Confocal Reconstruction Research Articles

Desiccation Tolerance in the Tardigrade Richtersius coronifer Relies on Muscle Mediated Structural Reorganization

Life unfolds within a framework of constraining abiotic factors, yet some organisms are adapted to handle large fluctuations in physical and chemical parameters. Tardigrades are microscopic ecdysozoans well known for their ability to endure hostile conditions, such as complete desiccation – a phenomenon called anhydrobiosis. During dehydration, anhydrobiotic animals undergo a series of anatomical changes. Whether this reorganization is an essential regulated event mediated by active controlled processes, or merely a passive result of the dehydration process, has not been clearly determined. Here, we investigate parameters pivotal to the formation of the so-called "tun", a state that in tardigrades and rotifers marks the entrance into anhydrobiosis. Estimation of body volume in the eutardigrade Richtersius coronifer reveals an 87 % reduction in volume from the hydrated active state to the dehydrated tun state, underlining the structural stress associated with entering anhydrobiosis. Survival experiments with pharmacological inhibitors of mitochondrial energy production and muscle contractions show that i) mitochondrial energy production is a prerequisite for surviving desiccation, ii) uncoupling the mitochondria abolishes tun formation, and iii) inhibiting the musculature impairs the ability to form viable tuns. We moreover provide a comparative analysis of the structural changes involved in tun formation, using a combination of cytochemistry, confocal laser scanning microscopy and 3D reconstructions as well as scanning electron microscopy. Our data reveal that the musculature mediates a structural reorganization vital for anhydrobiotic survival, and furthermore that maintaining structural integrity is essential for resumption of life following rehydration.

Effect of Modified Nonequilibrium Plasma with Chlorhexidine Digluconate against Endodontic Biofilms In Vitro

IntroductionNonequilibrium plasma has been reported to effectively kill Enterococcus faecalis in endodontic biofilm compared with chlorhexidine digluconate (CHX). The purpose of this study was to evaluate the antimicrobial in vitro activity of modified nonequilibrium plasma with CHX against E. faecalis and multispecies biofilms on bovine dentin discs. MethodsSterile bovine dentin discs were incubated with E. faecalis or a mixture of bacteria from human dental root canal infections to form 1- and 3-week-old biofilms. The specimens were subjected to nonequilibrium plasma, modified nonequilibrium plasma with CHX, and 2% CHX for 2- and 5-minute exposure. After treatment, the biofilms were stained with viability dyes and examined by confocal laser scanning microscopy and 3-dimensional reconstruction analysis. The proportions of bacterial cells killed by the treatments were calculated. ResultsThe 3-dimensional reconstruction images showed that 1- and 3-week-old biofilms adhered to bovine dentin discs. The proportions of dead cells increased significantly with the longer exposure in each treatment group (P < .05). Modified nonequilibrium plasma was the most effective in killing bacteria in E. faecalis and multispecies biofilms at both 2 and 5 minutes (P < .05). No significant difference was detected between nonequilibrium plasma and CHX groups (P > .05). Significantly more cells were killed in 1-week-old biofilms than in 3-week-old biofilms in all groups (P < .05). ConclusionsThe modified nonequilibrium plasma killed more bacteria than conventional nonequilibrium plasma and 2% CHX in E. faecalis and multispecies endodontic biofilms in vitro and thus shows promise as an additional tool in infection control during endodontic treatment.

Confocal 3-dimensional reconstruction can serve as a useful tool to quantify oxidative stress induced oocyte spindle damage

Oxidative stress has been shown to mediate damage to metaphase II (MII) mouse oocyte microtubule (MT) and chromosome (CH) structure; markers of oocyte quality. Our long term goal is to utilize confocal 3-Dimensional (3-D) imaging as a tool to: 1) quantitate spindle damage; 2) elucidate the mechanism by which reactive oxygen species (ROS) mediate spindle damage in inflammatory states and aging. The objective of this study was to utilize confocal 3-D visualization to assess peroxynitrite (ONOO-) mediated damage to oocyte spindle.

