Previous Electron Microscopic Studies Research Articles

Tracking Initial Fe(II)-Driven Ferrihydrite Transformations: A Mössbauer Spectroscopy and Isotope Investigation.

Transformation of nanocrystalline ferrihydrite to more stable microcrystalline Fe(III) oxides is rapidly accelerated under reducing conditions with aqueous Fe(II) present. While the major steps of Fe(II)-catalyzed ferrihydrite transformation are known, processes in the initial phase that lead to nucleation and the growth of product minerals remain unclear. To track ferrihydrite-Fe(II) interactions during this initial phase, we used Fe isotopes, Mössbauer spectroscopy, and extractions to monitor the structural, magnetic, and isotope composition changes of ferrihydrite within ∼30 min of Fe(II) exposure. We observed rapid isotope mixing between aqueous Fe(II) and ferrihydrite during this initial lag phase. Our findings from Mössbauer spectroscopy indicate that a more magnetically ordered Fe(III) phase initially forms that is distinct from ferrihydrite and bulk crystalline transformation products. The signature of this phase is consistent with the early stage emergence of lepidocrocite-like lamellae observed in previous transmission electron microscopy studies. Its signature is furthermore removed by xylenol extraction of Fe(III), the same approach used to identify a chemically labile form of Fe(III) resulting from Fe(II) contact that is correlated to the ultimate emergence of crystalline product phases detectable by X-ray diffraction. Our work indicates that the mineralogical changes in the initial lag phase of Fh transformation initiated by Fe(II)-Fh electron transfer are critical to understanding ferrihydrite behavior in soils and sediments, particularly with regard to metal uptake and release.

Composition and electronic structure of {rm SiO}_{rm x}/{rm TiO}_{rm y}/Al passivating carrier selective contacts on n-type silicon solar cells

Carrier-selective and passivating SiO_{rm x}/TiO_{rm y} heterocontacts are an attractive alternative to conventional contacts due to their high efficiency potentials combined with relatively simple processing schemes. It is widely accepted that post deposition annealing is necessary to obtain high photovoltaic efficiencies, especially for full area aluminum metallized contacts. Despite some previous high-level electron microscopy studies, the picture of atomic-scale processes underlying this improvement seems to be incomplete. In this work, we apply nanoscale electron microscopy techniques to macroscopically well-characterized solar cells with SiO_{rm x}/TiO_{rm y}/Al rear contacts on n-type silicon. Macroscopically, annealed solar cells show a tremendous decrease of series resistance and improved interface passivation. Analyzing the microscopic composition and electronic structure of the contacts, we find that partial intermixing of the SiO_{rm x} and TiO_{rm y} layers occurs due to annealing, leading to an apparent thickness reduction of the passivating SiO_{rm x}. However, the electronic structure of the layers remains clearly distinct. Hence, we conclude that the key to obtain highly efficient SiO_{rm x}/TiO_{rm y}/Al contacts is to tailor the processing such that the excellent chemical interface passivation of a SiO_{rm x} layer is achieved for a layer thin enough to allow efficient tunneling through the layer. Furthermore, we discuss the impact of aluminum metallization on the above mentioned processes.

Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses.

Ribbon synapses of cochlear inner hair cells (IHCs) are specialized to indefatigably transmit sound information at high rates. To understand the underlying mechanisms, structure-function analysis of the active zone (AZ) of these synapses is essential. Previous electron microscopy studies of synaptic vesicle (SV) dynamics at the IHC AZ used potassium stimulation, which limited the temporal resolution to minutes. Here, we established optogenetic IHC stimulation followed by quick freezing within milliseconds and electron tomography to study the ultrastructure of functional synapse states with good temporal resolution in mice. We characterized optogenetic IHC stimulation by patch-clamp recordings from IHCs and postsynaptic boutons revealing robust IHC depolarization and neurotransmitter release. Ultrastructurally, the number of docked SVs increased upon short (17-25 ms) and long (48-76 ms) light stimulation paradigms. We did not observe enlarged SVs or other morphological correlates of homotypic fusion events. Our results indicate a rapid recruitment of SVs to the docked state upon stimulation and suggest that univesicular release prevails as the quantal mechanism of exocytosis at IHC ribbon synapses.

