Development of the Drosophila mushroom bodies: elaboration,remodeling and spatial organization of dendrites in the calyx

One Drosophila mushroom body (MB) is derived from four indistinguishable cell lineages, development of which involves sequential generation of multiple distinct types of neurons. Differential labeling of distinct MB clones reveals that MB dendrites of different clonal origins are well mixed at the larval stage but become restricted to distinct spaces in adults. Interestingly, a small dendritic domain in the adult MB calyx remains as a fourfold structure that, similar to the entire larval calyx, receives dendritic inputs from all four MB clones. Mosaic analysis of single neurons demonstrates that MB neurons, which are born around pupal formation, acquire unique dendritic branching patterns and consistently project their primary dendrites into the fourfold dendritic domain. Distinct dendrite distribution patterns are also observed for other subtypes of MB neurons. In addition, pruning of larval dendrites during metamorphosis allows for establishment of adult-specific dendrite elaboration/distribution patterns. Taken together, subregional differences exist in the adult Drosophila MB calyx, where processing and integration of distinct types of sensory information begin.     

Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology

Neurons establish diverse dendritic morphologies during development, and a major challenge is to understand how these distinct developmental programs might relate to, and influence, neuronal function. Drosophila dendritic arborization (da) sensory neurons display class-specific dendritic morphology with extensive coverage of the body wall. To begin to build a basis for linking dendrite structure and function in this genetic system, we analyzed da neuron axon projections in embryonic and larval stages. We found that multiple parameters of axon morphology, including dorsoventral position, midline crossing and collateral branching, correlate with dendritic morphological class. We have identified a class-specific medial-lateral layering of axons in the central nervous system formed during embryonic development, which could allow different classes of da neurons to develop differential connectivity to second-order neurons. We have examined the effect of Robo family members on class-specific axon lamination, and have also taken a forward genetic approach to identify new genes involved in axon and dendrite development. For the latter, we screened the third chromosome at high resolution in vivo for mutations that affect class IV da neuron morphology. Several known loci, as well as putative novel mutations, were identified that contribute to sensory dendrite and/or axon patterning. This collection of mutants, together with anatomical data on dendrites and axons, should begin to permit studies of dendrite diversity in a combined developmental and functional context, and also provide a foundation for understanding shared and distinct mechanisms that control axon and dendrite morphology.     

Tiling of the Drosophila epidermis by multidendritic sensory neurons

Insect dendritic arborization (da) neurons provide an opportunity to examine how diverse dendrite morphologies and dendritic territories are established during development. We have examined the morphologies of Drosophila da neurons by using the MARCM (mosaic analysis with a repressible cell marker) system. We show that each of the 15 neurons per abdominal hemisegment spread dendrites to characteristic regions of the epidermis. We place these neurons into four distinct morphological classes distinguished primarily by their dendrite branching complexities. Some class assignments correlate with known proneural gene requirements as well as with central axonal projections. Our data indicate that cells within two morphological classes partition the body wall into distinct, non-overlapping territorial domains and thus are organized as separate tiled sensory systems. The dendritic domains of cells in different classes, by contrast, can overlap extensively. We have examined the cell-autonomous roles of starry night (stan) (also known as flamingo (fmi)) and sequoia (seq) in tiling. Neurons with these genes mutated generally terminate their dendritic fields at normal locations at the lateral margin and segment border, where they meet or approach the like dendrites of adjacent neurons. However, stan mutant neurons occasionally send sparsely branched processes beyond these territories that could potentially mix with adjacent like dendrites. Together, our data suggest that widespread tiling of the larval body wall involves interactions between growing dendritic processes and as yet unidentified signals that allow avoidance by like dendrites.     

