Multiple Dendrites Research Articles

Scanless volumetric imaging by selective access multifocal multiphoton microscopy.

Simultaneous, high-resolution imaging across a large number of synaptic and dendritic sites is critical for understanding how neurons receive and integrate signals. Yet, functional imaging that targets a large number of submicrometer-sized synaptic and dendritic locations poses significant technical challenges. We demonstrate a new parallelized approach to address such questions, increasing the signal-to-noise ratio by an order of magnitude compared to previous approaches. This selective access multifocal multiphoton microscopy uses a spatial light modulator to generate multifocal excitation in three dimensions (3D) and a Gaussian-Laguerre phase plate to simultaneously detect fluorescence from these spots throughout the volume. We test the performance of this system by simultaneously recording Ca2+ dynamics from cultured neurons at 98-118 locations distributed throughout a 3D volume. This is the first demonstration of 3D imaging in a "single shot" and permits synchronized monitoring of signal propagation across multiple different dendrites.

Neurotoxicity of the pesticide rotenone on neuronal polarization: a mechanistic approach.

Neurons are the most extensive and polarized cells that display a unique single long axon and multiple dendrites, which are compartments exhibiting structural and functional differences. Polarity occurs early in neuronal development and it is maintained by complex subcellular mechanisms throughout cell life. A well-defined and controlled spatio-temporal program of cellular and molecular events strictly regulates the formation of the axon and dendrites from a non-polarized cell. This event is critical for an adequate neuronal wiring and therefore for the normal functioning of the nervous system. Neuronal polarity is very sensitive to the harmful effects of different factors present in the environment. In this regard, rotenone is a crystalline, colorless and odorless isoflavone used as insecticide, piscicide and broad spectrum pesticide commonly used earlier in agriculture. In the present review we will summarize the toxicity mechanism caused by this pesticide in different neuronal cell types, focusing on a particular biological mechanism whereby rotenone could impair neuronal polarization in cultured hippocampal neurons. Recent advances suggest that the inhibition of axonogenesis produced by rotenone could be related with its effect on microtubule dynamics, the actin cytoskeleton and their regulatory pathways, particularly affecting the small RhoGTPase RhoA. Unveiling the mechanism by which rotenone produces neurotoxicity will be instrumental to understand the cellular mechanisms involved in neurodegenerative diseases influenced by this environmental pollutant, which may lead to research focused on the design of new therapeutic strategies.

CAMSAP3 maintains neuronal polarity through regulation of microtubule stability

The molecular mechanisms that guide each neuron to become polarized, forming a single axon and multiple dendrites, remain unknown. Here we show that CAMSAP3 (calmodulin-regulated spectrin-associated protein 3), a protein that regulates the minus-end dynamics of microtubules, plays a key role in maintaining neuronal polarity. In mouse hippocampal neurons, CAMSAP3 was enriched in axons. Although axonal microtubules were generally acetylated, CAMSAP3 was preferentially localized along a less-acetylated fraction of the microtubules. CAMSAP3-mutated neurons often exhibited supernumerary axons, along with an increased number of neurites having nocodazole-resistant/acetylated microtubules compared with wild-type neurons. Analysis using cell lines showed that CAMSAP3 depletion promoted tubulin acetylation, and conversely, mild overexpression of CAMSAP3 inhibited it, suggesting that CAMSAP3 works to retain nonacetylated microtubules. In contrast, CAMSAP2, a protein related to CAMSAP3, was detected along all neurites, and its loss did not affect neuronal polarity, nor did it cause increased tubulin acetylation. Depletion of α-tubulin acetyltransferase-1 (αTAT1), the key enzyme for tubulin acetylation, abolished CAMSAP3 loss-dependent multiple-axon formation. These observations suggest that CAMSAP3 sustains a nonacetylated pool of microtubules in axons, interfering with the action of αTAT1, and this process is important to maintain neuronal polarity.