Clustering of tetrameric influenza M2 peptides in lipid bilayers investigated by 19F solid-state NMR

The influenza M2 protein forms a drug-targeted tetrameric proton channel to mediate virus uncoating, and carries out membrane scission to enable virus release. While the proton channel function of M2 has been extensively studied, the mechanism by which M2 catalyzes membrane scission is still not well understood. Previous fluorescence and electron microscopy studies indicated that M2 tetramers concentrate at the neck of the budding virus in the host plasma membrane. However, molecular evidence for this clustering is scarce. Here, we use 19F solid-state NMR to investigate M2 clustering in phospholipid bilayers. By mixing equimolar amounts of 4F-Phe47 labeled M2 peptide and CF3-Phe47 labeled M2 peptide and measuring F-CF3 cross peaks in 2D 19F19F correlation spectra, we show that M2 tetramers form nanometer-scale clusters in lipid bilayers. This clustering is stronger in cholesterol-containing membranes and phosphatidylethanolamine (PE) membranes than in cholesterol-free phosphatidylcholine and phosphatidylglycerol membranes. The observed correlation peaks indicate that Phe47 sidechains from different tetramers are less than ~2 nm apart. 1H19F correlation peaks between lipid chain protons and fluorinated Phe47 indicate that Phe47 is more deeply inserted into the lipid bilayer in the presence of cholesterol than in its absence, suggesting that Phe47 preferentially interacts with cholesterol. Static 31P NMR spectra indicate that M2 induces negative Gaussian curvature in the PE membrane. These results suggest that M2 tetramers cluster at cholesterol- and PE-rich regions of cell membranes to cause membrane curvature, which in turn can facilitate membrane scission in the last step of virus budding and release.

Evolution of dislocation loops and effect of annealing temperature on hydrogen-ion-implanted Fe-based binary alloys

Reduced activation ferritic/martensitic (RAFM) steels have been considered as a family of prime candidate structural materials for fusion reactors due to low radioactivity and good resistance to irradiation swelling. Various types of defects such as dislocation loops can form in these materials during irradiation. Effects of alloying elements in iron on the formation and migration of dislocation loops have been widely investigated. However, most studies dealt with interstitial-type dislocation loops in iron alloys, while very few focused on vacancy-type dislocation loops. Previous high voltage electron microscope (HVEM) studies from the authors' group have shown that interstitial loops are fully eliminated in hydrogen-ion-implanted α-Fe at 500 ℃, only vacancy loops remain and can achieve up to 100 nm in size. The addition of Ni in α-Fe can reduce the formation temperature of vacancy-type dislocation loops (<i>T</i><sub>c</sub>) to ~450 ℃, while the addition of Cr can increase the temperature to above 600 ℃. However, these experiments are usually difficult to perform due to the scarce resource of HVEM facilities. In this work, in-situ observations by conventional transmission electron microscope (CTEM, 200 kV) are systematically carried out on the hydrogen-ion-implanted α-Fe and Fe-based binary alloys (Fe-3wt.%Cr, Fe-1.4wt.%Ni and Fe-1.4wt.%Mn). The evolutions of morphology and average size of dislocation loops under different annealing temperatures are investigated. The formation temperatures of vacancy-type dislocation loops are determined from the change of average loop size with annealing temperature. The results are consistent with previous studies by HVEM. The effect of Mn atoms in α-Fe is similar to that of Cr atoms, which leads to <i>T</i><sub>c</sub> increase, and the addition of Ni in α-Fe can reduce <i>T</i><sub>c</sub>. Furthermore, the results of D thermal desorption spectrum analysis show that <i>T</i><sub>c</sub> is affected by the binding and release process of hydrogen isotopes to vacancies in α-Fe. Alloying element Ni promotes the binding and release of hydrogen isotopes to vacancies, which leads to<i> T</i><sub>c</sub> decrease. Cr and Mn inhibit the binding and release of hydrogen isotopes to vacancies, causing <i> T</i><sub>c</sub> to increase.

