Electron Microscopic Level Research Articles

Histological and Electron Microscope Staining for the Identification of Elastic Fiber Networks.

Elastic fibers are a major component of the extracellular matrix and are present in many tissues. Routine histology and standard electron microscopy procedures often do not allow for clear identification of elastic fibers making their organization and ultrastructure difficult to study. In this paper, we describe staining methods and procedures to enhance the contrast of elastin at both the light and electron microscope levels.

Light and transmission electron microscopy of Cepedea longa (Opalinidae) from Fejervarya limnocharis.

Cepedea longa Bezzenberger, 1904, collected from Fejervarya limnocharis (Amphibia, Anura, Ranidae) from Honghu Lake, Hubei Province, China in May–July 2016, is described at both light and transmission electron microscope levels. This is the first electron microscopic study of this species. Cepedea longa possesses a developed fibrillar skeletal system, composed of longitudinal fibrillar bands and transversal fibrils as well as numerous thin microfibrils dispersed in the endoplasm, which may play an important role in morphogenesis and offer some resilience to deformations of the cell. Longitudinal microfibrils are polarizing elements of kineties, bordering the somatic kineties on the left side and possibly responsible for kinetosome alignment. Two types of vesicles exist in the somatic cortex: globular endocytotic vesicles and flattened exocytotic vesicles. As to the nuclei of C. longa, a thick microfibrillar layer was observed to attach to the cytoplasmic face of the nuclear envelope. This fact suggests no necessary connection between the presence of this microfibrillar layer and the number of nuclei. In addition, some unknown tightly-packed microtubular structures in the nucleoplasm were observed for the first time in opalinids; neither their nature nor physiological significance is known. A detailed list of all reported Cepedea species is included.

Reduced noradrenergic innervation of ventral midbrain dopaminergic cell groups and the subthalamic nucleus in MPTP-treated parkinsonian monkeys

There is anatomical and functional evidence that ventral midbrain dopaminergic (DA) cell groups and the subthalamic nucleus (STN) receive noradrenergic innervation in rodents, but much less is known about these interactions in primates. Degeneration of NE neurons in the locus coeruleus (LC) and related brainstem NE cell groups is a well-established pathological feature of Parkinson's disease (PD), but the development of such pathology in animal models of PD has been inconsistent across species and laboratories. We recently demonstrated 30–40% neuronal loss in the LC, A5 and A6 NE cell groups of rhesus monkeys rendered parkinsonian by chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this study, we used dopamine-beta-hydroxylase (DβH) immunocytochemistry to assess the impact of this neuronal loss on the number of NE terminal-like varicosities in the substantia nigra pars compacta (SNC), ventral tegmental area (VTA), retrorubral field (RRF) and STN of MPTP-treated parkinsonian monkeys. Our findings reveal that the NE innervation of the ventral midbrain and STN of normal monkeys is heterogeneously distributed being far more extensive in the VTA, RRF and dorsal tier of the SNC than in the ventral SNC and STN. In parkinsonian monkeys, all regions underwent a significant (~50–70%) decrease in NE innervation. At the electron microscopic level, some DβH-positive terminals formed asymmetric axo-dendritic synapses in VTA and STN. These findings demonstrate that the VTA, RRF and SNCd are the main ventral midbrain targets of ascending NE inputs, and that these connections undergo a major break-down in chronically MPTP-treated parkinsonian monkeys. This severe degeneration of the ascending NE system may contribute to the pathophysiology of ventral midbrain and STN neurons in PD.

Cellular and Subcellular Localization of the RGS7/Gβ5/R7BP Complex in the Cerebellar Cortex.

