Embryonic Origins of a Motor System: Motor Dendrites Form a Myotopic Map in Drosophila

The organisational principles of locomotor networks are less well understood than those of many sensory systems, where in-growing axon terminals form a central map of peripheral characteristics. Using the neuromuscular system of the Drosophila embryo as a model and retrograde tracing and genetic methods, we have uncovered principles underlying the organisation of the motor system. We find that dendritic arbors of motor neurons, rather than their cell bodies, are partitioned into domains to form a myotopic map, which represents centrally the distribution of body wall muscles peripherally. While muscles are segmental, the myotopic map is parasegmental in organisation. It forms by an active process of dendritic growth independent of the presence of target muscles, proper differentiation of glial cells, or (in its initial partitioning) competitive interactions between adjacent dendritic domains. The arrangement of motor neuron dendrites into a myotopic map represents a first layer of organisation in the motor system. This is likely to be mirrored, at least in part, by endings of higher-order neurons from central pattern-generating circuits, which converge onto the motor neuron dendrites. These findings will greatly simplify the task of understanding how a locomotor system is assembled. Our results suggest that the cues that organise the myotopic map may be laid down early in development as the embryo subdivides into parasegmental units.     

Midline Signalling Systems Direct the Formation of a Neural Map by Dendritic Targeting in the Drosophila Motor System

A fundamental strategy for organising connections in the nervous system is the formation of neural maps. Map formation has been most intensively studied in sensory systems where the central arrangement of axon terminals reflects the distribution of sensory neuron cell bodies in the periphery or the sensory modality. This straightforward link between anatomy and function has facilitated tremendous progress in identifying cellular and molecular mechanisms that underpin map development. Much less is known about the way in which networks that underlie locomotion are organised. We recently showed that in the Drosophila embryo, dendrites of motorneurons form a neural map, being arranged topographically in the antero-posterior axis to represent the distribution of their target muscles in the periphery. However, the way in which a dendritic myotopic map forms has not been resolved and whether postsynaptic dendrites are involved in establishing sets of connections has been relatively little explored. In this study, we show that motorneurons also form a myotopic map in a second neuropile axis, with respect to the ventral midline, and they achieve this by targeting their dendrites to distinct medio-lateral territories. We demonstrate that this map is “hard-wired”; that is, it forms in the absence of excitatory synaptic inputs or when presynaptic terminals have been displaced. We show that the midline signalling systems Slit/Robo and Netrin/Frazzled are the main molecular mechanisms that underlie dendritic targeting with respect to the midline. Robo and Frazzled are required cell-autonomously in motorneurons and the balance of their opposite actions determines the dendritic target territory. A quantitative analysis shows that dendritic morphology emerges as guidance cue receptors determine the distribution of the available dendrites, whose total length and branching frequency are specified by other cell intrinsic programmes. Our results suggest that the formation of dendritic myotopic maps in response to midline guidance cues may be a conserved strategy for organising connections in motor systems. We further propose that sets of connections may be specified, at least to a degree, by global patterning systems that deliver pre- and postsynaptic partner terminals to common “meeting regions.”     

Dendritic Targeting in the Leg Neuropil of Drosophila: The Role of Midline Signalling Molecules in Generating a Myotopic Map

Neural maps are emergent, highly ordered structures that are essential for organizing and presenting synaptic information. Within the embryonic nervous system of Drosophila motoneuron dendrites are organized topographically as a myotopic map that reflects their pattern of innervation in the muscle field. Here we reveal that this fundamental organizational principle exists in adult Drosophila, where the dendrites of leg motoneurons also generate a myotopic map. A single postembryonic neuroblast sequentially generates different leg motoneuron subtypes, starting with those innervating proximal targets and medial neuropil regions and producing progeny that innervate distal muscle targets and lateral neuropil later in the lineage. Thus the cellular distinctions in peripheral targets and central dendritic domains, which make up the myotopic map, are linked to the birth-order of these motoneurons. Our developmental analysis of dendrite growth reveals that this myotopic map is generated by targeting. We demonstrate that the medio-lateral positioning of motoneuron dendrites in the leg neuropil is controlled by the midline signalling systems Slit-Robo and Netrin-Fra. These results reveal that dendritic targeting plays a major role in the formation of myotopic maps and suggests that the coordinate spatial control of both pre- and postsynaptic elements by global neuropilar signals may be an important mechanism for establishing the specificity of synaptic connections.     

