Multiple Dendrites Research Articles

Twin experiments and detailed investigation of data assimilation system for columnar dendrite growth in thin film

Accurately predicting dendrite growth during alloy solidification is crucial for enhancing the quality of metallic products. Recently, data assimilation has emerged as a promising tool for integrating two cutting-edge methods for studying dendritic growth, viz. in situ X-ray observation experiments and phase-field (PF) simulations, to elucidate important parameter(s) of the simulation and produce a “digital twin” of a growing dendrite. This study was conducted to evaluate the performance of a data assimilation system, employing an ensemble Kalman filter, through systematic twin experiments focused on columnar dendrite growth in a thin film of a binary alloy. The results show that the data assimilation system effectively estimates multiple parameters and dendrite morphology simultaneously. Furthermore, two approaches—domain decomposition method and parameter estimation method from a part of the domain—to reduce computational costs are investigated. Both methods can effectively reduce the computational cost of data assimilation. Additionally, data assimilation using voxel observation data of varying resolutions, mimicking actual X-ray images, is performed. The study findings discourage the direct use of voxel data owing to incompatible PF simulations. PF relaxation computation is explored as a solution for generating observation data with smooth PF profiles. The results show that while relaxation computation enhances the estimation accuracy for high-resolution voxel data, its efficacy diminishes for low-resolution data. These detailed investigations of data assimilation provide valuable insights for utilizing actual in situ X-ray observation data in dendrite growth studies.

Rab6-Mediated Polarized Transport of Synaptic Vesicle Precursors Is Essential for the Establishment of Neuronal Polarity and Brain Formation.

Neurons are highly polarized cells that are composed of a single axon and multiple dendrites. Axon-dendrite polarity is essential for proper tissue formation and brain functions. Intracellular protein transport plays an important role in the establishment of neuronal polarity. However, the regulatory mechanism of polarized transport remains unclear. Here, we show that Rab6, a small GTPase that acts on the regulation of intracellular vesicular trafficking, plays key roles in neuronal polarization and brain development. Central nervous system-specific Rab6a/b double knock-out (Rab6 DKO) mice of both sexes exhibit severe dysplasia of the neocortex and the cerebellum. In the Rab6 DKO neocortex, impaired axonal extension of neurons results in hypoplasia of the intermediate zone. In vitro, deletion of Rab6a and Rab6b in cultured neurons from both sexes causes the abnormal accumulation of synaptic vesicle precursors (SVPs) adjacent to the Golgi apparatus, which leads to defects in axonal extension and the loss of axon-dendrite polarity. Moreover, Rab6 DKO causes significant expansion of lysosomes in the soma in neurons. Overall, our results reveal that Rab6-mediated polarized transport of SVPs is crucial for neuronal polarization and subsequent brain formation.

Kinesin Regulation in the Proximal Axon is Essential for Dendrite-selective Transport.

Neurons are polarized and typically extend multiple dendrites and one axon. To maintain polarity, vesicles carrying dendritic proteins are arrested upon entering the axon. To determine whether kinesin regulation is required for terminating anterograde axonal transport, we overexpressed the dendrite-selective kinesin KIF13A. This caused mistargeting of dendrite-selective vesicles to the axon and a loss of dendritic polarity. Polarity was not disrupted if the kinase MARK2/Par1b was coexpressed. MARK2/Par1b is concentrated in the proximal axon, where it maintains dendritic polarity-likely by phosphorylating S1371 of KIF13A, which lies in a canonical 14-3-3 binding motif. We probed for interactions of KIF13A with 14-3-3 isoforms and found that 14-3-3β and 14-3-3ζ bound KIF13A. Disruption of MARK2 or 14-3-3 activity by small molecule inhibitors caused a loss of dendritic polarity. These data show that kinesin regulation is integral for dendrite-selective transport. We propose a new model in which KIF13A that moves dendrite-selective vesicles in the proximal axon is phosphorylated by MARK2. Phosphorylated KIF13A is then recognized by 14-3-3, which causes dissociation of KIF13A from the vesicle and termination of transport. These findings define a new paradigm for the regulation of vesicle transport by localized kinesin tail phosphorylation, to restrict dendrite-selective vesicles from entering the axon.

In Vitro and In Vivo Analysis of Neuronal Polarity.