Principles of branch dynamics governing shape characteristics of cerebellar Purkinje cell dendrites

Neurons develop dendritic arbors in cell type-specific patterns. Using growing Purkinje cells in culture as a model, we performed a long-term time-lapse observation of dendrite branch dynamics to understand the rules that govern the characteristic space-filling dendrites. We found that dendrite architecture was sculpted by a combination of reproducible dynamic processes, including constant tip elongation, stochastic terminal branching, and retraction triggered by contacts between growing dendrites. Inhibition of protein kinase C/protein kinase D signaling prevented branch retraction and significantly altered the characteristic morphology of long proximal segments. A computer simulation of dendrite branch dynamics using simple parameters from experimental measurements reproduced the time-dependent changes in the dendrite configuration in live Purkinje cells. Furthermore, perturbation analysis to parameters in silico validated the important contribution of dendritic retraction in the formation of the characteristic morphology. We present an approach using live imaging and computer simulations to clarify the fundamental mechanisms of dendrite patterning in the developing brain.     

Microtubule nucleation and organization in dendrites

Dendrite branching is an essential process for building complex nervous systems. It determines the number, distribution and integration of inputs into a neuron, and is regulated to create the diverse dendrite arbor branching patterns characteristic of different neuron types. The microtubule cytoskeleton is critical to provide structure and exert force during dendrite branching. It also supports the functional requirements of dendrites, reflected by differential microtubule architectural organization between neuron types, illustrated here for sensory neurons. Both anterograde and retrograde microtubule polymerization occur within growing dendrites, and recent studies indicate that branching is enhanced by anterograde microtubule polymerization events in nascent branches. The polarities of microtubule polymerization events are regulated by the position and orientation of microtubule nucleation events in the dendrite arbor. Golgi outposts are a primary microtubule nucleation center in dendrites and share common nucleation machinery with the centrosome. In addition, pre-existing dendrite microtubules may act as nucleation sites. We discuss how balancing the activities of distinct nucleation machineries within the growing dendrite can alter microtubule polymerization polarity and dendrite branching, and how regulating this balance can generate neuron type-specific morphologies.     

Differentiation of Apical and Basal Dendrites in Pyramidal Cells and Granule Cells in Dissociated Hippocampal Cultures

Hippocampal pyramidal cells and dentate granule cells develop morphologically distinct dendritic arbors, yet also share some common features. Both cell types form a long apical dendrite which extends from the apex of the cell soma, while short basal dendrites are developed only in pyramidal cells. Using quantitative morphometric analyses of mouse hippocampal cultures, we evaluated the differences in dendritic arborization patterns between pyramidal and granule cells. Furthermore, we observed and described the final apical dendrite determination during dendritic polarization by time-lapse imaging. Pyramidal and granule cells in culture exhibited similar dendritic patterns with a single principal dendrite and several minor dendrites so that the cell types were not readily distinguished by appearance. While basal dendrites in granule cells are normally degraded by adulthood in vivo, cultured granule cells retained their minor dendrites. Asymmetric growth of a single principal dendrite harboring the Golgi was observed in both cell types soon after the onset of dendritic growth. Time-lapse imaging revealed that up until the second week in culture, final principal dendrite designation was not stabilized, but was frequently replaced by other minor dendrites. Before dendritic polarity was stabilized, the Golgi moved dynamically within the soma and was repeatedly repositioned at newly emerging principal dendrites. Our results suggest that polarized growth of the apical dendrite is regulated by cell intrinsic programs, while regression of basal dendrites requires cue(s) from the extracellular environment in the dentate gyrus. The apical dendrite designation is determined from among multiple growing dendrites of young developing neurons.     

The Morphological Identity of Insect Dendrites

Dendrite morphology, a neuron's anatomical fingerprint, is a neuroscientist's asset in unveiling organizational principles in the brain. However, the genetic program encoding the morphological identity of a single dendrite remains a mystery. In order to obtain a formal understanding of dendritic branching, we studied distributions of morphological parameters in a group of four individually identifiable neurons of the fly visual system. We found that parameters relating to the branching topology were similar throughout all cells. Only parameters relating to the area covered by the dendrite were cell type specific. With these areas, artificial dendrites were grown based on optimization principles minimizing the amount of wiring and maximizing synaptic democracy. Although the same branching rule was used for all cells, this yielded dendritic structures virtually indistinguishable from their real counterparts. From these principles we derived a fully-automated model-based neuron reconstruction procedure validating the artificial branching rule. In conclusion, we suggest that the genetic program implementing neuronal branching could be constant in all cells whereas the one responsible for the dendrite spanning field should be cell specific.     