Patterning and organization of motor neuron dendrites in the Drosophila larva

Precise patterns of motor neuron connectivity depend on the proper establishment and positioning of the dendritic arbor. However, how different motor neurons orient their dendrites to selectively establish synaptic connectivity is not well understood. The Drosophila neuromuscular system provides a simple model to investigate the underlying organizational principles by which distinct subclasses of motor neurons orient their dendrites within the central neuropil. Here we used genetic mosaic techniques to characterize the diverse dendritic morphologies of individual motor neurons from five main nerve branches (ISN, ISNb, ISNd, SNa, and SNc) in the Drosophila larva. We found that motor neurons from different nerve branches project their dendrites to largely stereotyped mediolateral domains in the dorsal region of the neuropil providing full coverage of the receptive territory. Furthermore, dendrites from different motor neurons overlap extensively, regardless of subclass, suggesting that repulsive dendrite-dendrite interactions between motor neurons do not influence the mediolateral positioning of dendritic fields. The anatomical data in this study provide important information regarding how different subclasses of motor neurons organize their dendrites and establishes a foundation for the investigation of the mechanisms that control synaptic connectivity in the Drosophila motor circuit.     

Microtubules have opposite orientation in axons and dendrites of Drosophila neurons

In vertebrate neurons, axons have a uniform arrangement of microtubules with plus ends distal to the cell body (plus-end-out), and dendrites have equal numbers of plus- and minus-end-out microtubules. To determine whether microtubule orientation is a conserved feature of axons and dendrites, we analyzed microtubule orientation in invertebrate neurons. Using microtubule plus end dynamics, we mapped microtubule orientation in Drosophila sensory neurons, interneurons, and motor neurons. As expected, all axonal microtubules have plus-end-out orientation. However, in proximal dendrites of all classes of neuron, approximately 90% of dendritic microtubules were oriented with minus ends distal to the cell body. This result suggests that minus-end-out, rather than mixed orientation, microtubules are the signature of the dendritic microtubule cytoskeleton. Surprisingly, our map of microtubule orientation predicts that there are no tracks for direct cargo transport between the cell body and dendrites in unipolar neurons. We confirm this prediction, and validate the completeness of our map, by imaging endosome movements in motor neurons. As predicted by our map, endosomes travel smoothly between the cell body and axon, but they cannot move directly between the cell body and dendrites.     

Present State and Future Perspectives of Prostaglandins as a Differentiation Factor in Motor Neurons

Spinal motor neurons have the longest axons that innervate the skeletal muscles of the central nervous system. Motor neuron diseases caused by spinal motor neuron cell death are incurable due to the unique and irreplaceable nature of their neural circuits. Understanding the mechanisms of neurogenesis, neuritogenesis, and synaptogenesis in motor neurons will allow investigators to develop new in vitro models and regenerative therapies for motor neuron diseases. In particular, small molecules can directly reprogram and convert into neural stem cells and neurons, and promote neuron-like cell differentiation. Prostaglandins are known to have a role in the differentiation and tissue regeneration of several cell types and organs. However, the involvement of prostaglandins in the differentiation of motor neurons from neural stem cells is poorly understood. The general cell line used in research on motor neuron diseases is the mouse neuroblastoma and spinal motor neuron fusion cell line NSC-34. Recently, our laboratory reported that prostaglandin E₂ and prostaglandin D₂ enhanced the conversion of NSC-34 cells into motor neuron-like cells with neurite outgrowth. Moreover, we found that prostaglandin E₂-differentiated NSC-34 cells had physiological and electrophysiological properties of mature motor neurons. In this review article, we provide contemporary evidence on the effects of prostaglandins, particularly prostaglandin E₂ and prostaglandin D₂, on differentiation and neural conversion. We also discuss the potential of prostaglandins as candidates for the development of new therapeutic drugs for motor neuron diseases.

     

Early functional impairment of sensory-motor connectivity in a mouse model of spinal muscular atrophy

To define alterations of neuronal connectivity that occur during motor neuron degeneration, we characterized the function and structure of spinal circuitry in spinal muscular atrophy (SMA) model mice. SMA motor neurons show reduced proprioceptive reflexes that correlate with decreased number and function of synapses on motor neuron somata and proximal dendrites. These abnormalities occur at an early stage of disease in motor neurons innervating proximal hindlimb muscles and medial motor neurons innervating axial muscles, but only at end-stage disease in motor neurons innervating distal hindlimb muscles. Motor neuron loss follows afferent synapse loss with the same temporal and topographical pattern. Trichostatin A, which improves motor behavior and survival of SMA mice, partially restores spinal reflexes, illustrating the reversibility of these synaptic defects. Deafferentation of motor neurons is an early event in SMA and may be a primary cause of motor dysfunction that is amenable to therapeutic intervention.     