Neuronal development is characterized by the unidirectional flow of signal from the axon to the dendrites via synapses. Neuronal polarization is a critical step during development that allows the specification of the different neuronal processes as a single axon and multiple dendrites both structurally and functionally, allowing the unidirectional flow of information. Along with extrinsic and intrinsic signaling, a whole network of molecular complexes involved in positive and negative feedback loops play a major role in this critical distinction of neuronal processes. As a result, neuronal morphology is drastically altered during establishment of polarity. In this chapter, we discuss how we can analyze the morphological alterations of neurons in vitro in culture to assess the development and polarity status of the neuron. We also discuss how these studies can be conducted in vivo, where polarity studies pose a greater challenge with promising results for addressing multiple pathological conditions. Our experimental model is limited to rodent hippocampal/cortical neurons in culture and cortical neurons in brain tissues, which are well-characterized model systems for understanding neuronal polarization.

Krupple-like factor 4 (KLF4) methylation signature in host cell in active viral keratitis with epithelial manifestation

HSV1 presents as epithelial or stromal keratitis or keratouveitis and can lead to sight-threatening complications. KLF4, a critical transcription factor, and regulator of cell growth and differentiation, is essential in corneal epithelium stratification and homeostasis. Here, we want to understand the epigenetic modification specifically the methylation status of KLF4 in epithelium samples of HSV1 keratitis patients. After obtaining consent, epithelial scrapes were collected from 7 patients with clinically diagnosed HSV1 keratitis and 7 control samples (patients undergoing photorefractive keratectomy). Genomic DNA was isolated from the collected samples using the Qiagen DNeasy Kit. Subsequently, bisulfite modification was performed. The bisulphite-modified DNA was then subjected to PCR amplification using specific primers designed to target the KLF4, ACTB gene region, allowing for the amplification of methylated and unmethylated DNA sequences. The amplified DNA products were separated and visualized on a 3% agarose gel. KLF4 hypermethylation was found in 6 out of 7 (85.71%) eyes with viral keratitis, while 1 eye showed hypomethylation compared to PRK samples. Out of these 6, there were 2 each of epithelial dendritic keratitis, epithelial geographical keratitis, and neurotrophic keratitis. The patient with hypomethylated KLF4 had a recurrent case of HSV1 keratitis with multiple dendrites and associated vesicular lesions of the lip along with a history of fever. KLF4 hypermethylation in most viral keratitis cases indicated the under functioning of KLF4 and could indicate a potential association between KLF4 hypermethylation and the development or progression of HSV1 keratitis.

Microglia are dispensable for developmental dendrite pruning of mitral cells in mice.

During early development, neurons in the brain often form excess synaptic connections. Later, they strengthen some connections while eliminating others to build functional neuronal circuits. In the olfactory bulb, a mitral cell initially extends multiple dendrites to multiple glomeruli but eventually forms a single primary dendrite through the activity-dependent dendrite pruning process. Recent studies have reported that microglia facilitate synapse pruning during the circuit remodeling in some systems. It has remained unclear whether microglia are involved in the activity-dependent dendrite pruning in the developing brains. Here, we examined whether microglia are required for the developmental dendrite pruning of mitral cells in mice. To deplete microglia in the fetal brain, we treated mice with a CSF1R inhibitor, PLX5622, from pregnancy. Microglia were reduced by >90% in mice treated with PLX5622. However, dendrite pruning of mitral cells was not significantly affected. Moreover, we found no significant differences in the number, density, and size of excitatory synapses formed in mitral cell dendrites. We also found no evidence for the role of microglia in the activity-dependent dendrite remodeling of layer 4 neurons in the barrel cortex. In contrast, the density of excitatory synapses (dendritic spines) in granule cells in the olfactory bulb was significantly increased in mice treated with PLX5622 at P6, suggesting a role for the regulation of dendritic spines. Our results indicate that microglia do not play a critical role in activity-dependent dendrite pruning at the neurite level during early postnatal development in mice.Significance StatementSynapse elimination is essential for activity-dependent circuit remodeling in the developing brains of mammals. Recent studies suggested that microglia play a critical role in the synapse elimination. This study found that microglia are dispensable for the activity-dependent dendrite pruning in developing mitral cells and layer 4 neurons in the barrel cortex. Thus, microglia are not critical for activity-dependent dendrite pruning at the neurite level during normal developmental process.