The Effects of Cocaine Self-Administration on Dendritic Spine Density in the Rat Hippocampus Are Dependent on Genetic Background

Chronic exposure to cocaine induces modifications to neurons in the brain regions involved in addiction. Hence, we evaluated cocaine-induced changes in the hippocampal CA1 field in Fischer 344 (F344) and Lewis (LEW) rats, 2 strains that have been widely used to study genetic predisposition to drug addiction, by combining intracellular Lucifer yellow injection with confocal microscopy reconstruction of labeled neurons. Specifically, we examined the effects of cocaine self-administration on the structure, size, and branching complexity of the apical dendrites of CA1 pyramidal neurons. In addition, we quantified spine density in the collaterals of the apical dendritic arbors of these neurons. We found differences between these strains in several morphological parameters. For example, CA1 apical dendrites were more branched and complex in LEW than in F344 rats, while the spine density in the collateral dendrites of the apical dendritic arbors was greater in F344 rats. Interestingly, cocaine self-administration in LEW rats augmented the spine density, an effect that was not observed in the F344 strain. These results reveal significant structural differences in CA1 pyramidal cells between these strains and indicate that cocaine self-administration has a distinct effect on neuron morphology in the hippocampus of rats with different genetic backgrounds.

Predicting the location of the axon initial segment using spike waveform analysis: simulations of retinal ganglion cell physiology

There are 16 morphologically defined classes of rat retinal ganglion cells (RGCs). Most commonly, they are classified on the basis of several criteria including: soma size, dendritic field diameter, the dendritic branching pattern and the depth of stratification in the inner plexiform layer. Recently, it has also been shown that the intrinsic physiological properties of each rat RGC type vary enormously. Using multicompartment models of RGC types we investigated whether the location of the axon initial segment (AIS), the site of greatest sodium channel density and lowest voltage threshold, can be predicted by measurements of spike waveform made at the soma. The action potential waveform in many neurons consists of several components, which can be determined by examining the first and second derivatives of the membrane potential. In this study, we focus on this technique as an objective method to analyze the action potential waveform for different morphological RGC types. In addition, we analyze the features of the phase plot, which shows the rate of change of the membrane potential against the membrane potential itself. Phase plot analysis allows the measurement of subtle differences in the action potential waveform such as the initial segment-soma/dendritic break (ISSD), which corresponds to the early rising phase of the action potential. When the recording is made at the soma, the presence of the ISSD in the phase plot indicates that a low threshold region (i.e. the AIS) is further away from the soma. Rat RGCs were characterized electrophysiologically using standard whole cell patch clamp recording techniques. Data were acquired at 20 kHz using custom software developed in LabView (National Instruments). Spontaneous spikes and spikes evoked by just-threshold current were used for analysis. For each of the recordings, the amplitude and time of the trough between the peaks in the second-order derivatives were analyzed. After three dimensional confocal reconstruction of each recorded cell (Zeiss PASCAL) it was classified morphologically into one of the 16 predefined types. Multicompartment models of real retinal ganglion cells were constructed from 3D rendering confocal reconstructions and their physiology was simulated using the Hodgkin-Huxley formalism in the NEURON environment. Sodium channel density in the AIS and its distance from the soma were systematically varied and the effects on the phase plot analyzed. Simulations showed that the further the AIS was from the soma, the more pronounced the ISSD break, resulting in a larger break with a deeper trough between the two peaks in the phase plot. This result allows us to predict the location of the AIS based on recordings of the impulse waveform. In addition, we found that the density of sodium channels in the AIS affects spike propagation into the soma. We observed that decreasing sodium conductance in the AIS, required two spikes to occur in the AIS in order to evoke a somatic spike. This was also observed experimentally, in particular in C4 cells. Further analysis of individual RGC spike waveforms demonstrated that certain RGC types could be reliably identified using their spike waveforms.

Quantitative analysis of cardiac tissue including fibroblasts using three-dimensional confocal microscopy and image reconstruction: towards a basis for electrophysiological modeling.