A member of regulator of G-protein signaling family, RGS7, is an essential modulator of signaling through GABAB receptors. RGS7 functions as a macromolecular complex with type 5 G protein β (Gβ5) and R7 binding protein (R7BP) to control the localization and function of the resultant heterotrimeric complexes. Here, we used co-immunoprecipitation, in situ hybridization, histoblot and immunohistochemical techniques at the light and electron microscopic level to advance understanding of RGS7-Gβ5-R7BP complexes in the central nervous system, focusing on distinct neuronal populations in the cerebellar cortex. Histoblot analysis showed that RGS7, Gβ5 and R7BP proteins were widely expressed in the brain, with mostly an overlapping pattern and showing a high expression level in the molecular layer of the cerebellar cortex. Co-immunoprecipitation experiments established that the RGS7/Gβ5 forms complexes with R7BP in the cerebellum. At the cellular level, RGS7 and R7BP mRNAs were expressed at the highest level in Purkinje cells (PCs) and Golgi cells, and at low levels in granule cells. Immunohistochemistry confirmed that labeling for RGS7, Gβ5 and R7BP were present in the three neuronal populations and concentrated in dendrites and spines. At the electron microscopic level, immunolabeling for RGS7, Gβ5 and R7BP proteins was found both at postsynaptic and presynaptic sites and showed similar distribution patterns. Immunoreactivity for the three proteins was mostly localized along the extrasynaptic plasma membrane of dendritic shafts and spines of PCs and to a lesser extent, in axon terminals (AT) establishing excitatory synapses. Quantitative analysis of immunogold particles for RGS7, Gβ5 and R7BP revealed that they are non-uniformly distributed along the surface of PCs, and show enrichment around excitatory synapses on dendritic spines. We further report that deletion of R7BP in mice reduced the targeting of both RGS7 and Gβ5 to the plasma membrane. Altogether, these data support the existence of macromolecular complexes composed of RGS7-Gβ5-R7BP in PCs. The location at post- and pre-synaptic sites in PCs spines-parallel fiber synapses suggests their involvement in the modulation of glutamatergic neurotransmission in the cerebellar cortex.

Immunohistochemical localization of SNARE core proteins in intrapulpal and intradentinal nerve fibers of rat molar teeth

ObjectiveThe present study was designed to elucidate whether three soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) core proteins, syntaxin-1, synaptosomal-associated protein of 25kDa (SNAP-25), and vesicle-associated membrane protein-2 (VAMP-2), are present in the dental pulp of the rat molar at both the light and electron microscopic levels. DesignImmunohistochemistry for protein gene product 9.5 (PGP 9.5), a pan-neuronal marker, syntaxin-1, SNAP-25, and VAMP-2 was performed on decalcified rat molars for light and electron microscopic analyses. Double-immunolabeling of PGP 9.5 and the SNARE core proteins, as well as combinations of the SNARE core proteins, was also carried out. ResultsPGP 9.5-immunoreactive nerve fibers ran toward the coronal region, ramified at the subodontoblast layer, and formed the subodontoblastic nerve plexus. Most nerve fibers penetrated the predentin and dentin along the dentinal tubules. Most, if not all, nerve fibers displayed immunoreactivity for syntaxin-1, SNAP-25, and VAMP-2. Immunoelectron microscopic analyses confirmed the presence of immunoreactivity for the SNARE core proteins within the intradental axonal elements. ConclusionsThe present findings suggest that, since SNARE core proteins participate in the docking and exocytosis of synaptic vesicles in the central nervous system, they may contribute to vesicle exocytosis from the dental nerve fibers even though there are no apparent synapses.

Histological analysis of arteriovenous anastomosis-like vessels established in the corpus luteum of cows during luteolysis.