Control of dendritic field formation in Drosophila: the roles of flamingo and competition between homologous neurons

Neurons elaborate dendrites with stereotypic branching patterns, thereby defining their receptive fields. These branching patterns may arise from properties intrinsic to the neurons or competition between neighboring neurons. Genetic and laser ablation studies reported here reveal that different multiple dendritic neurons in the same dorsal cluster in the Drosophila embryonic PNS do not compete with one another for dendritic fields. In contrast, when dendrites from homologous neurons in the two hemisegments meet at the dorsal midline in larval stages, they appear to repel each other. The formation of normal dendritic fields and the competition between dendrites of homologous neurons require the proper expression level of Flamingo, a G protein-coupled receptor-like protein, in embryonic neurons. Whereas Flamingo functions downstream of Frizzled in specifying planar polarity, Flamingo-dependent dendritic outgrowth is independent of Frizzled.     

A novel forward genetic screen for identifying mutations affecting larval neuronal dendrite development in Drosophila melanogaster

Vertebrate and invertebrate dendrites are information-processing compartments that can be found on both central and peripheral neurons. Elucidating the molecular underpinnings of information processing in the nervous system ultimately requires an understanding of the genetic pathways that regulate dendrite formation and maintenance. Despite the importance of dendrite development, few forward genetic approaches have been used to analyze the latest stages of dendrite development, including the formation of F-actin-rich dendritic filopodia or dendritic spines. We developed a forward genetic screen utilizing transgenic Drosophila second instar larvae expressing an actin, green fluorescent protein (GFP) fusion protein (actin::GFP) in subsets of sensory neurons. Utilizing this fluorescent transgenic reporter, we conducted a forward genetic screen of >4000 mutagenized chromosomes bearing lethal mutations that affected multiple aspects of larval dendrite development. We isolated 13 mutations on the X and second chromosomes composing 11 complementation groups affecting dendrite outgrowth/branching, dendritic filopodia formation, or actin::GFP localization within dendrites in vivo. In a fortuitous observation, we observed that the structure of dendritic arborization (da) neuron dendritic filopodia changes in response to a changing environment.     

Receptor‐dependent regulation of dendrite formation of noradrenaline and dopamine in non‐gabaergic cerebral cortical neurons

The present study characterized the receptor‐dependent regulation of dendrite formation of noradrenaline (NA) and dopamine (DA) in cultured neurons obtained from embryonic day 16 rat cerebral cortex. Morphological diversity of cortical dendrites was analyzed on various features: dendrite initiation, dendrite outgrowth, and dendrite branching. Using a combination of immunocytochemical markers of dendrites and GABAergic neurons, we focused on the dendrite morphology of non‐GABAergic neurons. Our results showed that (1) NA inhibited the dendrite branching, (2) β adrenergic receptor (β‐AR) agonist inhibited the dendrite initiation, while promoted the dendrite outgrowth, (3) β1‐AR and β2‐AR were present in all the cultured neurons, and both agonists inhibited the dendrite initiation, while only β1‐AR agonist induced the dendrite branching; (4) DA inhibited the dendrite outgrowth, (5) D1 receptor agonist inhibited the dendrite initiation, while promoted the dendrite branching. In conclusion, this study compared the effects of NA, DA and their receptors and showed that NA and DA regulate different features on the dendrite formation of non‐GABAergic cortical neurons, depending on the receptors. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 73: 370–383, 2013     

Features of Active Electrical States of Asymmetrical Dendrites of Brainstem Motoneurons during Generation of Stochastic Implulse Patterns: a Simulation Study