Effect of thermal anisotropy on binary alloy dendrite growth

A numerical model to study the effect of thermal anisotropy on binary alloy dendrite growth is presented. The model is based on the volume averaged enthalpy method with explicit surface tension anisotropy for crystal orientation. Thermal anisotropy is incorporated using anisotropic thermal conductivity in the energy equation. This is done by splitting the anisotropic conductivity into two parts, equivalent isotropic conductivity and anisotropic departure source term, enabling the use of a conventional isotropic solver to model anisotropic heat transfer. The proposed model is applied to study the effect of thermal anisotropy ratio on tip velocity, aspect ratio and equivalent radius of an equiaxed grain growing in an undercooled binary alloy melt. It is found that the thermal energy stored in the grain during solidification plays an important role in interface evolution, and thus anisotropic conductivity in the solid affects the grain morphology. There is a consistent increase in the aspect ratio of grains with increase in thermal anisotropy ratio, although the grain volume remains almost invariant. Due to unequal growth rates of the perpendicular arms, severe distortion of the solid crystal is seen at higher thermal anisotropy ratios. The model is further extended to study the growth of multiple dendrites in order to simulate microstructure evolution with thermal anisotropy. It is observed that thermal anisotropy significantly affects the grain morphology at low grain density but has a smaller influence at high grain density as compared to other governing factors such as solute transport.

Of what use is connectomics? A personal perspective on the Drosophila connectome.

The brain is a network of neurons and its biological output is behaviour. This is an exciting age, with a growing acknowledgement that the comprehensive compilation of synaptic circuits densely reconstructed in the brains of model species is now both technologically feasible and a scientifically enabling possibility in neurobiology, much as 30 years ago genomics was in molecular biology and genetics. Implemented by huge advances in electron microscope technology, especially focused ion beam-scanning electron microscope (FIB-SEM) milling (see Glossary), image capture and alignment, and computer-aided reconstruction of neuron morphologies, enormous progress has been made in the last decade in the detailed knowledge of the actual synaptic circuits formed by real neurons, in various brain regions of the fly Drosophila It is useful to distinguish synaptic pathways that are major, with 100 or more presynaptic contacts, from those that are minor, with fewer than about 10; most neurites are both presynaptic and postsynaptic, and all synaptic sites have multiple postsynaptic dendrites. Work on Drosophila has spearheaded these advances because cell numbers are manageable, and neuron classes are morphologically discrete and genetically identifiable, many confirmed by reporters. Recent advances are destined within the next few years to reveal the complete connectome in an adult fly, paralleling advances in the larval brain that offer the same prospect possibly within an even shorter time frame. The final amendment and validation of segmented bodies by human proof-readers remains the most time-consuming step, however. The value of a complete connectome in Drosophila is that, by targeting to specific neurons transgenes that either silence or activate morphologically identified circuits, and then identifying the resulting behavioural outcome, we can determine the causal mechanism for behaviour from its loss or gain. More importantly, the connectome reveals hitherto unsuspected pathways, leading us to seek novel behaviours for these. Circuit information will eventually be required to understand how differences between brains underlie differences in behaviour, and especially to herald yet more advanced connectomic strategies for the vertebrate brain, with an eventual prospect of understanding cognitive disorders having a connectomic basis. Connectomes also help us to identify common synaptic circuits in different species and thus to reveal an evolutionary progression in candidate pathways.

The loss of the kinases SadA and SadB results in early neuronal apoptosis and a reduced number of progenitors.

The neurons that form the mammalian neocortex originate from progenitor cells in the ventricular (VZ) and subventricular zone (SVZ). Newborn neurons are multipolar but become bipolar during their migration from the germinal layers to the cortical plate (CP) by forming a leading process and an axon that extends in the intermediate zone (IZ). Once they settle in the CP, neurons assume a highly polarized morphology with a single axon and multiple dendrites. The AMPK-related kinases SadA and SadB are intrinsic factors that are essential for axon formation during neuronal development downstream of Lkb1. The knockout of both genes encoding Sad kinases (Sada and Sadb) results not only in a loss of axons but also a decrease in the size of the cortical plate. The defect in axon formation has been linked to a function of Sad kinases in the regulation of microtubule binding proteins. However, the causes for the reduced size of the cortical plate in the Sada-/-;Sadb-/- knockout remain to be analyzed in detail. Here we show that neuronal cell death is increased and the number of neural progenitors is decreased in the Sada-/-;Sadb-/- CP. The reduced number of progenitors is a non-cell autonomous defect since they do not express Sad kinases. These defects are restricted to the neocortex while the hippocampus remains unaffected.

Principles that govern competition or co-existence in Rho-GTPase driven polarization.