Electrophysiological modeling of cardiac tissue is commonly based on functional and structural properties measured in experiments. Our knowledge of these properties is incomplete, in particular their remodeling in disease. Here, we introduce a methodology for quantitative tissue characterization based on fluorescent labeling, 3-D scanning confocal microscopy, image processing and reconstruction of tissue micro-structure at sub-micrometer resolution. We applied this methodology to normal rabbit ventricular tissue and tissue from hearts with myocardial infarction. Our analysis revealed that the volume fraction of fibroblasts increased from 4.83±0.42% (mean ± standard deviation) in normal tissue up to 6.51±0.38% in myocardium from infarcted hearts. The myocyte volume fraction decreased from 76.20±9.89% in normal to 73.48±8.02% adjacent to the infarct. Numerical field calculations on 3-D reconstructions of the extracellular space yielded an extracellular longitudinal conductivity of 0.264±0.082 S/m with an anisotropy ratio of 2.095±1.11 in normal tissue. Adjacent to the infarct, the longitudinal conductivity increased up to 0.400±0.051 S/m, but the anisotropy ratio decreased to 1.295±0.09. Our study indicates an increased density of gap junctions proximal to both fibroblasts and myocytes in infarcted versus normal tissue, supporting previous hypotheses of electrical coupling of fibroblasts and myocytes in infarcted hearts. We suggest that the presented methodology provides an important contribution to modeling normal and diseased tissue. Applications of the methodology include the clinical characterization of disease-associated remodeling.

Anin VivoMethod for Visualizing Flow Dynamics of Cells within Corneal Lymphatics

Monitoring the trafficking of specific cell populations within lymphatics could improve our understanding of processes such as transplant rejection and cancer metastasis. Current methods, however, lack appropriate image resolution for single-cell analysis or are incompatible with in vivo and longitudinal monitoring of lymphatics in their native state. We therefore sought to achieve high-resolution live imaging of the dynamic behavior of cells within lymph vessels in the rat cornea. Inflammatory angiogenesis was induced by suture placement in corneas of Wistar rats. Pre- and up to 3 weeks post-induction, corneas were noninvasively examined by laser-scanning in vivo corneal confocal microscopy (IVCM) using only endogenous contrast. Lymph vessels and the cells harbored therein were documented by still images, real-time video, and 3D confocal stack reconstruction of live tissue. In vivo, conjunctival and corneal lymphatics were morphologically distinct, those with corneal location being one-quarter the diameter of those in the conjunctiva (p<0.001). Cells were recruited to initially empty pre-existing lymph vessels during the first day of inflammation and maintained a dense occupation of vessels for up to 7 days. A diverse population of cells (diameter range: 1.5-27.5 μm) with varying morphology was observed, and exhibited variable flow patterns and were transported singly and in clusters of at least 2-9 adherent cells. The in vivo microscopic technique presented enables lymph vessels and cell trafficking to be studied in high resolution in a minimally-perturbed physiologic milieu.

Mapping the Mechanome of Live Stem Cells Using a Novel Method to Measure Local Strain Fields In Situ at the Fluid-Cell Interface

During mesenchymal condensation, the initial step of skeletogenesis, transduction of minute mechanical forces to the nucleus is associated with up or down-regulation of genes, ultimately resulting in formation of the skeletal template and appropriate cell lineage commitment. The summation of these biophysical cues affects the cell's shape and fate. Here, we predict and measure surface strain, in live stem cells, in response to controlled delivery of stresses, providing a platform to direct short-term structure - function relationships and long-term fate decisions. We measure local strains on stem cell surfaces using fluorescent microbeads coated with Concanavalin A. During delivery of controlled mechanical stresses, 4-Dimensional (x,y,z,t) displacements of the bound beads are measured as surface strains using confocal microscopy and image reconstruction. Similarly, micro-particle image velocimetry (μ-piv) is used to track flow fields with fluorescent microspheres. The measured flow velocity gradient is used to calculate stress imparted by fluid drag at the surface of the cell. We compare strain measured on cell surfaces with those predicted computationally using parametric estimates of the cell's elastic and shear modulus. Finally, cross-correlating stress - strain data to measures of gene transcription marking lineage commitment enables us to create stress - strain - fate maps, for live stem cells in situ. The studies show significant correlations between live stem cell stress - strain relationships and lineage commitment. The method presented here provides a novel means to probe the live stem cell's mechanome, enabling mechanistic studies of the role of mechanics in lineage commitment as it unfolds.