Climbing fiber multi-innervation of mouse Purkinje dendrites with arborization common to human.

Canonically, each Purkinje cell (PC) in the adult cerebellum receives only one climbing fiber (CF) from the inferior olive. Underlying current theories of cerebellar function is the notion that this highly conserved one-to-one relationship renders Purkinje dendrites into a single computational compartment. However, we discovered that multiple primary dendrites are a near-universal morphological feature in humans. Using tract tracing, immunolabeling, and in vitro electrophysiology, we found that in mice ~25% of mature multibranched cells receive more than one CF input. Two-photon calcium imaging in vivo revealed that separate dendrites can exhibit distinct response properties to sensory stimulation, indicating that some multibranched cells integrate functionally independent CF-receptive fields. These findings indicate that PCs are morphologically and functionally more diverse than previously thought.

A mathematical study of the efficacy of possible negative feedback pathways involved in neuronal polarization

Neuronal polarization, a process wherein nascent neurons develop a single long axon and multiple short dendrites, can occur within in vitro cell cultures without environmental cues. This is an apparently random process in which one of several short processes, called neurites, grows to become long, while the others remain short. In this study, we propose a minimum model for neurite growth, which involves bistability and random excitations reflecting actin waves. Positive feedback is needed to produce the bistability, while negative feedback is required to ensure that no more than one neurite wins the winner-takes-all contest. By applying the negative feedback to different aspects of the neurite growth process, we demonstrate that targeting the negative feedback to the excitation amplitude results in the most persistent polarization. Also, we demonstrate that there are optimal ranges of values for the neurite count, and for the excitation rate and amplitude that best maintain the polarization. Finally, we show that a previously published model for neuronal polarization based on competition for limited resources shares key features with our best-performing minimal model: bistability and negative feedback targeted to the size of random excitations.

SNAP: a structure-based neuron morphology reconstruction automatic pruning pipeline.

Neuron morphology analysis is an essential component of neuron cell-type definition. Morphology reconstruction represents a bottleneck in high-throughput morphology analysis workflow, and erroneous extra reconstruction owing to noise and entanglements in dense neuron regions restricts the usability of automated reconstruction results. We propose SNAP, a structure-based neuron morphology reconstruction pruning pipeline, to improve the usability of results by reducing erroneous extra reconstruction and splitting entangled neurons. For the four different types of erroneous extra segments in reconstruction (caused by noise in the background, entanglement with dendrites of close-by neurons, entanglement with axons of other neurons, and entanglement within the same neuron), SNAP incorporates specific statistical structure information into rules for erroneous extra segment detection and achieves pruning and multiple dendrite splitting. Experimental results show that this pipeline accomplishes pruning with satisfactory precision and recall. It also demonstrates good multiple neuron-splitting performance. As an effective tool for post-processing reconstruction, SNAP can facilitate neuron morphology analysis.

Development of a data assimilation system for the investigation of the dendrite solidification process by integrating in situ X-ray imaging and phase-field simulation

The dendrite solidification process has been observed and simulated using state-of-the-art techniques, such as time-resolved X-ray tomography (4D-CT) and high-performance phase-field (PF) simulations. 4D-CT has enabled the direct observation of the 3D dendrite growth in opaque alloys. However, the spatiotemporal resolution is not sufficient for investigating fast phenomena because a 3D solidification structure is obtained using hundreds of transmission images during the 180° rotation of a sample. High-performance PF simulations have enabled the simulation of multiple 3D dendrite growth phenomena. However, the material properties required in PF solutions of alloys are often unavailable. Therefore, integrating in situ X-ray observations with PF simulations using data assimilation is a promising approach for simultaneously solving these issues. In this study, we developed a data assimilation system with an ensemble Kalman filter, in which the solid fraction along the thickness of a sample was used as observation data to enable data assimilation using X-ray transmission images. The performance of the developed data assimilation system was evaluated via twin experiments for columnar dendrite growth during the directional solidification of a binary alloy in a thin film. The results showed that data assimilation using the solid fraction as observation data estimated the material properties and solidification morphologies with reasonable accuracy.

Synaptic connectivity of the TRPV1-positive trigeminal afferents in the rat lateral parabrachial nucleus.