Rho-GTPases are master regulators of polarity establishment and cell morphology. Positive feedback enables concentration of Rho-GTPases into clusters at the cell cortex, from where they regulate the cytoskeleton. Different cell types reproducibly generate either one (e.g. the front of a migrating cell) or several clusters (e.g. the multiple dendrites of a neuron), but the mechanistic basis for unipolar or multipolar outcomes is unclear. The design principles of Rho-GTPase circuits are captured by two-component reaction-diffusion models based on conserved aspects of Rho-GTPase biochemistry. Some such models display rapid winner-takes-all competition between clusters, yielding a unipolar outcome. Other models allow prolonged co-existence of clusters. We investigate the behavior of a simple class of models and show that while the timescale of competition varies enormously depending on model parameters, a single factor explains a large majority of this variation. The dominant factor concerns the degree to which the maximal active GTPase concentration in a cluster approaches a “saturation point” determined by model parameters. We suggest that both saturation and the effect of saturation on competition reflect fundamental properties of the Rho-GTPase polarity machinery, regardless of the specific feedback mechanism, which predict whether the system will generate unipolar or multipolar outcomes.

Phase-field lattice Boltzmann simulations of multiple dendrite growth with motion, collision, and coalescence and subsequent grain growth

In the formation of a typical equiaxed structure during the solidification of metals and alloys, multiple equiaxed dendrites typically grow with motion, collision, and coalescence and subsequently grain growth occurs after the formation of grain boundaries. In this study, we develop a phase-field lattice Boltzmann model that can simulate these complex formation processes involving equiaxed structures. In this model, multiple dendrites are represented by employing multiple phase-field variables, and the formation of grain boundaries is modeled by simply introducing an interaction term between the phase-field variables. Liquid flow is computed using the lattice Boltzmann method, and the motion of a solid is described by solving the equations of motion. Collision-coalescence representation in the present model was validated by performing simulations of collisions between two circular objects. Furthermore, grain growth was validated through static and dynamic conditions in a simple three-grain system. Good agreements with theoretical solutions were obtained for both cases. Finally, using the developed model, a series of formation processes of multiple-dendrite growth with motion, collision, and coalescence and the subsequent grain growth are successfully performed for the first time.

Phase-field Modeling for Accurate Prediction of Equiaxed Structure with Considering Motion of Multiple Dendrites

Phase-field Modeling for Accurate Prediction of Equiaxed Structure with Considering Motion of Multiple Dendrites

Discovery of long-range inhibitory signaling to ensure single axon formation

A long-standing question in neurodevelopment is how neurons develop a single axon and multiple dendrites from common immature neurites. Long-range inhibitory signaling from the growing axon is hypothesized to prevent outgrowth of other immature neurites and to differentiate them into dendrites, but the existence and nature of this inhibitory signaling remains unknown. Here, we demonstrate that axonal growth triggered by neurotrophin-3 remotely inhibits neurite outgrowth through long-range Ca2+ waves, which are delivered from the growing axon to the cell body. These Ca2+ waves increase RhoA activity in the cell body through calcium/calmodulin-dependent protein kinase I. Optogenetic control of Rho-kinase combined with computational modeling reveals that active Rho-kinase diffuses to growing other immature neurites and inhibits their outgrowth. Mechanistically, calmodulin-dependent protein kinase I phosphorylates a RhoA-specific GEF, GEF-H1, whose phosphorylation enhances its GEF activity. Thus, our results reveal that long-range inhibitory signaling mediated by Ca2+ wave is responsible for neuronal polarization.

Differentiation of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neurons in Mouse Hippocampal Slice Cultures.

Potential clinical applications of neurons derived from human induced pluripotent stem cells (hiPSC-neurons) for drug screening and transplantation therapies have received considerable attention. However, it remains unclear whether and how transplanted hiPSC-neurons are incorporated into pre-existing neural circuits. Here we developed a co-culture system of hiPSC-neurons and mouse hippocampal slices to examine the differentiation of hiPSC-neurons in pre-existing neural circuits. hiPSC-neurons transplanted in mouse hippocampal slices expressed the hippocampal neuron-specific markers HuB and Prox1 after 7 days of culture, while those markers were scarcely expressed in hiPSC-neurons cultured on glass dishes. Furthermore, hiPSC-neurons transplanted in the dentate gyrus (DG) of slice cultures grew to exhibit dentate granule cell-like morphologies, including besom-shaped dendrites. Similarly, hiPSC-neurons transplanted in the CA1 region of slice cultures grew to exhibit CA1 pyramidal cell-like morphologies, including primary apical and multiple basal dendrites with synaptic spines. Additionally, these cells projected axons toward the entorhinal cortex (EC) as observed in vivo. These data suggest that hiPSC-neurons were anatomically integrated into pre-existing neural circuits in a region-specific manner. Thus, the co-culture system will be useful for the study of efficient strategies to differentiate transplanted hiPSC-neurons.