Serotonin immunoreactive interneurons in the brain of the Remipedia: new insights into the phylogenetic affinities of an enigmatic crustacean taxon

BackgroundRemipedia, a group of homonomously segmented, cave-dwelling, eyeless arthropods have been regarded as basal crustaceans in most early morphological and taxonomic studies. However, molecular sequence information together with the discovery of a highly differentiated brain led to a reconsideration of their phylogenetic position. Various conflicting hypotheses have been proposed including the claim for a basal position of Remipedia up to a close relationship with Malacostraca or Hexapoda. To provide new morphological characters that may allow phylogenetic insights, we have analyzed the architecture of the remipede brain in more detail using immunocytochemistry (serotonin, acetylated α-tubulin, synapsin) combined with confocal laser-scanning microscopy and image reconstruction techniques. This approach allows for a comprehensive neuroanatomical comparison with other crustacean and hexapod taxa.ResultsThe dominant structures of the brain are the deutocerebral olfactory neuropils, which are linked by the olfactory globular tracts to the protocerebral hemiellipsoid bodies. The olfactory globular tracts form a characteristic chiasm in the center of the brain. In Speleonectes tulumensis, each brain hemisphere contains about 120 serotonin immunoreactive neurons, which are distributed in distinct cell groups supplying fine, profusely branching neurites to 16 neuropilar domains. The olfactory neuropil comprises more than 300 spherical olfactory glomeruli arranged in sublobes. Eight serotonin immunoreactive neurons homogeneously innervate the olfactory glomeruli. In the protocerebrum, serotonin immunoreactivity revealed several structures, which, based on their position and connectivity resemble a central complex comprising a central body, a protocerebral bridge, W-, X-, Y-, Z-tracts, and lateral accessory lobes.ConclusionsThe brain of Remipedia shows several plesiomorphic features shared with other Mandibulata, such as deutocerebral olfactory neuropils with a glomerular organization, innervations by serotonin immunoreactive interneurons, and connections to protocerebral neuropils. Also, we provided tentative evidence for W-, X-, Y-, Z-tracts in the remipedian central complex like in the brain of Malacostraca, and Hexapoda. Furthermore, Remipedia display several synapomorphies with Malacostraca supporting a sister group relationship between both taxa. These homologies include a chiasm of the olfactory globular tract, which connects the olfactory neuropils with the lateral protocerebrum and the presence of hemiellipsoid bodies. Even though a growing number of molecular investigations unites Remipedia and Cephalocarida, our neuroanatomical comparison does not provide support for such a sister group relationship.

Interstitial cells in the primate gastrointestinal tract

Kit immunohistochemistry and confocal reconstructions have provided detailed 3-dimensional images of ICC networks throughout the gastrointestinal (GI) tract. Morphological criteria have been used to establish that different classes of ICC exist within the GI tract and physiological studies have shown that these classes have distinct physiological roles in GI motility. Structural studies have focused predominately on rodent models and less information is available on whether similar classes of ICC exist within the GI tracts of humans or non-human primates. Using Kit immunohistochemistry and confocal imaging, we examined the 3-dimensional structure of ICC throughout the GI tract of cynomolgus monkeys. Whole or flat mounts and cryostat sections were used to examine ICC networks in the lower esophageal sphincter (LES), stomach, small intestine and colon. Anti-histamine antibodies were used to distinguish ICC from mast cells in the lamina propria. Kit labeling identified complex networks of ICC populations throughout the non-human primate GI tract that have structural characteristics similar to that described for ICC populations in rodent models. ICC-MY formed anastomosing networks in the myenteric plexus region. ICC-IM were interposed between smooth muscle cells in the stomach and colon and were concentrated within the deep muscular plexus (ICC-DMP) of the intestine. ICC-SEP were found in septal regions of the antrum that separated circular muscle bundles. Spindle-shaped histamine(+) mast cells were found in the lamina propria throughout the GI tract. Since similar sub-populations of ICC exist within the GI tract of primates and rodents and the use of rodents to study the functional roles of different classes of ICC is warranted.

Modeling predicts that parameters shaping action potentials and synaptic responses differ in pyramidal neurons of the visual and prefrontal cortices