Recent studies have shown a direct projection of nociceptive trigeminal afferents into the lateral parabrachial nucleus (LPBN). Information about the synaptic connectivity of these afferents may help understand how orofacial nociception is processed in the LPBN, which is known to be involved primarily in the affective aspect of pain. To address this issue, we investigated the synapses of the transient receptor potential vanilloid 1-positive (TRPV1+) trigeminal afferent terminals in the LPBN by immunostaining and serial section electron microscopy. TRPV1 + afferents arising from the ascending trigeminal tract issued axons and terminals (boutons) in the LPBN. TRPV1+ boutons formed synapses of asymmetric type with dendritic shafts and spines. Almost all (98.3%) TRPV1+ boutons formed synapses with one (82.6%) or two postsynaptic dendrites, suggesting that, at a single bouton level, the orofacial nociceptive information is predominantly transmitted to a single postsynaptic neuron with a small degree of synaptic divergence. A small fraction (14.9%) of the TRPV1+ boutons formed synapses with dendritic spines. None of the TRPV1+ boutons were involved in axoaxonic synapses. Conversely, in the trigeminal caudal nucleus (Vc), TRPV1+ boutons often formed synapses with multiple postsynaptic dendrites and were involved in axoaxonic synapses. Number of dendritic spine and total number of postsynaptic dendrites per TRPV1+ bouton were significantly fewer in the LPBN than Vc. Thus, the synaptic connectivity of the TRPV1+ boutons in the LPBN differed significantly from that in the Vc, suggesting that the TRPV1-mediated orofacial nociception is relayed to the LPBN in a distinctively different manner than in the Vc.

Large-scale phase-field simulations for dendrite growth: A review on current status and future perspective

The current status of large-scale phase-field (PF) simulations for dendrite growth is reviewed by focusing on the study conducted by our group. The discussion includes the competitive growth of multiple columnar dendrites, dendrite growth with liquid flow and solid motion, permeability prediction, and cross-scale simulations using the PF method. All PF simulations introduced here were executed using a graphics processing unit (GPU) or a GPU supercomputer to significantly accelerate the PF simulations. Finally, the future perspectives of large-scale dendrite-growth PF simulations are discussed.

Neuronal mitochondrial morphology is significantly affected by both fixative and oxygen level during perfusion.

Neurons in the brain have a uniquely polarized structure consisting of multiple dendrites and a single axon generated from a cell body. Interestingly, intracellular mitochondria also show strikingly polarized morphologies along the dendrites and axons: in cortical pyramidal neurons (PNs), dendritic mitochondria have a long and tubular shape, while axonal mitochondria are small and circular. Mitochondria play important roles in each compartment of the neuron by generating adenosine triphosphate (ATP) and buffering calcium, thereby affecting synaptic transmission and neuronal development. In addition, mitochondrial shape, and thereby function, is dynamically altered by environmental stressors such as oxidative stress or in various neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. Although the importance of altered mitochondrial shape has been claimed by multiple studies, methods for studying this stress-sensitive organelle have not been standardized. Here we address pertinent steps that influence mitochondrial morphology during experimental processes. We demonstrate that fixative solutions containing only paraformaldehyde (PFA), or that introduce hypoxic conditions during the procedure, induce dramatic fragmentation of mitochondria both in vitro and in vivo. This disruption was not observed following the use of glutaraldehyde (GA) addition or oxygen supplementation, respectively. Finally, using pre-formed fibril α-synuclein treated neurons, we show fixative choice can alter experimental outcomes. Specifically, α-synuclein-induced mitochondrial remodeling could not be observed with PFA only fixation as fixation itself caused mitochondrial fragmentation. Our study provides optimized methods for examining mitochondrial morphology in neurons and demonstrates that fixation conditions are critical when investigating the underlying cellular mechanisms involving mitochondria in physiological and neurodegenerative disease models.

Cells of the Central Nervous System: An Overview of Their Structure and Function.