Trace Element Conundrum of Natural Quasicrystals

We report laser ablation inductively coupled plasma mass spectrometry measurements of the trace element contents of the two naturally occurring quasicrystalline minerals, Al63Cu24Fe13 icosahedrite and Al71Ni24Fe5 decagonite, from their type locality in the Khatyrka meteorite. The isolated quasicrystal fragments were mounted separately from any matrix and are larger than the laser beam diameter. When the elements are sorted in order of volatility, a systematic and unique pattern emerges in both bulk natural quasicrystal specimens. They are highly depleted compared to primitive solar system materials (chondritic meteorites) in moderately refractory elements (those with 50% condensation temperatures near 1350–1300 K; V, Co, Mg, Cr) and significantly enriched in moderately volatile elements (those with 50% condensation temperatures between 1250 and 500 K; Sb, B, Ag, Sn, Bi). We compare the chondrite-normalized trace element patterns and ratios of the quasicrystals to those of scoriaceous cosmic spherules and other meteoritic components. The nonmonotonic shapes of the chondrite-normalized trace element patterns in both icosahedrite and decagonite are incompatible with a single condensation process from the gas of the solar nebula. Previous transmission electron microscopy studies show that the natural quasicrystals contain 3–5 vol % of silicate and oxide nanoparticle inclusions, which we consider to be the main host of the measured trace elements. On this basis, we construct a three-stage model for the formation of the quasicrystals and their inclusions: a high-temperature condensation stage and a low-temperature vapor-fractionation stage to make nanoparticles, followed by a third stage that leads to the formation of quasicrystals incorporating the two different types of nanoparticles and their incorporation into the CV chondrite parent body of the Khatyrka meteorite.

On the atomic structure of Pt(1 1 1)/γ-Al2O3(1 1 1) interfaces and the changes in their interfacial energy with temperature and oxygen pressure

Metal/metal-oxide interfaces are important in many applications including metal coatings, sensing, and catalysis. Here weinvestigate and predict the structure and chemical bonding of the interface between γ-Al2O3and Pt using density functional theory. Previous high-resolution transmission electron microscopy studies showed atomically flat interfaces between the (111) planes of Pt and γ-Al2O3;this interfacial orientation is studied herein. A simulatedtemperature–pressure equilibrium configurationdiagram shows howthe stability of the interfaces changes with temperature and oxygen pressure.Of the three interfacial terminations (O, Al, Al2) the oxygen-terminated γ-Al2O3(111) is the most stable at atmospheric conditions. The stability of this interface is attributed to the strong electrostatic interaction between the Pt and the oxygen atoms at the oxygen-terminated interface. This work provides essential insights into the atomic structure and the origins of the interactions at the interface between transition metals and metal-oxides, as well as interfacial configuration at a range of conditions.

Plasmodium falciparum parasites exit the infected erythrocyte after haemolysis with saponin and streptolysin O.

Malaria is caused by unicellular parasites of the genus Plasmodium, which reside in erythrocytes during the clinically relevant stage of infection. To separate parasite from host cell material, haemolytic agents such as saponin are widely used. Previous electron microscopy studies on saponin-treated parasites reported both, parasites enclosed by the erythrocyte membrane and liberated from the host cell. These ambiguous reports prompted us to investigate haemolysis by live-cell time-lapse microscopy. Using either saponin or streptolysin O to lyse Plasmodium falciparum-infected erythrocytes, we found that ring-stage parasites efficiently exit the erythrocyte upon haemolysis. For late-stage parasites, we found that only approximately half were freed, supporting the previous electron microscopy studies. Immunofluorescence imaging indicated that freed parasites were surrounded by the parasitophorous vacuolar membrane. These results may be of interest for future work using haemolytic agents to enrich for parasite material.

The dynamics of ultrastructural changes during Entamoeba invadens encystation.

Entamoeba histolytica infection causes amoebiasis, which is a global public health problem. The major route of infection is oral ingestion of E. histolytica cysts, cysts being the sole form responsible for host-to-host transmission. Cysts are produced by cell differentiation from proliferative trophozoites in a process termed 'encystation'. Therefore, encystation is an important process from a medical as well as a biological perspective. Previous electron microscopy studies have shown the ultrastructure of precysts and mature cysts; however, the dynamics of ultrastructural changes during encystation were ambiguous. Here, we analysed a series of Entamoeba invadens encysting cells by transmission electron microscopy. Entamoeba invadens is a model for encystation and the cells were prepared by short interval time course sampling from in vitro encystation-inducing cultures. We related sampled cells to stage conversion, which was monitored in the overall population by flow cytometry. The present approach revealed the dynamics of ultrastructure changes during E. invadens encystation. Importantly, the results indicate a functional linkage of processes that are crucial in encystation, such as glycogen accumulation and cyst wall formation. Hence, this study provides a reference for studying sequential molecular events during Entamoeba encystation.