BackgroundThe mechanisms regulating the function and regression of the corpus luteum (CL) have not yet been elucidated in detail. The regressed CL of cows was previously reported to be filled with unusual vessels like arteriovenous anastomosis (AVA); however how these vessels are being established during luteolysis remains unknown.MethodsThe bovine CL at different luteal stages and regressing bovine CL induced by prostaglandin F2α (PGF) were histologically analyzed using light and electron microscopic levels. The changes in mRNA expression of genes encoding α-smooth muscle actin (SMA; Acta2) and transforming growth factor β1 (Tgfb1) in luteal tissues were analyzed by quantitative RT-PCR.ResultsAVA-like vessels appeared in the regressed CL with a diameter less than 1.5 cm in which no functional luteal cells and macrophages were observed. Epithelioid cells in the AVA-like vessel wall were immunoreactive for SMA, and the lumen of the vessels were narrow. Immunoreaction for SMA was found in the tunica media of typical arteries and arterioles, and pericytes around capillary vessel. Cells with elongated cytoplasmic processes ―resident fibroblasts expressing vimentin― distributed in the CL parenchyma without any association with blood vessels are also immunoreactive for SMA, and accumulated around arteries and arterioles during the late-luteal stage. In the regressed CL, walls of arteries and arterioles consisted of more than two layers of epithelioid cells positive for both SMA and desmin, suggesting that they are myofibroblasts transformed from fibroblasts. The percentage of the area positive for SMA and the mRNA expression of Acta2 were significantly increased in the regressed CL; however, they did not alter when a luteolytic dose of PGF was injected in vivo and collected within 24 h after the injection. On the other hand, Tgfb1, a known regulator for myofibroblast transformation, was significantly increased in PGF-induced regressing CL as well as in the CL during the late-luteal stage.ConclusionsSMA-positive myofibroblasts accumulates around the arteries and arterioles to form AVA-like vessels during luteolysis in cows. PGF indirectly regulates myofibroblast transformation through enhancing the expression of TGFβ1. These peculiar AVA-like vessels may be involved in the regulation of blood flow in the bovine CL during luteolysis.

Developmental Injury to the Cerebellar Cortex Following Hydroxyurea Treatment in Early Postnatal Life: An Immunohistochemical and Electron Microscopic Study.

Postnatal development of the cerebellar cortex was studied in rats administered with a single dose (2mg/g) of the cytotoxic agent hydroxyurea (HU) on postnatal day (P) 9 and collected at appropriate times ranging from 6h to 45days. Quantification of several parameters such as the density of pyknotic, mitotic, BrdU-positive, and vimentin-stained cells revealed that HU compromises the survival of the external granular layer (EGL) cells. Moreover, vimentin immunocytochemistry revealed overexpression and thicker immunoreactive glial processes in HU-treated rats. On the other hand, we also show that HU leads to the activation of apoptotic cellular events, resulting in a substantial number of dying EGL cells, as revealed by TUNEL staining and at the electron microscope level. Additionally, we quantified several features of the cerebellar cortex of rats exposed to HU in early postnatal life and collected in adulthood. Data analysis indicated that the analyzed parameters were less pronounced in rats administered with this agent. Moreover, we observed several alterations in the cerebellar cortex cytoarchitecture of rats injected with HU. Anomalies included ectopic placement of Purkinje cells and abnormities in the dendritic arbor of these macroneurons. Ectopic granule cells were also found in the molecular layer. These findings provide a clue for investigating the mechanisms of HU-induced toxicity during the development of the central nervous system. Our results also suggest that it is essential to avoid underestimating the adverse effects of this hydroxylated analog of urea when administered during early postnatal life.

The karyosphere capsule in oocytes of hibernating frogs Rana temporaria contains actin, lamins, and SnRNP

The nuclei of late vitellogenic oocytes of hibernating frogs Rana temporaria were studied. During this period of oogenesis, chromosomes are inactivated and surrounded by a fibrillar karyosphere capsule. Formation of the karyosphere capsule in grass frog oocytes has been investigated in detail at the light and electron microscopic levels, but the molecular composition of the capsule remains uncertain. Immunofluorescent staining of whole-mount preparations of oocyte nuclei revealed that the karyosphere capsule contained actin, lamins A, C, and B and snRNPs proteins. A putative role of these proteins in formation of the karyosphere capsule is discussed.

In vivo Whole-Cell Recordings Combined with Electron Microscopy Reveal Unexpected Morphological and Physiological Properties in the Lateral Nucleus of the Trapezoid Body in the Auditory Brainstem.