On models of motoneurons of the n. abducens nucleus with reconstructed dendritic arborizations having an active membrane, we investigated features of the relationships between passive transfer properties and dynamics of excitation states of asymmetrical dendrites during generation of complex periodical and stochastic impulse patterns (output neuronal codes). Various patterns were obtained by varying the intensity of tonic synaptic excitation homogeneously distributed over the dendrites. The electrical states of sites belonging to branches of the same dendrite or different dendrites were compared. For this comparison, branches were selected, which, according to the earlier performed cluster analysis, were assigned to the groups (electrotonic clusters) with a high and a low effectiveness of passive transfer of the somatopetal current. The selection took into account features of the dendritic structure of neurons of the exemined type. These were: (i) the presence of groups of the asymmetrical branches differing from each other according to their belonging to different clusters (high or low transfer effectiveness) in different dendrites, and (ii) the presence of branches belonging to different dendrites characterized by significantly different orientations in three-dimensional space of the brainstem within each electrical cluster. Comparative analysis showed that, in a given dendrite during generation of a complex periodical pattern, the asymmetrical branches belonging to high- or low-efficiency clusters were characterized by being in different states (high or low depolarization) in different phases of generation of repeated sequences of action potentials (APs). This relationship was consistent with those previously detected in neurons of other types and in other specimens of neurons of the above-mentioned type. During generation of such periodical spike patterns, the branches of different dendrites belonging to the same electrotonic cluster were in similar states. Similar relationships between the states of the branches of the same dendrite belonging to different clusters were also observed during generation of complex stochastic (non-periodical) impulse patterns. In the latter case, however, the essential feature was that the branches of different dendrites belonging to the same electrotonic cluster were often in opposite states. Thus, the number of combinations of discrete electrical states of asymmetrical parts of the dendritic arborization was much greater. Probably, it is precisely this circumstance that determined the quasi-stochastic nature of the output impulse pattern.     

The control of dendrite development

Dendrite development is an important and unsolved problem in neuroscience. The nervous system is composed of a vast number of neurons with strikingly different morphology. Neurons are highly polarized cells with distinct subcellular compartments, including one or multiple dendritic processes arising from the cell body, and a single, extended axon. Communications between neurons involve synapses formed between axons of the presynaptic neurons and dendrites of the postsynaptic neurons. Extensive studies over the past decade have identified many molecules underlying axonal outgrowth and pathfinding. In contrast, the control of dendrite development is still much less well understood. However, recent progress has begun to shed light on the molecular mechanisms that orchestrate dendrite growth, arborization, and guidance.     

Control of dendritic branching and tiling by the Tricornered-kinase/Furry signaling pathway in Drosophila sensory neurons

To cover the receptive field completely but without redundancy, neurons of certain functional groups exhibit tiling of their dendrites via dendritic repulsion. Here we show that two evolutionarily conserved proteins, the Tricornered (Trc) kinase and Furry (Fry), are essential for tiling and branching control of Drosophila sensory neuron dendrites. Dendrites of fry and trc mutants display excessive terminal branching and fail to avoid homologous dendritic branches, resulting in significant overlap of the dendritic fields. Trc control of dendritic branching involves regulation of RacGTPase, a pathway distinct from the action of Trc in tiling. Timelapse analysis further reveals a specific loss of the ability of growing dendrites to turn away from nearby dendritic branches in fry mutants, suggestive of a defect in like-repels-like avoidance. Thus, the Trc/Fry signaling pathway plays a key role in patterning dendritic fields by promoting avoidance between homologous dendrites as well as by limiting dendritic branching.     

Differentiated neurons retain the capacity to generate axons from dendrites

Cutting the axon of a morphologically polarized neuron (stage 3) close to the cell body causes another neurite to grow as an axon [1-3]. Stage 3 neurons still lack molecular segregation of axonal and dendritic proteins, however. Axonal and dendritic compartments acquire their distinct composition at stage 4 (4-5days in culture), when proteins such as the microtubule-associated protein 2 (MAP-2) and the glutamate receptor subunit GluR1 localize to the dendrites and disappear from the axon [4,5]. We investigated whether cultured hippocampal neurons retained axon/dendrite plasticity after axons and dendrites have created their distinct cytoskeletal architecture and acquired their specific membrane composition. We found that axotomy of stage 4 neurons transformed a dendrite into an axon. Using axonal and dendritic markers, we tested whether cytoskeletal changes could cause similar transformations, and found that actin depolymerization induced multiple axons in unpolarized neurons. Moreover, depletion of actin filaments from both morphologically and molecularly polarized cells also resulted in the growth of multiple axons from pre-existing dendrites. These results imply that dendrites retain the potential to become axons even after molecular segregation has occurred and that the dendritic fate depends on the integrity of the actin cytoskeleton.