Structure Determination of Er Doped Ti-Al-Nb Alloy by Neutron Diffraction Analysis.

Neutron diffraction experiments with both conventional powder diffraction setup and under in-situ compressive loading were conducted to investigate the structural origins of large strength and toughness enhancements in Ti-16Al-27Nb alloy after Er addition. The primary phase is determined to be the ordered B2 structure form, in agreement with the previous electron microscopy study. Lattice strains of {210} and {100} planes were measured as a function of applied stress, and elastic anisotropy was found for both, and strong plastic nonlinearity was discovered for (210) reflection. The grain refinement during plastic deformation was proposed by both the 2D diffraction intensity distribution and SEM observations, while stress-induced martensitic phase transition was not observed in this study. It is believed that the activation of different slip systems and grain refinement might be the structural origin of the novel mechanical properties of this alloy.

Impaired Myelination in the Aging Primate Brain: Quantification using a Combination of Novel Label‐Free Spectral Confocal Reflectance (SCoRe) Microscopy and Fluorescent Immunohistochemistry (IHC)

IntroductionNormal aging in the rhesus monkey is characterized by cognitive decline in learning, memory, and executive function. Monkeys are free of frank neurodegenerative diseases like Alzheimer's, and cortical neurons do not degenerate. Instead, age‐related myelin pathology accumulates and correlates with cognitive decline. Hence, quantifying myelination and assessing individual myelin sheaths is paramount to the study of normal aging. One of the challenges in understanding white matter pathology is the absence of suitable methods to measure myelin dynamics in the primate brain. While electron microscopy has identified ballooning degeneration and loss of myelin sheaths at the ultrastructural level, it has limited applicability to examination of large brain regions and requires unique fixation procedures that make other analyses difficult. Similarly, traditional myelin stains are very difficult to quantify and regular immunohistochemical markers fail to show the pathology directly. As an alternative, we investigated using the novel, label‐free microscopy method SCoRe (spectral confocal reflectance microscopy) in combination with IHC markers of normal myelin to measure myelin integrity in the aging primate brain.MethodsUsing SCoRe and IHC in brain tissue of 10 behaviorally tested adult rhesus monkeys (5 young and 5 old), we examined myelination in the cingulum bundle and cingulate gyrus of the frontal lobe, two regions previously identified for their involvement in cognitive functions compromised in aging. SCoRe was used to visualize individual myelinated axons in combination with a paranodal marker (anti‐Caspr) and axonal marker (anti‐neurofilament H—NFH). Specific measurements were made of the internode length of myelinated axons (anti‐Caspr + SCoRe) and percent of myelinated fibers (anti‐NFH + SCoRe). We also assessed remyelination efforts using a novel, selective antibody (BCAS1) marking actively myelinating oligodendrocytes (anti‐BCAS + anti‐CC1).ResultsWe found that the percentage of myelinated fibers decreases in the aging brain, which is consistent with previous findings in mouse, non‐human primate, and human. We also report an age‐related decrease in internode length and an increase in paranodes that support previous electron microscopy studies in the non‐human primate brain. Additionally, there is a decrease in the number of actively myelinating oligodendrocytes in the aging brain, corroborating previous data showing impaired remyelination with age.ConclusionsThese results demonstrate that we can use label‐free confocal microscopy techniques combined with IHC to quantify age‐related changes in myelin in the brain. Furthermore, these results suggest that myelin disturbances may be exacerbated (or caused) by a decrease in actively myelinating oligodendrocytes and provide an area for further research into the mechanisms of white matter pathology. This combination of techniques should be widely applicable in the study of white matter pathology that develops in health and disease.Support or Funding Information[Supported by NIH Grants R01‐AG04364 and R21‐061678]This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Therapeutic potential of intravesical injections of platelet-rich plasma in the treatment of lower urinary tract disorders due to regenerative deficiency.