The lateral nucleus of the trapezoid body (LNTB) is a prominent nucleus in the superior olivary complex in mammals including humans. Its physiology in vivo is poorly understood due to a paucity of recordings. It is thought to provide a glycinergic projection to the medial superior olive (MSO) with an important role in binaural processing and sound localization. We combined in vivo patch clamp recordings with labeling of individual neurons in the Mongolian gerbil. Labeling of the recorded neurons allowed us to relate physiological properties to anatomy at the light and electron microscopic level. We identified a population of quite dorsally located neurons with surprisingly large dendritic trees on which most of the synaptic input impinges. In most neurons, one or more of these dendrites run through and are then medial to the MSO. These neurons were often binaural and could even show sensitivity to interaural time differences (ITDs) of stimulus fine structure or envelope. Moreover, a subpopulation showed enhanced phase-locking to tones delivered in the tuning curve tail. We propose that these neurons constitute the gerbil main LNTB (mLNTB). In contrast, a smaller sample of neurons was identified that was located more ventrally and that we designate to be in posteroventral LNTB (pvLNTB). These cells receive large somatic excitatory terminals from globular bushy cells. We also identified previously undescribed synaptic inputs from the lateral superior olive. pvLNTB neurons are usually monaural, display a primary-like-with-notch response to ipsilateral short tones at CF and can phase-lock to low frequency tones. We conclude that mLNTB contains a population of neurons with extended dendritic trees where most of the synaptic input is found, that can show enhanced phase-locking and sensitivity to ITD. pvLNTB cells, presumed to provide glycinergic input to the MSO, get large somatic globular bushy synaptic inputs and are typically monaural with short tone responses similar to their primary input from the cochlear nucleus.

Functional conservation of the lncRNA NEAT1 in the ancestrally diverged marsupial lineage: Evidence for NEAT1 expression and associated paraspeckle assembly during late gestation in the opossum Monodelphis domestica

ABSTRACTLong non-coding RNAs (lncRNAs) are widely expressed and play various roles in cell homeostasis. However, because of their low conservation at the sequence level, recapitulating lncRNA evolutionary history is often challenging. While performing an ultrastructural analysis of viral particles present in uterine glands of gestating opossum females, we serendipitously noticed the presence of numerous structures similar to paraspeckles, nuclear bodies which in human and mouse cells are assembled around an architectural NEAT1/MENϵ/β lncRNA. Here, using an opossum kidney (OK) cell line, we confirmed by immuno-electron microscopy the presence of paraspeckles in marsupials. We then identified the orthologous opossum NEAT1 gene which, although poorly conserved at the sequence level, displays NEAT1 characteristic features such as short and long isoforms expressed from a unique promoter and for the latter an RNase P cleavage site at its 3′-end. Combining tissue-specific qRT-PCR, in situ hybridization at the optical and electron microscopic levels, we show that (i) NEAT1 is paraspeckle-associated in opossum (ii) NEAT1 expression is strongly induced in late gestation in uterine/placental extracts (iii) NEAT1 induction occurs in the uterine gland nuclei in which paraspeckles were detected. Finally, treatment of OK cells with proteasome inhibitors induces paraspeckle assembly, as previously observed in human cells. Altogether, these results demonstrate that paraspeckles are tissue-specific, stress-responding nuclear bodies in marsupials, illustrating their structural and functional continuity over 200 My of evolution throughout the mammalian lineage. In contrast, the rapid evolution of the NEAT1 transcripts highlights the relaxed constraint that, despite functional conservation, is exerted on this lncRNA.

Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa.

Freezing of boar spermatozoa includes the cryoprotectant glycerol, but renders low cryosurvival, owing to major changes in osmolarity during freezing/thawing. We hypothesize that aquaporins (AQPs) 7 and 9 adapt their membrane domain location to these osmotic challenges, thus maintaining sperm homeostasis. Western blotting (WB) and immunocytochemistry (ICC) at light and electron microscope levels with several commercial primary antibodies and protocols explored AQP location on cauda epididymal and ejaculated spermatozoa (from different fractions of the ejaculate), unprocessed, extended, chilled and frozen-thawed. Although differences in WB and ICC labelling were seen among antibodies, AQP-7 was conspicuously located in the entire tail and cytoplasmic droplet in caudal spermatozoa, being restricted to the mid-piece and principal piece domains in ejaculated spermatozoa. AQP-9 was mainly localized in the sperm head in both caudal and ejaculated spermatozoa. While unaffected by chilling (+5°C), freezing and thawing of ejaculated spermatozoa clearly relocated the head labelling of AQP-7, but not that of AQP-9. In vitro mimicking of cell membrane expansion during quick thawing maintained the localization of AQP-9 but relocated AQP-7 towards the acrosome. AQP-7, but not AQP-9, appears as a relevant marker for non-empirical studies of sperm handling.