Pyramidal neurons in the prefrontal cortex integrate inputs that are greater in number and diversity than pyramidal neurons of the primary visual cortex [1]. We have recently characterized the morphology and physiology of layer 3 neurons from area 46 of the dorsolateral prefrontal cortex (dlPFC) and from visual area V1 in the rhesus monkey. Ultra-high resolution confocal imaging and 3D reconstruction revealed that the dendritic arbors of V1 neurons are much smaller and less complex than those of the dlPFC, and possess far fewer dendritic spines. Physiologically, whole cell patch clamp recordings demonstrated that V1 neurons have twice as high mean input resistance and significantly increased action potential firing rates compared to dlPFC neurons. Further, AMPAR-mediated spontaneous excitatory postsynaptic currents (sEPSCs) exhibit significantly faster kinetics and smaller mean amplitudes in V1 compared to dlPFC neurons. We have used compartment modeling with the NEURON simulation environment [2] to investigate the extent to which morphology accounts for the observed physiological differences. We first applied a relatively simple model, including only Hodgkin-Huxley sodium and potassium currents [3], to representative 3D reconstructions of neurons from dlPFC and V1. This model predicts that morphology alone leads to major differences in the attenuation of electrical signals coming into, and leaving, the soma in neurons from the two brain areas. It also predicts that morphology largely accounts for the increased excitability of V1 cells, but not the differences in kinetics and amplitudes of sEPSCs. The model predicts that parameters controlling passive voltage spread, the density of Hodgkin-Huxley channels, and synaptic inputs likely differ between dlPFC and V1 neurons. Yet, the simple model on which these predictions are based reproduces neither the H-current dependent ‘sag’ response during hyperpolarization, nor spike frequency adaptation, both of which are observed in dlPFC and V1 neurons. We have extended the model to include the H current [4], plus a calcium-dependent potassium channel with an associated one-pool source of intracellular calcium [5]. We have tuned the model with a combination of manual and automatic optimization techniques to match the resting potential, input resistance, and overall firing rates of the recorded dlPFC and V1 neurons. As in the simpler model, we find that morphology alone cannot account for observed physiological differences. Therefore, we predict that parameters shaping action potentials and synaptic input differ between dlPFC and V1.

Formation of bacterial streamers during filtration in microfluidic systems

Bacterial behavior during filtration is complex and is influenced by numerous factors. The aim of this paper is to report on experiments designed to make progress in the understanding of bacterial transfer in filters and membranes. Polydimethylsiloxane (PDMS) microsystems were built to allow direct dynamic observation of bacterial transfer across different microchannel geometries mimicking filtration processes. When filtering Escherichia coli suspensions in such devices, the bacteria accumulated in the downstream zone of the filter forming long streamers undulating in the flow. Confocal microscopy and 3D reconstruction of streamers showed how the streamers are connected to the filter and how they form in the stream. Streamer development was found to be influenced by the flow configuration and the presence of connections or tortuosity between channels. Experiments showed that streamer formation was greatest in a filtration system composed of staggered arrays of squares 10 μm apart.

Identification of radial glia-like cells in the adult mouse olfactory bulb

Immature neurons migrate tangentially within the rostral migratory stream (RMS) to the adult olfactory bulb (OB), then radially to their final positions as granule and periglomerular neurons; the controls over this transition are not well understood. Using adult transgenic mice with the human GFAP promoter driving expression of enhanced GFP, we identified a population of radial glia-like cells that we term adult olfactory radial glia-like cells (AORGs). AORGs have large, round somas and simple, radially oriented processes. Confocal reconstructions indicate that AORGs variably express typical radial glial markers, only rarely express mouse GFAP, and do not express astroglial, oligodendroglial, neuronal, or tanycyte markers. Electron microscopy provides further supporting evidence that AORGs are not immature neurons. Developmental analyses indicate that AORGs are present as early as P1, and are generated through adulthood. Tracing studies show that AORGs are not born in the SVZa, suggesting that they are born either in the RMS or the OB. Migrating immature neurons from the adult SVZa are closely apposed to AORGs during radial migration in vivo and in vitro. Taken together, these data indicate a newly-identified population of radial glia-like cells in the adult OB that might function uniquely in neuronal radial migration during adult OB neurogenesis.

Branchial ionocyte organization and ion-transport protein expression in juvenile alewives acclimated to freshwater or seawater