The bladder urothelium plays an important role of barrier function to prevent influx of urinary toxic substance and bacteria. When there is insult to the urinary bladder, the urothelium will start to regenerate on injury. However, several factors might affect the regenerative function of bladder urothelium, including aging, chronic inflammation, and system diseases such as diabetes and chronic kidney diseases (CKDs). Impairment of bladder mucosal regenerative function might result in defective urothelial cell differentiation as well as barrier function, which might be the underlying pathophysiology of interstitial cystitis/bladder pain syndrome (IC/BPS) and recurrent bacterial cystitis. Our previous immunohistochemistry (IHC) study and electron microscopic study revealed that the loss of normal umbrella cells and defective junction proteins in IC/BPS and recurrent cystitis. Platelet-rich plasma (PRP) has been previously used in many medical aspects as regenerative medicine therapy. PRP is rich in many growth factors and cytokines which modulate the process of inflammation and regeneration in the wound healing process. Recent pilot studies have shown that intravesical PRP injections improve IC symptoms and yield a success rate of 70% at 3 months after treatment. The results highly suggest that PRP injection could improve urothelial regenerative function and reduce chronic inflammation in IC patients. This article reviews recently published researches on the urothelial dysfunction biomarkers, urothelial cell differentiation, and urinary regenerative and inflammatory proteins in patients with IC/BPS or recurrent bacterial cystitis. The pathophysiology of the insufficient urothelial regeneration and differentiation; and chronic inflammation may induce urothelial dysfunction and further affect the regenerative ability of the diseased bladder urothelium in IC/BPS and recurrent bacterial cystitis are discussed.

Impact of F1Fo-ATP-synthase dimer assembly factors on mitochondrial function and organismic aging.

In aerobic organisms, mitochondrial F1Fo-ATP-synthase is the major site of ATP production. Beside this fundamental role, the protein complex is involved in shaping and maintenance of cristae. Previous electron microscopic studies identified the dissociation of F1Fo-ATP-synthase dimers and oligomers during organismic aging correlating with a massive remodeling of the mitochondrial inner membrane. Here we report results aimed to experimentally proof this impact and to obtain further insights into the control of these processes. We focused on the role of the two dimer assembly factors PaATPE and PaATPG of the aging model Podospora anserina. Ablation of either protein strongly affects mitochondrial function and leads to an accumulation of senescence markers demonstrating that the inhibition of dimer formation negatively influences vital functions and accelerates organismic aging. Our data validate a model that links mitochondrial membrane remodeling to aging and identify specific molecular components triggering this process.

Cocaine increases dopaminergic connectivity in the nucleus accumbens

The development of addictive behavior is associated with functional and structural plasticity in the mesocorticolimbic pathway. Increased connectivity upon cocaine administration has been inferred from increases in dendritic spine density, but without observations of presynaptic elements. Recently, we established a method that enables analyses of both dendritic spines and glutamatergic boutons and presented evidence that cocaine induces changes in striatal connectivity. As the pharmacological and behavioral effects of cocaine directly implicate dopaminergic neurons and their afferents, a remaining question is whether dopaminergic striatal innervations also undergo structural plasticity. To address this issue, we generated transgenic mice in which the fluorophore tdTomato is expressed under the promoter of the dopamine transporter gene. In these mice, specific labeling of dopaminergic boutons was observed in the striatum. Of note, the accordance of our results for control mice with previous electron microscopy studies confirms that our method can be used to decipher the spatial organization of boutons in relation to dendritic elements. Following repeated cocaine administration that led to behavioral locomotor sensitization, an increased density of dopaminergic boutons was observed 1day later in the nucleus accumbens shell specifically, and not in other striatal regions. Combined labeling of dopaminergic boutons and striatal dendrites showed that cocaine significantly increased the percentage of dendritic spines associated with a dopaminergic bouton. Our results show that chronic cocaine administration induces structural plasticity of dopaminergic boutons that could participate in dopamine-dependent neuronal adaptations in the striatum.

