these subcortical nuclei of the mouse forebrain. Point process theory and graph signal processing techniques identified three topological principles of organization. First, cholinergic interneuronal distance is not uniform across brain regions. Specifically, in the septum, globus pallidus, nucleus accumbens, and the caudate-putamen, the cholinergic neurons were clustered compared with a uniform random distribution. In contrast, in the nucleus basalis, the cholinergic neurons had a spatial distribution of greater regularity than a uniform random distribution. Second, a quarter of the caudate-putamen is composed of axonal bundles, yet the spatial distribution of cholinergic neurons remained clustered when axonal bundles were accounted for. However, comparison with an inhomogeneous Poisson distribution showed that the nucleus basalis and caudate-putamen findings could be explained by density gradients in those structures. Third, the number of cholinergic neurons varies as a function of the volume of a specific brain region but cell body volume is constant across regions. The results of the present investigation provide topographic descriptions of cholinergic somata distribution and axonal conduits, and demonstrate spatial differences in cognitive control networks. The study provides a comprehensive digital database of the total population of ChAT-positive neurons in the reported structures, with the x,y,z coordinates of each neuron at micrometer resolution. This information is important for future digital cellular atlases and computational models of the forebrain cholinergic system enabling models based on actual spatial geometry.

Three-dimensional imaging for the analysis of human epidermal melanocytes.

Three-dimensional (3-D) analysis of human epidermal melanocytes is required for deeper understanding of melanocytic disorders. The purpose of this study was to standardize 3-D imaging and quantification for the evaluation of epidermal melanocytes. The epidermal specimen was obtained using the suction blister method from a patient with melanocytic nevus on the forearm skin. Cutaneous ACT-PRESTO, the tissue-clearing and labeling technique, was subsequently performed. With the 3-D image analysis program, morphological reconstruction and quantification of selected perilesional and melanocytic nevus areas were possible. The region of melanocytic nevus showed higher numbers of total melanocytic dendrites and similar numbers of cell bodies compared with perilesional area. In addition, the mean area and volume of cell bodies increased in the melanocytic nevus area compared with the results in the perilesional area. The 3-D evaluation method of human epidermal melanocytes can be applied to investigate novel pathologies related to hyper- or hypo-pigmentary disorders.

P16.05 Implementation of a novel ex-vivo brain slice model to study human glioblastoma and glioma-associated microglia

Abstract BACKGROUND Glioblastoma multiforme is a highly malignant brain tumor with a devastating prognosis. Resection followed by radio-chemotherapy leads to an overall survival of only 15 months. Up to 40% of the tumor mass consist of tumor-associated microglia and macrophages (TAMs). These cells were shown to promote tumor growth and invasiveness in many murine glioma models. The interaction between TAMs and tumor cells is crucial for tumor progression and includes several known pathways. Still, murine glioma models only partially mirror the human tumor microenvironment. Several known genes, which are highly upregulated in human glioma and TAMs are only expressed in human tissue and not in mice. To further investigate some of these genes, we aimed at establishing a humanized ex-vivo brain slice model, in which human TAMs and human glioma cells can be studied in a standardized manner. MATERIAL AND METHODS We used 250 micrometer thick murine brain slices, which were depleted of intrinsic microglia by applying clodoronated liposomes. Next, we inoculated human glioma cells (originating from the cell lines mCherryU87, mCherryU251MG, mCherryLN229 and several patient derived cells lines) with or without human microglia derived from induced pluripotent stem cells (iPSCs). Slices were cultivated for 7 to 14 days. Next, we performed a detailed analysis of microglia morphology (sphericity, cell body volume, process length and branching pattern) and tumor volume. RESULTS Clodronation efficacy was high, depending on duration of treatment and length of cultivation. iPSCs and tumor cells integrated into the slice very well. The presence of tumor cells led to an increased sphericity of iPSC-dervied microglia and to an increased cell body volume. Branching pattern and process length did not differ between both conditions. Tumor volume was significantly larger when iPSC-derived microglia were present. This was found in various glioma cells lines and also in patient derived cells. CONCLUSION The newly established humanized ex-vivo brain slice system was shown to be feasible. The method successfully allows to study the interaction between human TAMs and tumor cells. Microglia foster tumor growth not only in murine glioma models, but also in a human paradigm. The humanized ex-vivo brain slice model therefore is the optimal basis to study the role human-specific genes in TAM-glioma interaction.