The central nervous system is the last major organ system in the vertebrate body to yield its cellular structure, due to the complexity of its cells and their interactions. The fundamental unit of the nervous system is the neuron, which forms complex circuits that receive and integrate information and generate adaptive responses. Each neuron is composed of an input domain consisting of multiple dendrites along with the cell body, which is also responsible for the majority of macromolecule synthesis for the cell. The output domain is the axon which is a singular extension from the cell body that propagates the action potential to the synapse, where signals pass from one neuron to another. Facilitating these functions are cohorts of supporting cells consisting of astrocytes, oligodendrocytes and microglia along with NG2 cells and ependymal cells. Astrocytes have a dazzling array of functions including physical support, maintenance of homeostasis, development and integration of synaptic activity. Oligodendrocytes form the myelin sheath which surrounds axons and enables rapid conduction of the nerve impulse. Microglia are the resident immune cells, providing immune surveillance and remodeling of neuronal circuits during development and trauma. All these cells function in concert with each other, producing the remarkably diverse functions of the nervous system.

Application of phase field model coupled with convective effects in binary alloy directional solidification and roll casting processes

Based on the Kim-Kim-Suzuki (KKS) phase field model coupled with the thermodynamic parameters, the transformation process from columnar dendrites to equiaxed crystals during directional solidification of aluminium alloy was simulated, and the effects of phase field parameters on the growth morphology and dendrite segregation were discussed. Furthermore, considering the effect of the microcosmic flow field, the convection influence gradient term is introduced into KKS formula near the solid-liquid interface, and the phase field model considering flow field was applied to the inherent convective environment of the actual roll casting process, also the multiple dendrites growth behavior of magnesium alloy under the action of microscopic convection was further explored. When coupling calculation of microscopic velocity field and pressure field, the staggered grid method was used to deal with the complex interface. The combined solution of Marker in Cell (MAC) algorithm and phase field discrete calculation was realized. In order to further describe the influence of convection on the solidification process, the roll casting experiments are used to verify the impact growth of multiple dendrites under convection. The results show that the dendrites undergo solute remelting and the dendrites melt into equiaxed crystals, showing the phenomenon of Columnar to Equiaxed Transition (CET).

Numerical simulation on fluid flow behavior during 3-dimensional dendrite growth with random preference angle

The flow behavior of three-dimensional (3D) dendrite growth with random preferred angle under natural convection was studied by using the Lattice Boltzmann-Cellular Automata (LB-CA) method with dynamic and static grids. In this model, the temperature field, flow field and solute field calculated by Lattice Boltzmann method (LBM) and dendrite growth calculated by CA method were carried out in static and dynamic grids respectively, and the coupling between LBM and CA was performed by interpolation of calculation parameters between dynamic and static grids. Results show that the asymmetry of solid phase distribution makes the streamline distribution more complex. At the initial stage of multiple dendrites growth, the fluid flow is relatively free. When dendrites grow close to each other, the fluid flow is blocked and can only flow along the gap between dendrites. During the wall equiaxed-columnar-central equiaxed crystals transformation (ECET) process, dense eddy current is formed at the wall equiaxed crystals at first. Then, when the wall equiaxed crystals gradually develop into columnar crystals, the eddy current moves with the solid-liquid interface. When the central equiaxed crystals are formed, the eddy current at the front of the columnar crystals gradually disappears. New eddies appear as the central equiaxed crystal grows.

Cellular automata-lattice Boltzmann simulation of multi-dendrite motion under convection based on dynamic grid technology

A cellular automata-lattice Boltzmann coupling model with dynamic grids for simulating the preferential growth and movement of multiple dendrites under convection of binary alloy melt. In this model, dendritic growth is computed using the cellular automata method, and flow of the melt is computed using the lattice Boltzmann method, and the motion of the dendrite is described by solving the equation of motion. To deal with the motion of the dendrite, each dendrite is given a local dynamic grid, which can move with the motion of the dendrite. The correctness of this model in dealing with the motion of solids in fluids by simulating the settlement of a cylinder in a vertical channel. Then, dendrites of three different preferred growth directions grow under pure diffusion are simulated to prove that the model can also solve the grid dependence of dendritic growth in the Cartesian grid. Finally, this model is used to stimulate the growth of a single dendrite under Couette flow, and the settlement of multiple dendrites under gravity, and the growing motion of multiple dendrites under Poiseuille flow. The simulation results show that the dendritic morphology can be well maintained when this model is used to deal with dendritic movement, and the melt convection and motion will promote the dendritic growth in the direction of motion.

Dendritic Morphology of an Inhibitory Retinal Interneuron Enables Simultaneous Local and Global Synaptic Integration.