The alewife (Alosa pseudoharengus) is a clupeid that undergoes larval and juvenile development in freshwater preceding marine habitation. The purpose of this study was to investigate osmoregulatory mechanisms in alewives that permit homeostasis in different salinities. To this end, we measured physiological, branchial biochemical and cellular responses in juvenile alewives acclimated to freshwater (0.5 p.p.t.) or seawater (35.0 p.p.t.). Plasma chloride concentration was higher in seawater-acclimated than freshwater-acclimated individuals (141 mmol l(-1) vs 134 mmol l(-1)), but the hematocrit remained unchanged. In seawater-acclimated individuals, branchial Na(+)/K(+)-ATPase (NKA) activity was higher by 75%. Western blot analysis indicated that the abundance of the NKA α-subunit and a Na(+)/K(+)/2Cl(-) cotransporter (NKCC1) were greater in seawater-acclimated individuals by 40% and 200%, respectively. NKA and NKCC1 were localized on the basolateral surface and tubular network of ionocytes in both acclimation groups. Immunohistochemical labeling for the cystic fibrosis transmembrane conductance regulator (CFTR) was restricted to the apical crypt of ionocytes in seawater-acclimated individuals, whereas sodium/hydrogen exchanger 3 (NHE3) labeling was present on the apical surface of ionocytes in both acclimation groups. Ionocytes were concentrated on the trailing edge of the gill filament, evenly distributed along the proximal 75% of the filamental axis and reduced distally. Ionocyte size and number on the gill filament were not affected by salinity; however, the number of lamellar ionocytes was significantly lower in seawater-acclimated fish. Confocal z-series reconstructions revealed that mature ionocytes in seawater-acclimated alewives occurred in multicellular complexes. These complexes might reduce paracellular Na(+) resistance, hence facilitating Na(+) extrusion in hypo-osmoregulating juvenile alewives after seaward migration.

Development of the nervous system in Phoronopsis harmeri (Lophotrochozoa, Phoronida) reveals both deuterostome- and trochozoan-like features

BackgroundInferences concerning the evolution of invertebrate nervous systems are often hampered by the lack of a solid data base for little known but phylogenetically crucial taxa. In order to contribute to the discussion concerning the ancestral neural pattern of the Lophotrochozoa (a major clade that includes a number of phyla that exhibit a ciliated larva in their life cycle), we investigated neurogenesis in Phoronopsis harmeri, a member of the poorly studied Phoronida, by using antibody staining against serotonin and FMRFamide in combination with confocal microscopy and 3D reconstruction software.ResultsThe larva of Phoronopsis harmeri exhibits a highly complex nervous system, including an apical organ that consists of four different neural cell types, such as numerous serotonin-like immunoreactive flask-shaped cells. In addition, serotonin- and FMRFamide-like immunoreactive bi- or multipolar perikarya that give rise to a tentacular neurite bundle which innervates the postoral ciliated band are found. The preoral ciliated band is innervated by marginal serotonin-like as well as FMRFamide-like immunoreactive neurite bundles. The telotroch is innervated by two neurite bundles. The oral field is the most densely innervated area and contains ventral and ventro-lateral neurite bundles as well as several groups of perikarya. The digestive system is innervated by both serotonin- and FMRFamide-like immunoreactive neurites and perikarya. Importantly, older larvae of P. harmeri show a paired ventral neurite bundle with serial commissures and perikarya.ConclusionsSerotonin-like flask-shaped cells such as the ones described herein for Phoronopsis harmeri are found in the majority of lophotrochozoan larvae and therefore most likely belong to the ground pattern of the last common lophotrochozoan ancestor. The finding of a transitory paired ventral neurite bundle with serially repeated commissures that disappears during metamorphosis suggests that such a structure was part of the “ur-phoronid” nervous system, but was lost in the adult stage, probably due to its acquired sessile benthic lifestyle.

Comparison of Scanning Acoustic Microscopy and Histology Images in Characterizing Surface Irregularities Among Engineered Human Oral Mucosal Tissues