Neuropeptides as potential modulators of behavioral transitions in the ant Cataglyphis nodus.

Age-related behavioral plasticity is a major prerequisite for the ecological success of insect societies. Although ecological aspects of behavioral flexibility have been targeted in many studies, the underlying intrinsic mechanisms controlling the diverse changes in behavior along the individual life history of social insects are not completely understood. Recently, the neuropeptides allatostatin-A, corazonin, and tachykinin have been associated with the regulation of behavioral transitions in social insects. Here, we investigated changes in brain localization and expression of these neuropeptides following major behavioral transitions in Cataglyphis nodus ants. Our immunohistochemical analyses in the brain revealed that the overall branching pattern of neurons immunoreactive (ir) for the three neuropeptides is largely independent of the behavioral stages. Numerous allatostatin-A- and tachykinin-ir neurons innervate primary sensory neuropils and high-order integration centers of the brain. In contrast, the number of corazonergic neurons is restricted to only four neurons per brain hemisphere with cell bodies located in the pars lateralis and axons extending to the medial protocerebrum and the retrocerebral complex. Most interestingly, the cell-body volumes of these neurons are significantly increased in foragers compared to freshly eclosed ants and interior workers. Quantification of mRNA expression levels revealed a stage-related change in the expression of allatostatin-A and corazonin mRNA in the brain. Given the presence of the neuropeptides in major control centers of the brain and the neurohemal organs, these mRNA-changes strongly suggest an important modulatory role of both neuropeptides in the behavioral maturation of Cataglyphis ants.

TAMI-02. ALTERATIONS IN C-X3-C MOTIF CHEMOKINE RECEPTOR 1 (CX3CR1) EXPRESSION INFLUENCE MICROGLIAL AND MACROPHAGE RESPONSE IN DIFFERENT STEPS OF CEREBRAL METASTASIS FORMATION

Abstract Metastasis to the brain is a frequent complication in lung cancer and is still associated with a dismal prognosis. Current treatment strategies not only target tumor cells but also focus on cells of the tumor microenvironment like tumor associated microglia/macrophages (TAMs). The interactions between tumor cells and TAMs during different steps of cerebral metastasis formation of lung cancer brain metastasis are poorly characterized. Moreover, the role of CX3CR1 in this process remains unclear. We established a syngeneic cerebral metastasis mouse model by combining a chronic cranial window and two-photon laser scanning microscopy (TPLSM), which allows the tracking of single fluorescent metastasizing tumor cells and the tumor microenvironment on a cellular resolution in vivoover time for a period of weeks. Transgenic CX3CR1 proficient and deficient mice (CX3CRGFP/wt and CX3CR1GFP/GFP) were injected with red fluorescent Lewis lung carcinoma cells. During different steps of metastasis formation (extravasation, formation of micro- and macrometastasis) the density and cell body volume of TAMs, their interaction with tumor cells and possible influence on the fate of single metastatic tumor cells were investigated using serial TPLSM. We found that during metastasis formation TAM density was significantly lower in CX3CR1 deficient mice. However, activation as assessed by TAM morphology did not differ in the absence of CX3CR1. Strikingly, CX3CR1 deficiency was associated with a significant increase of tumor cells successfully extravasating the cerebral vasculature. However, subsequent steps (mirco- and macrometastasis formation) were observed less frequent in CX3CR1 deficient mice. In summary, our results highlight a complex role of CX3CR1 for TAMs during cerebral metastasis formation, indicating anti-tumorous properties of CX3CR1 at early steps and possible pro-tumorous effects at later stages (micro- and macrometastasis formation).