MiR-338-3p regulates neuronal maturation and suppresses glioblastoma proliferation.

Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to learning, memory, and antidepressant efficacy. MicroRNAs (miRNAs) have been previously shown to play an important role in the regulation of neuronal development and neurogenesis in the dentate gyrus via modulation of gene expression. However, this mode of regulation is both incompletely described in the literature thus far and highly multifactorial. In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus. Comparison of miR-338-3p expression with neuronal markers of maturity indicates miR-338-3p is expressed most highly in the mature neuron. We also designed a viral “sponge” to knock down in vivo expression of miR-338-3p. When miR-338-3p is knocked down, neurons sprout multiple primary dendrites that branch off of the soma in a disorganized manner, cellular proliferation is upregulated, and neoplasms form spontaneously in vivo. Additionally, miR-338-3p overexpression in glioblastoma cell lines slows their proliferation in vitro. Further, low miR-338-3p expression is associated with increased mortality and disease progression in patients with glioblastoma. These data identify miR-338-3p as a clinically relevant tumor suppressor in glioblastoma.

GPU-accelerated three-dimensional phase-field simulation of dendrite growth in a nickel-based superalloy

The microstructure formation of a nickel-based superalloy during solidification in three dimensions was investigated using the phase-field method. To accelerate the large-scale phase-field simulation, a parallel computing approach was developed using the graphic processing unit (GPU), and the limitation of insufficient GPU memory was circumvented by employing an asynchronous concurrent algorithm. The performance of the GPU-based parallel computing method was tested and the results demonstrate that a maximum performance of 774.292 GFLOPS (giga floating-point operations per second) can be obtained using a single NVIDIA GTX1080 GPU. In simulations of isothermal solidification, the microstructure evolution of a single and multiple dendrites under different undercooling levels was shown in detail. During the solidification, the dendrite tip growth velocity and fraction solid were recorded and then analyzed. In simulations of directional solidification, the formation of primary dendrite arms under different temperature gradients was investigated, and the simulated microstructure was in good agreement with experimental observations. Additionally, the distribution of primary dendrite arm spacing was quantitatively analyzed by Voronoi tessellation. Finally, simulation of polycrystalline growth in directional solidification was conducted to study the dendrite competitive growth. The unusual overgrowth phenomenon was observed in the initial growth stage, while as the solidification process proceeded, the dendrites with small inclination angles were more likely to overgrow the dendrites with large inclination angles.

Modeling of coupled motion and growth interaction of equiaxed dendritic crystals in a binary alloy during solidification

Motion of growing dendrites is a common phenomenon during solidification but often neglected in numerical simulations because of the complicate underlying multiphysics. Here a phase-field model incorporating dendrite-melt two-phase flow is proposed for simulating the dynamically interacted process. The proposed model circumvents complexity to resolve dendritic growth, natural convection and solid motion simultaneously. Simulations are performed for single and multiple dendritic growth of an Al-based alloy in a gravity environment. Computing results of an isolated dendrite settling down in the convective supersaturated melt shows that solid motion is able to overwhelm solutal convection and causes a rather different growth morphology from the stationary dendrite that considers natural convection alone. The simulated tip growth dynamics are correlated with a modified boundary layer model in the presence of melt flow, which well accounts for the variation of tip velocity with flow direction. Polycrystalline simulations reveal that the motion of dendrites accelerates the occurrence of growth impingement which causes the behaviors of multiple dendrites are distinct from that of single dendrite, including growth dynamics, morphology evolution and movement path. These polycrystalline simulations provide a primary understanding of the sedimentation of crystals and resulting chemical homogeneity in industrial ingots.

Neuronal polarization: From spatiotemporal signaling to cytoskeletal dynamics.

Neuronal polarization establishes distinct molecular structures to generate a single axon and multiple dendrites. Studies over the past years indicate that this efficient separation is brought about by a network of feedback loops. Axonal growth seems to play a major role in fueling those feedback loops and thereby stabilizing neuronal polarity. Indeed, various effectors involved in feedback loops are pivotal for axonal growth by ultimately acting on the actin and microtubule cytoskeleton. These effectors have key roles in interconnecting actin and microtubule dynamics - a mechanism crucial to commanding the growth of axons. We propose a model connecting signaling with cytoskeletal dynamics and neurite growth to better describe the underlying processes involved in neuronal polarization. We will discuss the current views on feedback loops and highlight the current limits of our understanding.