Organization of motor neurons innervating the proboscis musculature in Drosophila melanogaster meigen (diptera : Drosophilidae)

The present study addresses the question as to how the motor neurons involved in feeding in Drosophila melanogaster Meigen (Diptera : Drosophilidae) are organized. The motor neurons have been visualized both by Golgi-silver impregnation and by intramuscular injection of horseradish peroxidase, and analyzed in light of the existing information on taste sensory system and the feeding behaviour. The motor neurons have been broadly classified into the following types: labial nerve motor neurons, pharyngeal nerve motor neurons, and accessory pharyngeal nerve motor neurons, depending on the nerve through which their axons exit. The arborization of all the motor neurons is confined to the suboesophageal ganglion (SOG). All of them have predominantly ipsilateral and some contralateral arborizations. Their dendrites predominantly occupy the ventral region of the neuropil of the SOG and partially overlap the taste sensory projections, thereby providing an opportunity for interaction with the taste sensory input. The pharyngeal motor neurons arborize more extensively in the ventral tritocerebram, anteroventral. and mid-ventral neuropil, whereas the dendritic fields of labial motor neurons are confined to the mid-ventral neuropil. There is a functional segregation in motor neuron organization: cibarial muscles involved in sucking are innervated by pharyngeal motor neurons, while the proboscis muscles involved in positioning, of the proboscis are innervated by labial motor neurons. We have also observed projections of the stomodaeal nerve in the tritocerebrum.     

Dorsal longitudinal stretch receptor of Drosophila melanogaster larva - fine structure and maturation

Insects possess two types of sensory neurons: ciliated type I sensory neurons that innervate external sensory organs and chordotonal organs, and type II sensory neurons that form a subepidermal plexus or innervate stretch receptors. Among stretch receptors, a dorsel longitudinal stretch receptor is highly conserved in insects, being found in all insect orders investigated. Here we describe the topology and anatomical structure of this receptor in the fruit fly embryo and larva using transmission electron microscopy and single cell staining for fluorescence microscopy. The receptor is composed of the dorsal bipolar dendrite neuron, which arises from an archetypal cell lineage, its sister glial cell and the peripheral glial cell accompanying the nerve. The neuron is situated among the muscles in the dorsal body wall on the intersegmental nerve. Its two dendrites stretch the length of the segment to the segmental folds. The neuron is wrapped by both glial cells and surrounded by a common basal lamina, which fans out at the dendritic tips to attach them to the epidermal cells at the segmental borders.     

Distribution of Motor Cortical Neuron Synaptic Terminals on Monkey Parvocellular Red Nucleus Neurons

We determined the location of 54 horseradish peroxidase (HRP)-labeled motor cortical neuron synaptic terminals on 17 parvocellular neurons in the monkey red nucleus. Synaptic terminals and their postsynaptic elements were identified and reconstructed, using light- and electron-microscopic techniques, from serial thick and thin sections. Terminals were found on proximal and distal dendrites of small and medium-sized parvocellular neurons, where they formed excitatory synapses. Some were 180 μm from cell somata. Approximately half of the labeled terminals, aside from those located at dendritic origins, were situated strategically at or near dendritic branch points. Since monkey parvocellular neurons show little activity during movement, the obvious next question is this: How and in what way does motor cortex influence these cells?     

Motor neurons and the sense of place

Seventy years ago George Romanes began to document the anatomical organization of the spinal motor system, uncovering a multilayered topographic plan that links the clustering and settling position of motor neurons to the spatial arrangement and biomechanical features of limb muscles. To this day, these findings have provided a structural foundation for analysis of the neural control of movement and serve as?a guide for studies to explore mechanisms that direct the wiring of spinal motor circuits. In this brief essay we outline the?core of Romanes's findings and place them in the context of recent studies that begin to provide insight?into molecular programs that assign motor pool position and to resolve how motor neuron position shapes circuit assembly. Romanes's findings reveal how and why neuronal positioning contributes to sensory-motor connectivity and may have relevance to circuit organization in other regions of the central nervous system.     

Long-range neuronal circuits underlying the interaction between sensory and motor cortex

In the rodent vibrissal system, active sensation and sensorimotor integration are mediated in part by connections between barrel cortex and vibrissal motor cortex. Little is known about how these structures interact at the level of neurons. We used Channelrhodopsin-2 (ChR2) expression, combined with anterograde and retrograde labeling, to map connections between barrel cortex and pyramidal neurons in mouse motor cortex. Barrel cortex axons preferentially targeted upper layer (L2/3, L5A) neurons in motor cortex; input to neurons projecting back to barrel cortex was particularly strong. Barrel cortex input to deeper layers (L5B, L6) of motor cortex, including neurons projecting to the brainstem, was weak, despite pronounced geometric overlap of dendrites with axons from barrel cortex. Neurons in different layers received barrel cortex input within stereotyped dendritic domains. The cortico-cortical neurons in superficial layers of motor cortex thus couple motor and sensory signals and might mediate sensorimotor integration and motor learning.     