Disrupted Coordination of Hypoglossal Motor Control in a Mouse Model of Pediatric Dysphagia in DiGeorge/22q11.2 Deletion Syndrome.

We asked whether the physiological and morphologic properties of hypoglossal motor neurons (CNXII MNs) that innervate protruder or retractor tongue muscles are disrupted in neonatal LgDel mice that carry a heterozygous deletion parallel to that associated with DiGeorge/22q11.2 deletion syndrome (22q11.2DS). Disrupted coordination of tongue movement in LgDel mouse pups may contribute to suckling, feeding, and swallowing (S/F/S) disruptions that parallel pediatric dysphagia in infants and toddlers with 22q11.2DS. Using an in vitro rhythmically active medullary slice preparation, we found spontaneous firing as well as IPSC frequency differed significantly in neonatal LgDel versus wild-type (WT) protruder and retractor CNXII MNs that were identified by retrograde tracing from their target muscles. In response to respiration-related activity, initiation and decay of transiently increased firing in WT protruder MNs is delayed in LgDel, accompanied by altered excitatory/inhibitory (E/I) balance. In addition, LgDel retractor MNs have a transient increase in firing with diminished IPSC frequency that is not seen in WT. There were no significant differences in cell body volume of either XII class in WT and LgDel. Sholl analysis showed the total numbers of dendritic intersections (at 50- and 90-μm radii from the cell soma) were significantly greater for LgDel versus WT retractor MNs. Thus, the physiological, synaptic and cellular properties of distinct classes of CNXII MNs that coordinate tongue movement in neonatal WT mice are altered in LgDel. Such changes could contribute to sub-optimal coordination of S/F/S that underlies pediatric dysphagia in 22q11.2DS.

Atlas of Human Retinal Pigment Epithelium Organelles Significant for Clinical Imaging.

PurposeTo quantify organelles impacting imaging in the cell body and intact apical processes of human retinal pigment epithelium (RPE), including melanosomes, lipofuscin–melanolipofuscin (LM), mitochondria, and nuclei.MethodsA normal perifovea of a 21-year-old white male was preserved after rapid organ recovery. An aligned image stack was generated using serial block-face scanning electron microscopy and was annotated by expert readers (TrakEM, ImageJ). Acquired measures included cell body and nuclear volume (n = 17); organelle count in apical processes (n = 17) and cell bodies (n = 8); distance of cell body organelles along a normalized apical–basal axis (n = 8); and dimensions of organelle-bounding boxes in apical processes in selected subsamples of cell bodies and apical processes.ResultsIn 2661 sections through 17 cells, apical processes contained 65 ± 24 melanosomes in mononucleate (n = 15) and 131 ± 28 in binucleate cells (n = 2). Cell bodies contained 681 ± 153 LM and 734 ± 170 mitochondria. LM was excluded from the basal quartile, and mitochondria from the apical quartile. Lengths of melanosomes, LM, and mitochondria, respectively were 2305 ± 528, 1320 ± 574, and 1195 ± 294 nm. The ratio of cell body to nucleus volume was 4.6 ± 0.4. LM and mitochondria covered 75% and 63%, respectively, of the retinal imaging plane.ConclusionsAmong RPE signal sources for optical coherence tomography, LM and mitochondria are the most numerous reflective cell body organelles. These and our published data show that most melanosomes are in apical processes. Overlapping LM and previously mitochondria cushions may support multiple reflective bands in cell bodies. This atlas of subcellular reflectivity sources can inform development of advanced optical coherence tomography technologies.