Phase-field simulation of equiaxed structure formation with growth, motion and collision of multiple dendrites

Phase-field simulation of equiaxed structure formation with growth, motion and collision of multiple dendrites

Of microtubules and memory: implications for microtubule dynamics in dendrites and spines.

Microtubules (MTs) are cytoskeletal polymers composed of repeating subunits of tubulin that are ubiquitously expressed in eukaryotic cells. They undergo a stochastic process of polymerization and depolymerization from their plus ends termed dynamic instability. MT dynamics is an ongoing process in all cell types and has been the target for the development of several useful anticancer drugs, which compromise rapidly dividing cells. Recent studies also suggest that MT dynamics may be particularly important in neurons, which develop a highly polarized morphology, consisting of a single axon and multiple dendrites that persist throughout adulthood. MTs are especially dynamic in dendrites and have recently been shown to polymerize directly into dendritic spines, the postsynaptic compartment of excitatory neurons in the CNS. These transient polymerization events into dendritic spines have been demonstrated to play important roles in synaptic plasticity in cultured neurons. Recent studies also suggest that MT dynamics in the adult brain function in the essential process of learning and memory and may be compromised in degenerative diseases, such as Alzheimer’s disease. This raises the possibility of targeting MT dynamics in the design of new therapeutic agents.

An approach to variable-order prediction via multiple distal dendrites of neurons

In this paper, we proposed an extended version of binary code selection algorithm (BCSA) for the variable-order prediction by introducing multiple distal dendrites into BCSA. The proposed model of artificial neurons has a single proximal dendrite to receive the feed-forward inputs (sequences) from the world and multiple distal dendrites to receive the horizontal inputs from nearby neurons. During training, each distal dendrite is able to remember the states of neurons activated at different time and store the temporal correlations. After training, each distal dendrite independently recalls the temporal correlations contained in sequences and makes a local prediction. The variable-order prediction can be obtained by combining these local predictions made by multiple distal dendrites. Experiments show that the proposed method outperforms BCSA and other methods, such as back-propagation networks and radial basis function networks, especially while processing complex sequences.

Ultrastructure of Chemosensilla on Antennae and Tarsi ofEctropis obliqua(Lepidoptera: Geometridae)

Tea geometrid Ectropis obliqua Prout (Lepidoptera: Geometridae) is a devastating defoliator throughout the tea plantations in China. To explore the putative functions of chemosensilla involved in host selection, the morphology and ultrastructure of antennal and tarsal sensilla in E. obliqua moths were visualized by scanning and transmission electron microscopy. Eight types (including 15 subtypes) of sensilla were identified. On antennae, sensilla trichodea (STR I and STR II) were the most abundant sensillum type characterized by porous cuticle and dendrites in the sensillum lymph. Sensilla chaetica (SCH I–SCH III) possessed a terminal pore and thick nonporous wall, with dendrites in the inner lymph cavity. Three subtypes of sensilla basiconica (SBA I–SBA III) and one type of sensilla auricillica were equipped with thin porous cuticular walls and multiple dendrites in the lymph. Sensilla styloconica were equipped with two or three dendrites that lacked cuticular pores. Sensilla coeloconica possessed four to seven dendrites and were double walled with spoke channels. Böhm bristles and sensilla squamiformia were also observed on the antennae. On tarsi, two subtypes of sensilla chaetica (SCH IV and SCH V) comprised the majority of chemosensilla, and are believed to function in gustatory perception. We summarize the validated functions of related sensilla, and propose potential functions of the corresponding sensilla in E. obliqua . These findings provide a working basis for investigating sensillum function and sensory mechanisms in this pest species.

Waves of actin and microtubule polymerization drive microtubule-based transport and neurite growth before single axon formation.

Many developing neurons transition through a multi-polar state with many competing neurites before assuming a unipolar state with one axon and multiple dendrites. Hallmarks of the multi-polar state are large fluctuations in microtubule-based transport into and outgrowth of different neurites, although what drives these fluctuations remains elusive. We show that actin waves, which stochastically migrate from the cell body towards neurite tips, direct microtubule-based transport during the multi-polar state. Our data argue for a mechanical control system whereby actin waves transiently widen the neurite shaft to allow increased microtubule polymerization to direct Kinesin-based transport and create bursts of neurite extension. Actin waves also require microtubule polymerization, arguing that positive feedback links these two components. We propose that actin waves create large stochastic fluctuations in microtubule-based transport and neurite outgrowth, promoting competition between neurites as they explore the environment until sufficient external cues can direct one to become the axon.