Amacrine cells, inhibitory interneurons of the retina, feature synaptic inputs and outputs in close proximity throughout their dendritic trees, making them notable exceptions to prototypical somato-dendritic integration with output transmitted via axonal action potentials. The extent of dendritic compartmentalization in amacrine cells with widely differing dendritic tree morphology, however, is largely unexplored. Combining compartmental modeling, dendritic Ca2+ imaging, targeted microiontophoresis and multielectrode patch-clamp recording (voltage and current clamp, capacitance measurement of exocytosis), we investigated integration in the AII amacrine cell, a narrow-field electrically coupled interneuron that participates in multiple, distinct microcircuits. Physiological experiments were performed with in vitro slices prepared from retinas of both male and female rats. We found that the morphology of the AII enables simultaneous local and global integration of inputs targeted to different dendritic regions. Local integration occurs within spatially restricted dendritic subunits and narrow time windows and is largely unaffected by the strength of electrical coupling. In contrast, global integration across the dendritic tree occurs over longer time periods and is markedly influenced by the strength of electrical coupling. These integrative properties enable AII amacrines to combine local control of synaptic plasticity with location-independent global integration. Dynamic inhibitory control of dendritic subunits is likely to be of general importance for amacrine cells, including cells with small dendritic trees, as well as for inhibitory interneurons in other regions of the CNS.SIGNIFICANCE STATEMENT Our understanding of synaptic integration is based on the prototypical morphology of a neuron with multiple dendrites and a single axon at opposing ends of a cell body. Many neurons, notably retinal amacrine cells, are exceptions to this arrangement, and display input and output synapses interspersed along their dendritic branches. In the large dendritic trees of some amacrine cells, such arrangements can give rise to multiple computational subunits. Other amacrine cells, with small dendritic trees, have been assumed to operate as single computational units. Here, we report the surprising result that despite a small dendritic tree, the AII amacrine cell simultaneously performs local integration of synaptic inputs (over smaller dendritic subregions) and global integration across the entire cell.

Ni nanodendrites prepared by a low-temperature process as electrocatalysts for hydrogen evolution reaction in alkaline solution

It is essential to explore high catalytic activity and stability inexpensive metal electrocatalysts for the hydrogen evolution reaction (HER) to achieve the hydrogen energy generation. Therefore, in this paper, we report a simple low-temperature method which is template-free and environmentally-friendly to fabricate the Ni nanodendrites (Ni NDs) manifesting the excellent HER catalytic performances. This method is a simple one-step reduction, and at 50 °C we obtain the Ni NDs50 that exhibit a low overpotential of 49 mV (10 mA cm−2) and a small Tafel slope of 32.70 mV dec−1 in 1.0 M KOH electrolyte. Besides, the Ni NDs50 exhibit the favorable stability for 22 h. The superior HER performance may be attributed to the exposure of more electrochemical surface areas due to the multiple branching dendrite structure. Hence, our research provides a simple method for large-scale preparing nickel-based electrocatalysts.

Microstructural transition of nanoparticle deposits from multiple dendrites to compact layer

Nanoparticle deposit has various microstructures from multiple dendrites to mesoporous compact structures depending on deposition conditions, and the required microstructures are different depending on its applications. There have been recent reports that the overall porosity of deposit can be predicted by a function of diffusive Knudsen number (KnD) and dimensionless translational energy (χF) of the particle. Unlike compact structures, however, dendritic structures consisting of multiple dendrites cannot be characterized solely by overall porosity and their formation mechanism is not known. In this study, various structures of deposit were produced in a wide range of deposition conditions (KnD: 10−1.5–101.5; χF: 10−3–103) using off-lattice Monte Carlo simulation, and the unique characteristics of the deposit could be visualized by presenting local fractional accumulations of pores as color-filled contours. Furthermore, a spatial autocorrelation was calculated for the contour plot in order to find a quantitative footprint of structural features of nanoparticle deposits, in compact vs dendritic structures. As a result, we successfully identified the dendritic-to-compact structural transition in a quantitative manner, for the first time. Lastly considering the deposition behavior of particles, we developed a new model capable of a priori prediction of the specific deposit structure among compact, dendritic, and transition structures, given the deposition condition in terms of KnD and χF.