Sex differences in the motor nucleus of cranial nerve IX-X in Xenopus laevis: a quantitative Golgi study

In the clawed frog (Xenopus laevis), motor neurons in cranial nerve nucleus IX-X control contraction of laryngeal muscles responsible for sexually dimorphic vocal behaviors. We examined sex differences in dendritic arbors of n.IX-X cells using the Golgi-Cox method. Three morphological classes of somal types (ovoid, triangular, and elongate) are present in similar frequencies in n.IX-X of both males and females. The male n.IX-X neuron is a more complex and hypertrophied version of the female n.IX-X cell. The number of primary dendrites is the same for both sexes, but males have more total dendritic segments. The overall dendritic length of male n.IX-X neurons is two to three times that of the female. Males have longer dendritic segments between all branch points. Male and female frogs differ in levels of circulating androgens; neurons of n.IX-X are targets for androgenic steroids. To determine if androgen can affect dendritic morphology in adult females, we examined Golgi-impregnated cells in n.IX-X from ovariectomized females treated with testosterone for 1 month. The total number of dendritic segments was reduced by androgen treatment due to reduction in the number of higher order dendritic segments; the number of primary dendritic segments was unchanged. Androgen treatment may induce resorption of higher order dendritic branches. The overall dendritic length of androgen-treated female n.IX-X neurons was unchanged, and dendritic segments were longer. Thus, although androgen can alter dendrites of n.IX-X cells in adult females, this short-term treatment does not produce a masculine dendritic architecture.     

The retrograde frequency response of passive dendritic trees constrains the nonlinear firing behaviour of a reduced neuron model

Our goal was to investigate how the propagation of alternating signals (i.e. AC), like action potentials, into the dendrites influenced nonlinear firing behaviour of motor neurons using a systematically reduced neuron model. A recently developed reduced modeling approach using only steady-current (i.e. DC) signaling was analytically expanded to retain features of the frequency-response analysis carried out in multicompartment anatomically reconstructed models. Bifurcation analysis of the extended model showed that the typically overlooked parameter of AC amplitude attenuation was positively correlated with the current threshold for the activation of a plateau potential in the dendrite. Within the multiparameter space map of the reduced model the region demonstrating "fully-bistable" firing was bounded by directional DC attenuation values that were negatively correlated to AC attenuation. Based on these results we conclude that analytically derived reduced models of dendritic trees should be fit on DC and AC signaling, as both are important biophysical parameters governing the nonlinear firing behaviour of motor neurons.     

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.     

Mice, the Motor System, and Human Motor Neuron Pathology

Motor neurons are among some of the most unusual cells in the body becaue of their immense size and their role as the critical link between the motor centers of the brain and the muscles. In addition to their intrinsic biological interest, it is vital that we gain a better understanding of these cells and their pathology, since motor neuron degenerative diseases are lethal disorders that affect young and old and are relatively common. For example, one form of spinal muscular atrophy (SMA) is the most common genetic killer of children in the developed world. Amyotrophic lateral sclerosis (ALS), another form of motor neuron degeneration, is the third most common neurodegenerative cause of adult death, after Alzheimer's disease and Parkinson's disease, and is significantly more common than multiple sclerosis (Motor Neurone Disease Association 1998). Currently, approximately 1 in 500 people in England and Wales who die have a form of motor neuron disease (Motor Neurone Disease Association 1998). Each year, 5000 Americans are diagnosed with ALS, and of these, 10% are under 40 years old. Mouse models of motor neuron degeneration are essential for understanding the causes and mechanisms of motor neuron pathology. These mice are yielding important information that will ultimately lead to treatments and potentially cures for these diseases. Received: 5 June 2000 / Accepted: 27 July 2000     

Disruption of peripheral target contact influences the development of identified central dendritic branches in a leech motor neuron in vivo

Retrograde signaling from target tissues has been shown to influence many aspects of neuronal development in a number of developmental systems. In these experiments using embryonic leeches (Hirudo medicinalis), we examined how depriving a neuron of contact with its peripheral target affects the development of the cell's central arborization. We focused our attention on the motor neuron cell 3, which normally stimulates dorsal longitudinal muscle fibers to contract. At different locations in the periphery and in embryos of several different stages, we cut the nerve containing the growing axon of cell 3. This surgery led to dramatic overgrowth of cell 3's central dendritic branches, which normally accept synaptic contacts from other neurons, including the inhibitory motor neuron cell 1. When cell 3's peripheral axon was cut relatively early in development, its overgrown central branches eventually retracted. However, cells that were disrupted later in development retained their overextended branches into adulthood. In addition, if the axon was cut close to the ganglion early in development, depriving the cell of contact with any dorsal tissues, the central branches failed to retract and were instead retained into adulthood. Unlike cell 3, the central branches of cell 1, which has the same peripheral target muscles as cell 3, remained unchanged following all axotomy protocols. These results suggest that in at least some neurons contact with peripheral targets can influence development of the central processes that normally mediate synaptic contacts.