Acoustic microscopy was used to monitor an ex vivo produced oral mucosal equivalent (EVPOME) developed on acellular cadaveric dermis (AlloDerm ®). As seeded cells adhered and grew, they filled in and smoothed out the surface irregularities, followed by the production of a keratinized protective outermost layer. If noninvasive in vitro ultrasonic monitoring of these cellular changes could be developed, then tissue cultivation could be adjusted in-process to account for biologic variations in the development of these stratified cell layers. Cultured keratinocytes (from freshly obtained oral mucosa) were harvested and seeded onto AlloDerm ® coated with human type IV collagen and cultured 11 days. EVPOMEs were imaged on the 11th day post-seeding using a scanning acoustic microscope (SAM) that consists of a single-element transducer: 61 MHz center frequency, 32 MHz bandwidth, 1.52 f-number. The specimen surface was determined by thresholding the magnitude of the signal at the first axial incidence of a value safely above noise: 20–40 dB above the signal for the water and 2-dimensional (2-D) ultrasonic images were created using confocal image reconstruction. A known area from each micrograph was divided into 12–40 even segments and examined for surface irregularities. These irregularities were quantified and one-way analysis of variance (ANOVA) and linear regression analysis were performed to correlate the surface profiles for both the AlloDerm ® and EVPOME specimens imaged by SAM. Histology micrographs of the AlloDerm ® and EVPOME specimens were also prepared and examined for surface irregularities. Unseeded AlloDerm ® averaged seven to nine surface changes per 400 μm. The number of changes in surface irregularities decreased to two to three per 400 μm on the mature EVPOMEs. The numbers of surface irregularities between the unseeded AlloDerm ® vs. developing EVPOME are similar for both histology and SAM 2-D B-scan images. For the EVPOME 2-D B-scan micrographs produced by SAM, the decrease in surface irregularities is indicative of the stratified epithelium formed by seeded oral keratinocytes; verified in the histology images between the AlloDerm ® and EVPOME. A near 1:1 linear correlation shows the similarities between the two imaging modalities. SAM demonstrates its ability to discern the cell development and differentiation occurring on the EVPOME devices. Unlike histology, SAM measurements are noninvasive and can be used to monitor tissue graft development without damaging any cells/tissues.

Striatal cellular properties conserved from lampreys to mammals

The striatum of the lamprey, the first vertebrate group to appear in evolution, shows striking similarities to that of mammals with respect to histochemical markers, afferent and efferent projections and the effect of dopamine depletion, which leads to hypokinetic motor symptoms. The cellular properties of lamprey striatal neurons were studied here using patch-clamp recordings in acute striatal slices. Sixty-five per cent of recorded neurons were characterised by a prominent inward rectification due to a K+ conductance of the Kir type. They had a ramping response with a long delay to the first action potential due to activation of a low-voltage-activated A-type K+ current. Many such inwardly rectifying neurons (IRNs) had a hyperpolarised resting membrane potential and some had spiny dendrites. The remaining 35% of the neurons (non-IRNs) represent a heterogeneous group, including some with characteristics similar to the fast-spiking interneuron of the mammalian striatum. They showed short-lasting, large after hyperpolarisations (AHPs) and discharged action potentials at high frequency. None of the recorded neurons were spontaneously active but they received GABAergic and glutamatergic synaptic input. The fact that most lamprey striatal neurons display inward rectification indicates that this is a conserved characteristic of striatal neurons throughout vertebrate phylogeny. This is a cellular property of critical importance for the operations of the striatum in mammals.

Antennal-Lobe Organization in Desert Ants of the Genus Cataglyphis

Desert ants of the genus Cataglyphis possess remarkable visual navigation capabilities. Although Cataglyphis species lack a trail pheromone system, Cataglyphis fortis employs olfactory cues for detecting nest and food sites. To investigate potential adaptations in primary olfactory centers of the brain of C. fortis, we analyzed olfactory glomeruli (odor processing units) in their antennal lobes and compared them to glomeruli in different Cataglyphis species. Using confocal imaging and 3D reconstruction, we analyzed the number, size and spatial arrangement of olfactory glomeruli in C. fortis, C.albicans, C.bicolor, C.rubra, and C.noda. Workers of all Cataglyphis species have smaller numbers of glomeruli (198–249) compared to those previously found in olfactory-guided ants. Analyses in 2 species of Formica – a genus closely related to Cataglyphis – revealed substantially higher numbers of olfactory glomeruli (c. 370), which is likely to reflect the importance of olfaction in these wood ant species. Comparisons between Cataglyphis species revealed 2 special features in C. fortis. First, with c. 198 C. fortis has the lowest number of glomeruli compared to all other species. Second, a conspicuously enlarged glomerulus is located close to the antennal nerve entrance. Males of C. fortis possess a significantly smaller number of glomeruli (c. 150) compared to female workers and queens. A prominent male-specific macroglomerulus likely to be involved in sex pheromone communication occupies a position different from that of the enlarged glomerulus in females. The behavioral significance of the enlarged glomerulus in female workers remains elusive. The fact that C. fortis inhabits microhabitats (salt pans) that are avoided by all other Cataglyphis species suggests that extreme ecological conditions may not only have resulted in adaptations of visual capabilities, but also in specializations of the olfactory system.

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro. Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.