Mechanisms of fine extinction band development in vein quartz: new insights from correlative light and electron microscopy

Fine extinction bands (FEBs) (also known as deformation lamellae) visible with polarized light microscopy in quartz consist of a range of nanostructures, inferring different formation processes. Previous transmission electron microscopy studies have shown that most FEB nanostructures in naturally deformed quartz are elongated subgrains formed by recovery of dislocation slip bands. Here we show that three types of FEB nanostructure occur in naturally deformed vein quartz from the low-grade metamorphic High-Ardenne slate belt (Belgium). Prismatic oriented FEBs are defined by bands of dislocation walls. Dauphine twin boundaries present along the FEB boundaries probably formed after FEB formation. In an example of two sub-rhombohedral oriented FEBs, developed as two sets in one grain, the finer FEB set consists of elongated subgrains, similar to FEBs described in previous transmission electron microscopy studies. The second wider FEB set consists of bands with different dislocation density and fluid-inclusion content. The wider FEB set is interpreted as bands with different plastic strain associated with the primary growth banding of the vein quartz grain. The nanometre-scale fluid inclusions are interpreted to have formed from structurally bounded hydroxyl groups that moreover facilitated formation of the elongate subgrains. Larger fluid inclusions aligned along FEBs are explained by fluid-inclusion redistribution along dislocation cores. The prismatic FEB nanostructure and the relation between FEBs and growth bands have not been recognized before, although related structures have been reported in experimentally deformed quartz.

Spatial distribution of synapses on tyrosine hydroxylase–expressing juxtaglomerular cells in the mouse olfactory glomerulus

AbstractOlfactory sensory axons converge in specific glomeruli where they form excitatory synapses onto dendrites of mitral/tufted (M/T) and juxtaglomerular (JG) cells, including periglomerular (PG), external tufted (ET), and superficial‐short axon cells. JG cells consist of heterogeneous subpopulations with different neurochemical, physiological, and morphological properties. Among JG cells, previous electron microscopic (EM) studies have shown that the majority of synaptic inputs to tyrosine hydroxylase (TH)‐immunoreactive neurons were asymmetrical synapses from olfactory nerve (ON) terminals. However, recent physiological results revealed that 70% of dopaminergic/γ‐aminobutyric acid (GABA)ergic neurons received polysynaptic inputs via ET cells, whereas the remaining 30% received monosynaptic ON inputs. To understand the discrepancies between EM and physiological data, we used serial EM analysis combined with confocal laser scanning microscope images to examine the spatial distribution of synapses on dendrites using mice expressing enhanced green fluorescent protein under the control of the TH promoter. The majority of synaptic inputs to TH‐expressing JG cells were from ON terminals, and they preferentially targeted distal dendrites from the soma. On the other hand, the numbers of non‐ON inputs were fewer and targeted proximal dendrites. Furthermore, individual TH‐expressing JG cells formed serial synapses, such as M/T→TH→another presumed M/T or ON→TH→presumed M/T, but not reciprocal synapses. Serotonergic fibers also associated with somatic regions of TH neurons, displaying non‐ON profiles. Thus, fewer proximal non‐ON synapses provide more effective inputs than large numbers of distal ON synapses and may occur on the physiologically characterized population of dopaminergic‐GABAergic neurons (70%) that receive their most effective inputs indirectly via an ON→ET→TH circuit. J. Comp. Neurol. 525:1059–1074, 2017. © 2016 Wiley Periodicals, Inc.

Spatial distribution of synapses on tyrosine hydroxylase-expressing juxtaglomerular cells in the mouse olfactory glomerulus.

Olfactory sensory axons converge in specific glomeruli where they form excitatory synapses onto dendrites of mitral/tufted (M/T) and juxtaglomerular (JG) cells, including periglomerular (PG), external tufted (ET), and superficial-short axon cells. JG cells consist of heterogeneous subpopulations with different neurochemical, physiological, and morphological properties. Among JG cells, previous electron microscopic (EM) studies have shown that the majority of synaptic inputs to tyrosine hydroxylase (TH)-immunoreactive neurons were asymmetrical synapses from olfactory nerve (ON) terminals. However, recent physiological results revealed that 70% of dopaminergic/γ-aminobutyric acid (GABA)ergic neurons received polysynaptic inputs via ET cells, whereas the remaining 30% received monosynaptic ON inputs. To understand the discrepancies between EM and physiological data, we used serial EM analysis combined with confocal laser scanning microscope images to examine the spatial distribution of synapses on dendrites using mice expressing enhanced green fluorescent protein under the control of the TH promoter. The majority of synaptic inputs to TH-expressing JG cells were from ON terminals, and they preferentially targeted distal dendrites from the soma. On the other hand, the numbers of non-ON inputs were fewer and targeted proximal dendrites. Furthermore, individual TH-expressing JG cells formed serial synapses, such as M/T→TH→another presumed M/T or ON→TH→presumed M/T, but not reciprocal synapses. Serotonergic fibers also associated with somatic regions of TH neurons, displaying non-ON profiles. Thus, fewer proximal non-ON synapses provide more effective inputs than large numbers of distal ON synapses and may occur on the physiologically characterized population of dopaminergic-GABAergic neurons (70%) that receive their most effective inputs indirectly via an ON→ET→TH circuit. J. Comp. Neurol. 525:1059-1074, 2017. © 2017 Wiley Periodicals, Inc.