Ultrastructure of Neoblasts in Turbellarian Geocentrophora wagini Timoshkin, 1984 (Lecithoepitheliata: Plathelminthes)

Acoelomorpha and Plathelminthes have a unique system of stem cells (neoblasts), which is believed to represent a common proliferative compartment of somatic and germ line cells. Meanwhile, on the electron-microscopic level, these cells are not studied in most taxa of Turbellaria. In this study, I describe the ultrastructure of neoblasts in Geocentrophora wagini Timoshkin, 1984 (Lecithoepitheliata, Plathelminthes), an endemic turbellarian from Lake Baikal. The neoblast-like cells showed a high nucleus/cytoplasm ratio. The cytoplasm revealed the features of undifferentiated cells. Particularly, there were free ribosomes and mitochondria, while other organelles were rare and did not occur in all the cells studied. Based on their cytoplasmic and nuclear organization, three main types of parenchymal neoblasts have been distinguished. In type 1, the nuclei had a complex, highly branched configuration. The peripheral heterochromatin was not developed. A large loose structure of fibrous nature and a tiny Golgi apparatus with several secretory granules could be observed in the type 1 neoblast cytoplasm. The nuclei of the type 2 neoblasts had much simpler outlines than those of type 1, despite some processes or invaginations. The poor development of peripheral condensed chromatin was observed and distribution density of the heterochromatin clumps tended to be slightly higher, as compared to type 1. A cluster of small dense granules or a little loose body occasionally could be seen in the proximity to nuclear membrane. Type 3 neoblasts had the most compact nuclei and their heterochromatin was seen as large, irregular clumps of extremely high electron density. Many of these clumps were connected with each other and with the nuclear membrane. The scarce cytoplasm contained only mitochondria and ribosomes. Undifferentiated cells in the gastrodermis were similar to the type 2 neoblasts in the parenchyma. For the first time outside Tricladida special structures were found in the neoblast cytoplasm. These loose fibrous bodies and clusters of granules are likely to be functionally identical to the planarian chromatoid bodies. The obtained results contribute to the comparative morphology of the stem cells in flatworms and basic Bilateria and confirm the heterogeneity of the proliferative compartment in Turbellaria.

Voluntary Exercise Induces Astrocytic Structural Plasticity in the Globus Pallidus.

Changes in astrocyte morphology are primarily attributed to the fine processes where intimate connections with neurons form the tripartite synapse and participate in neurotransmission. Recent evidence has shown that neurotransmission induces dynamic synaptic remodeling, suggesting that astrocytic fine processes may adapt their morphologies to the activity in their environment. To illustrate such a neuron-glia relationship in morphological detail, we employed a double transgenic Olig2CreER/WT; ROSA26-GAP43-EGFP mice, in which Olig2-lineage cells can be visualized and traced with membrane-targeted GFP. Although Olig2-lineage cells in the adult brain usually become mature oligodendrocytes or oligodendrocyte precursor cells with NG2-proteoglycan expression, we found a population of Olig2-lineage astrocytes with bushy morphology in several brain regions. The globus pallidus (GP) preferentially contains Olig2-lineage astrocytes. Since the GP exerts pivotal motor functions in the indirect pathway of the basal ganglionic circuit, we subjected the double transgenic mice to voluntary wheel running to activate the GP and examined morphological changes of Olig2-lineage astrocytes at both the light and electron microscopic levels. The double transgenic mice were divided into three groups: control group mice were kept in a cage with a locked running wheel for 3 weeks, Runner group were allowed free access to a running wheel for 3 weeks, and the Runner-Rest group took a sedentary 3-week rest after a 3-week running period. GFP immunofluorescence analysis and immunoelectron microscopy revealed that astrocytic fine processes elaborated complex arborization in the Runner mice, and reverted to simple morphology comparable to that of the Control group in the Runner-Rest group. Our results indicated that the fine processes of the Olig2-lineage astrocytes underwent plastic changes that correlated with overall running activities, suggesting that they actively participate in motor functions.