Effect of a binge-like dosing regimen of methamphetamine on dopamine levels and tyrosine hydroxylase expressing neurons in the rat brain

Methamphetamine, an amphetamine derivative, is a powerful psychomotor stimulant and commonly used drug of abuse. This study examined the effect of binge-like methamphetamine (MA) dosing (4 × 4 mg/kg, s.c., 2 h apart) on regional dopamine and dopaminergic metabolite levels in rat brain at a range of early time points after final dose (2–48 h). Body temperature was elevated when measured 2 h after the last dose. MA increased dopamine levels in the frontal cortex 2 and 24 h after the last dose. The dopamine level was also increased in the amygdala at 24 h. No change was observed in the striatum at any time point, but levels of the dopamine metabolite DOPAC were markedly reduced at 24 and 48 h. Tyrosine hydroxylase expression is induced downstream of dopamine activity, and it is the rate limiting enzyme in dopamine synthesis. The effect of MA binge-like dosing on the volume of tyrosine hydroxylase containing cell bodies and the area fraction of tyrosine hydroxylase containing fibres was also assessed. MA increased the area fraction of tyrosine hydroxylase fibres in the frontal cortex and reduced the volume of tyrosine hydroxylase containing cell bodies 2 h after last dose in the ventral tegmental area and the substantia nigra. An increase in cell body volume in the substantia nigra was observed 48 h after treatment. These findings collectively highlight the importance of the dopaminergic system in methamphetamine induced effects, identify the frontal cortex, amygdala and striatum as key regions that undergo early changes in response to binge-like methamphetamine dosing and provide evidence of time-dependent effects on the cell bodies and fibres of tyrosine hydroxylase expressing neurons.

Hypertrophy of nigral neurons in Torsin1A deletion (DYT1) carriers manifesting dystonia

ObjectiveTo individuate morphometric changes and prevalent types of intraneuronal inclusions in nigral neurons of DYT1 dystonia autopsy-brains. MethodsUsing precise methods of quantification, such as unbiased stereology, we measured cellular and subcellular volumes of neuromelanin-containing (pigmented) neurons in the substantia nigra (SN) of DYT1 carriers with and without manifestation of generalized dystonia (manif-DYT1 and non-manif-DYT1, respectively), non-DYT1 carriers manifesting generalized dystonia (manif-non-DYT1) patients, and age-matched control subjects (controls). A total of four DYT1 carriers (two manif-DYT1 and two non-manif-DYT1), six manif-non-DYT1 carriers, and six controls autopsy-brains were available for these neuropathological-morphometric analyses. The search of brain lesions was performed for: tau neurofibrillary tangles and neurites, extracellular β-amyloid deposits, Lewy bodies and neurites, TorsinA, Laminin A + C, Ubiquitin, p62, pTDP43 intraneuronal inclusions; and Negri, Bunina, Hirano, Marinesco, Nissl, and Buscaino bodies. ResultsAn increased mean cell body, nuclear, and nucleolar volume of nigral neurons in manif-DYT1 vs. non-manif-DYT1 (p < 0.0001), manif-non-DYT1 (p < 0.0001), and controls (p < 0.00001) was found. Increased nuclear and nucleolar volumes in manif-non-DYT1 vs. controls were also found. None of the considered possible intraneuronal lesions were more frequent or prevalent in nigral neurons of manif-DYT1 vs. all the other groups. ConclusionsUnbiased stereology-based measurements of nigral neurons enlargement in manif-DYT1 in the absence of intraneuronal inclusions or neurodegenerative processes, is novel. These findings suggest distinct pathogenetic mechanisms between manif-DYT1 vs. non-manif-DYT1 and manif-non-DYT1 dystonia, especially in terms of possible nigral dopaminergic abnormalities. These data could open new pathophysiologic views on specific dopamino-associated pathomechanisms related to the clinical manifestation of generalized dystonia.

From two to three dimensions: The importance of the third dimension for evaluating the limits to neuronal miniaturization in insects.