Excitatory/inhibitory balance of serotonergic axon connectivity in the brain

Serotonin neurons originate from the brainstem raphe nuclei and innervate the entire brain to regulate mood, emotion, sleep, appetite and aggression. Previous electron microscopy (EM) studies have revealed that 5-HT boutons directly contact several neuronal populations via asymmetrical (excitatory) or symmetrical (inhibitory) synapses. Additionally, 5-HT boutons sometimes form “triads” with the pre and postsynaptic components of asymmetrical or symmetrical synapses to modulate their activity. However, the exact proportion and distribution of 5-HT excitatory/inhibitory synapses and triads within the entire brain remains poorly described. Recently, we have published a novel semi-quantitative approach which combines fluorescent confocal microscopy and 3D reconstruction of 5-HT fibers apposed to excitatory and inhibitory neurochemical synapses (triads). Here, we review the similarities and differences in the distribution of 5-HT asymmetrical/symmetrical synapses observed in EM and the distribution of 5-HT excitatory/inhibitory triads quantified in our recent study. We further put into perspective the possible physiological role played by 5-HT triads in the regulation of glutamate and GABA signaling in these various brain regions.

Orientation Mapping and In Situ Annealing Applied for Characterization of Changes in Aluminium Alloys after Deformation

The paper presents application of combined in situ annealing and orientation mapping technique for investigation the microstructural changes in the aluminium alloy 6013 with bimodal distribution of the second phase particles during recovery and recrystallization processes. Information about grain distribution, misorientation between grains, size and shape of the grains at each stage of recrystallization process were obtained. Complexity of the experimental procedure is defined and discussed in order to avoid artificial results. Although the article described the advantages and disadvantages of those method used in transmission electron microscopy and scanning electron microscopy in the work the results from scanning electron microscopy/electron backscattering diraction in situ heating experiments are highlighted. Obtained data are in a good agreement with previous transmission electron microscopy and calorimetry studies. DOI: 10.12693/APhysPolA.130.979 PACS/topics: 68.37.Hk, 81.40.Lm, 81.70.‐q

Mapping the connectivity of serotonin transporter immunoreactive axons to excitatory and inhibitory neurochemical synapses in the mouse limbic brain

Serotonin neurons arise from the brainstem raphe nuclei and send their projections throughout the brain to release 5-HT which acts as a modulator of several neuronal populations. Previous electron microscopy studies in rats have morphologically determined the distribution of 5-HT release sites (boutons) in certain brain regions and have shown that 5-HT containing boutons form synaptic contacts that are either symmetric or asymmetric. In addition, 5-HT boutons can form synaptic triads with the pre- and postsynaptic specializations of either symmetrical or asymmetrical synapses. However, due to the labor intensive processing of serial sections required by electron microscopy, little is known about the neurochemical properties or the quantitative distribution of 5-HT triads within whole brain or discrete subregions. Therefore, we used a semi-automated approach that combines immunohistochemistry and high-resolution confocal microscopy to label serotonin transporter (SERT) immunoreactive axons and reconstruct in 3D their distribution within limbic brain regions. We also used antibodies against key pre- (synaptophysin) and postsynaptic components of excitatory (PSD95) or inhibitory (gephyrin) synapses to (1) identify putative 5-HTergic boutons within SERT immunoreactive axons and, (2) quantify their close apposition to neurochemical excitatory or inhibitory synapses. We provide a 5-HTergic axon density map and have determined the ratio of synaptic triads consisting of a 5-HT bouton in close proximity to either neurochemical excitatory or inhibitory synapses within different limbic brain areas. The ability to model and map changes in 5-HTergic axonal density and the formation of triadic connectivity within whole brain regions using this rapid and quantitative approach offers new possibilities for studying neuroplastic changes in the 5-HTergic pathway.