High-voltage electron microscopy tomography and structome analysis of unique spiral bacteria from the deep sea.

Structome analysis is a useful tool for identification of unknown microorganisms that cannot be cultured. In 2012, we discovered a unique deep-sea microorganism with a cell structure intermediate between those of prokaryotes and eukaryotes and described its features using freeze-substitution electron microscopy and structome analysis (quantitative and three-dimensional structural analysis of a whole cell at the electron microscopic level). We named it Myojin parakaryote Here we describe, using serial ultrathin sectioning and high-voltage electron microscopy tomography of freeze-substituted specimens, the structome analysis and 3D reconstruction of another unique spiral bacteria, found in the deep sea off the coast of Japan. The bacteria, which is named as 'Myojin spiral bacteria' after the discovery location and their morphology, had a total length of 1.768 ± 0.478 µm and a total diameter of 0.445 ± 0.050 µm, and showed either clockwise or counter-clockwise spiral. The cells had a cell surface membrane, thick fibrous layer, ribosomes and inner fibrous structures (most likely DNA). They had no flagella. The bacteria had 322 ± 119 ribosomes per cell. This ribosome number is only 1.2% of that of Escherichia coli and 19.3% of Mycobacterium tuberculosis and may reflect a very slow growth rate of this organism in the deep sea.

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain.

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Effects of Optogenetic Activation of Corticothalamic Terminals in the Motor Thalamus of Awake Monkeys.

This study provides the first analysis of the physiological effects of cortical inputs on the activity of neurons in the primate ventral motor thalamus using light activation of opsin-containing corticothalamic terminals in awake monkeys. We found that selective light activation of corticothalamic terminals in contact with distal dendrites of thalamocortical neurons and GABAergic interneurons elicits complex patterns of slowly developing excitatory and inhibitory effects in thalamic neurons of the basal ganglia- and cerebellar-receiving regions of the motor thalamus. Our observations suggest a modulatory (instead of a "driver") role of the corticothalamic system in the primate ventral motor thalamus.

Seasonally Changing Cryptochrome 1b Expression in the Retinal Ganglion Cells of a Migrating Passerine Bird.

Cryptochromes, blue-light absorbing proteins involved in the circadian clock, have been proposed to be the receptor molecules of the avian magnetic compass. In birds, several cryptochromes occur: Cryptochrome 2, Cryptochrome 4 and two splice products of Cryptochrome 1, Cry1a and Cry1b. With an antibody not distinguishing between the two splice products, Cryptochrome 1 had been detected in the retinal ganglion cells of garden warblers during migration. A recent study located Cry1a in the outer segments of UV/V-cones in the retina of domestic chickens and European robins, another migratory species. Here we report the presence of cryptochrome 1b (eCry1b) in retinal ganglion cells and displaced ganglion cells of European Robins, Erithacus rubecula. Immuno-histochemistry at the light microscopic and electron microscopic level showed eCry1b in the cell plasma, free in the cytosol as well as bound to membranes. This is supported by immuno-blotting. However, this applies only to robins in the migratory state. After the end of the migratory phase, the amount of eCry1b was markedly reduced and hardly detectable. In robins, the amount of eCry1b in the retinal ganglion cells varies with season: it appears to be strongly expressed only during the migratory period when the birds show nocturnal migratory restlessness. Since the avian magnetic compass does not seem to be restricted to the migratory phase, this seasonal variation makes a role of eCry1b in magnetoreception rather unlikely. Rather, it could be involved in physiological processes controlling migratory restlessness and thus enabling birds to perform their nocturnal flights.