Most studies dealing with the limits to miniaturization in insect brains have until now relied on information based on data collected in two dimensions: either histological sections imaged by light microscopy, or electron micrographs of single ultrathin sections imaged by transmission electron microscopy (TEM). To test the validity of transferring information gained from two-dimensional images to the third dimension, we examined a 3D image stack from serial-section TEM (ssTEM) of the optic neuropiles of the miniature parasitic wasp Trichogramma brassicae (Bezdenko, 1968). We reinvestigated the proposed lower limit of 2 µm for the diameters of neuronal somata and found average volumes of 6.5 μm3 for lamina cells and 3.8 μm3 for medulla cells. We likewise found a limiting factor for the volume of nuclei, which averages 41.9% and 49.2% of the cell body volume, respectively, but that in turn the compactness of heterochromatin was not a limiting factor in the minimal volume of the nuclei. Finally, we also found a minimum axon diameter of 98 nm that could nevertheless accommodate axoplasmic mitochondria. Incorporating the third dimension thus proves critically important in avoiding volumetric misinterpretations of these values. We discuss the limitations of analyzing the effects of miniaturization from profile data of neurons and demonstrate that miniaturization within the nervous system can lie beyond previously described limits and in some cases is already present in the optic lobe neurons of T. brassicae.

Degeneration of the osteocyte network in the C57BL/6 mouse model of aging.

Age-related bone loss and associated fracture risk are major problems in musculoskeletal health. Osteocytes have emerged as key regulators of bone mass and as a therapeutic target for preventing bone loss. As aging is associated with changes in the osteocyte lacunocanalicular system, we focused on the responsible cellular mechanisms in osteocytes. Bone phenotypic analysis was performed in young-(5mo) and aged-(22mo) C57BL/6 mice and changes in bone structure/geometry correlated with alterations in osteocyte parameters determined using novel multiplexed-3D-confocal imaging techniques. Age-related bone changes analogous to those in humans were observed, including increased cortical diameter, decreased cortical thickness, reduced trabecular BV/TV and cortical porosities. This was associated with a dramatic reduction in osteocyte dendrite number and cell density, particularly in females, where osteocyte dendricity decreased linearly from 5, 12, 18 to 22mo and correlated significantly with cortical bone parameters. Reduced dendricity preceded decreased osteocyte number, suggesting dendrite loss may trigger loss of viability. Age-related degeneration of osteocyte networks may impair bone anabolic responses to loading and gender differences in osteocyte cell body and lacunar fluid volumes we observed in aged mice may lead to gender-related differences in mechanosensitivity. Therapies to preserve osteocyte dendricity and viability may be beneficial for bone health in aging.

Rapid adaptive remote focusing microscope for sensing of volumetric neural activity.

The ability to record neural activity in the brain of a living organism at cellular resolution is of great importance for defining the neural circuit mechanisms that direct behavior. Here we present an adaptive two-photon microscope optimized for extraction of neural signals over volumes in intact Drosophila brains, even in the presence of specimen motion. High speed volume imaging was made possible through reduction of spatial resolution while maintaining the light collection efficiency of a high resolution, high numerical aperture microscope. This enabled simultaneous recording of odor-evoked calcium transients in a defined volume of mushroom body Kenyon cell bodies in a live fruit fly.

Structural changes in the inner ear over time studied in the experimentally deafened guinea pig.

Today a cochlear implant (CI) may significantly restore auditory function, even for people with a profound hearing loss. Because the efficacy of a CI is believed to depend mainly on the remaining population of spiral ganglion neurons (SGNs), it is important to understand the timeline of the degenerative process of the auditory neurons following deafness. Guinea pigs were transtympanically deafened with neomycin, verified by recording auditory brainstem responses (ABRs), and then sacrificed at different time points. Loss of SGNs as well as changes in cell body and nuclear volume were estimated. To study the effect of delayed treatment, a group of animals that had been deaf for 12 weeks was implanted with a stimulus electrode mimicking a CI, after which they received a 4‐week treatment with glial cell‐derived neurotrophic factor (GDNF). The electrical responsiveness of the SGNs was measured by recording electrically evoked ABRs. There was a rapid degeneration during the first 7 weeks, shown as a significant reduction of the SGN population. The degenerative process then slowed, and there was no difference in the amount of remaining neurons between weeks 7 and 18. © 2016 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.