Synaptic Organization of Perisomatic GABAergic Inputs onto the Principal Cells of the Mouse Basolateral Amygdala.

Spike generation is most effectively controlled by inhibitory inputs that target the perisomatic region of neurons. Despite the critical importance of this functional domain, very little is known about the organization of the GABAergic inputs contacting the perisomatic region of principal cells (PCs) in the basolateral amygdala. Using immunocytochemistry combined with in vitro single-cell labeling we determined the number and sources of GABAergic inputs of PCs at light and electron microscopic levels in mice. We found that the soma and proximal dendrites of PCs were innervated primarily by two neurochemically distinct basket cell types expressing parvalbumin (PVBC) or cholecystokinin and CB1 cannabinoid receptors (CCK/CB1BC). The innervation of the initial segment of PC axons was found to be parceled out by PVBCs and axo-axonic cells (AAC), as the majority of GABAergic inputs onto the region nearest to the soma (between 0 and 10 μm) originated from PVBCs, while the largest portion of the axon initial segment was innervated by AACs. Detailed morphological investigations revealed that the three perisomatic region-targeting interneuron types significantly differed in dendritic and axonal arborization properties. We found that, although individual PVBCs targeted PCs via more terminals than CCK/CB1BCs, similar numbers (15–17) of the two BC types converge onto single PCs, whereas fewer (6–7) AACs innervate the axon initial segment of single PCs. Furthermore, we estimated that a PVBC and a CCK/CB1BC may target 800–900 and 700–800 PCs, respectively, while an AAC can innervate 600–650 PCs. Thus, BCs and AACs innervate ~10 and 20% of PC population, respectively, within their axonal cloud. Our results collectively suggest, that these interneuron types may be differently affiliated within the local amygdalar microcircuits in order to fulfill specific functions in network operation during various brain states.

Paclitaxel and the dietary flavonoid fisetin: a synergistic combination that induces mitotic catastrophe and autophagic cell death in A549 non-small cell lung cancer cells

Background The use of the dietary polyphenols as chemosensitizing agents to enhance the efficacy of conventional cytostatic drugs has recently gained the attention of scientists and clinicians as a plausible approach for overcoming the limitations of chemotherapy (e.g. drug resistance and cytotoxicity). The aim of this study was to investigate whether a naturally occurring diet-based flavonoid, fisetin, at physiologically attainable concentrations, could act synergistically with clinically achievable doses of paclitaxel to produce growth inhibitory and/or pro-death effects on A549 non-small cell lung cancer cells, and if it does, what mechanisms might be involved.MethodsThe drug–drug interactions were analyzed based on the combination index method of Chou and Talalay and the data from MTT assays. To provide some insights into the mechanism underlying the synergistic action of fisetin and paclitaxel, selected morphological, biochemical and molecular parameters were examined, including the morphology of cell nuclei and mitotic spindles, the pattern of LC3-II immunostaining, the formation of autophagic vacuoles at the electron and fluorescence microscopic level, the disruption of cell membrane asymmetry/integrity, cell cycle progression and the expression level of LC3-II, Bax, Bcl-2 and caspase-3 mRNA.ResultsHere, we reported the first experimental evidence for the existence of synergism between fisetin and paclitaxel in the in vitro model of non-small cell lung cancer. This synergism was, at least partially, ascribed to the induction of mitotic catastrophe. The switch from the cytoprotective autophagy to the autophagic cell death was also implicated in the mechanism of the synergistic action of fisetin and paclitaxel in the A549 cells. In addition, we revealed that the synergism between fisetin and paclitaxel was cell line-specific as well as that fisetin synergizes with arsenic trioxide, but not with mitoxantrone and methotrexate in the A549 cells.ConclusionsOur results provide rationale for further testing of fisetin in the combination with paclitaxel or arsenic trioxide to obtain detailed insights into the mechanism of their synergistic action as well as to evaluate their toxicity towards normal cells in an animal model in vivo. We conclude that this study is potentially interesting for the development of novel chemotherapeutic approach to non-small cell lung cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0288-3) contains supplementary material, which is available to authorized users.