Integrins regulate repulsion-mediated dendritic patterning of drosophila sensory neurons by restricting dendrites in a 2D space

Dendrites of the same neuron usually avoid each other. Some neurons also repel similar neurons through dendrite-dendrite interaction to tile the receptive field. Nonoverlapping coverage based on such contact-dependent repulsion requires dendrites to compete for limited space. Here we show that Drosophila class IV dendritic arborization (da) neurons, which tile the larval body wall, grow their dendrites mainly in a 2D space on the extracellular matrix (ECM) secreted by the epidermis. Removing neuronal integrins or blocking epidermal laminin production causes dendrites to grow into the epidermis, suggesting that integrin-laminin interaction attaches dendrites to the ECM. We further show that some of the previously identified tiling mutants fail to confine dendrites in a 2D plane. Expansion of these mutant dendrites in three dimensions results in overlap of dendritic fields. Moreover, overexpression of integrins in these mutant neurons effectively reduces dendritic crossing and restores tiling, revealing an additional mechanism for tiling.     

Drosophila sensory neurons require Dscam for dendritic self-avoidance and proper dendritic field organization

A neuron's dendrites typically do not cross one another. This intrinsic self-avoidance mechanism ensures unambiguous processing of sensory or synaptic inputs. Moreover, some neurons respect the territory of others of the same type, a phenomenon known as tiling. Different types of neurons, however, often have overlapping dendritic fields. We found that Down's syndrome Cell Adhesion Molecule (Dscam) is required for dendritic self-avoidance of all four classes of Drosophila dendritic arborization (da) neurons. However, neighboring mutant class IV da neurons still exhibited tiling, suggesting that self-avoidance and tiling differ in their recognition and repulsion mechanisms. Introducing 1 of the 38,016 Dscam isoforms to da neurons in Dscam mutants was sufficient to significantly restore self-avoidance. Remarkably, expression of a common Dscam isoform in da neurons of different classes prevented their dendrites from sharing the same territory, suggesting that coexistence of dendritic fields of different neuronal classes requires divergent expression of Dscam isoforms.     

Extra spike formation in sensory neurons and the disruption of afferent spike patterning

The peculiar pseudounipolar geometry of primary sensory neurons can lead to ectopic generation of "extra spikes" in the region of the dorsal root ganglion potentially disrupting the fidelity of afferent signaling. We have used an explicit model of myelinated vertebrate sensory neurons to investigate the location and mechanism of extra spike formation, and its consequences for distortion of afferent impulse patterning. Extra spikes originate in the initial segment axon under conditions in which the soma spike becomes delayed and broadened. The broadened soma spike then re-excites membrane it has just passed over, initiating an extra spike which propagates outwards into the main conducting axon. Extra spike formation depends on cell geometry, electrical excitability, and the recent history of impulse activity. Extra spikes add to the impulse barrage traveling toward the spinal cord, but they also travel antidromically in the peripheral nerve colliding with and occluding normal orthodromic spikes. As a result there is no net increase in afferent spike number. However, extra spikes render firing more staccato by increasing the number of short and long interspike intervals in the train at the expense of intermediate intervals. There may also be more complex changes in the pattern of afferent spike trains, and hence in afferent signaling.     

EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons

Dendritic spines are the postsynaptic compartment of a neuronal synapse and are critical for synaptic connectivity and plasticity. A developmental precursor to dendritic spines, dendritic filopodia (DF), facilitate synapse formation by sampling the environment for suitable axon partners during neurodevelopment and learning. Despite the significance of the actin cytoskeleton in driving these dynamic protrusions, the actin elongation factors involved are not well characterized. We identified the Ena/VASP protein EVL as uniquely required for the morphogenesis and dynamics of DF. Using a combination of genetic and optogenetic manipulations, we demonstrated that EVL promotes protrusive motility through membrane-direct actin polymerization at DF tips. EVL forms a complex at nascent protrusions and DF tips with MIM/MTSS1, an I-BAR protein important for the initiation of DF. We proposed a model in which EVL cooperates with MIM to coalesce and elongate branched actin filaments, establishing the dynamic lamellipodia-like architecture of DF.     

Neuropeptides in sensory neurons

Substance P, somatostatin, VIP, CCK, angiotensin II, and bombesin have all been localized by immunohistochemical or radioimmunological means in neurons of sensory ganglia or in the dorsal horn of the spinal cord. Most of these neuropeptides have electrophysiological effects on spinal neurons and for substance P and somatostatin, these effects have been associated with particular sensory modalities. Newer investigations using the compound capsaicin are consistent with the hypothesis that substance P is an important neurochemical mediator of certain kinds of noxious peripheral stimuli. The newly described substance P antagonists promise to be important pharmacological tools for investigation of the long-neglected neurochemical bases of sensory neuron function. Elaboration of the roles of these sensory neuropeptides will no doubt shed light on many disease states in which there seems to be sensory neuron involvement.     

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.     

Painful channels in sensory neurons

Pain is an unpleasant sensation experienced when tissues are damaged. Thus, pain sensation in some way protects body from imminent threat or injury. Peripheral sensory nerves innervated to peripheral tissues initially respond to multiple forms of noxious or strong stimuli, such as heat, mechanical and chemical stimuli. In response to these stimuli, electrical signals for conducting the nociceptive neural signals through axons are generated. These action potentials are then conveyed to specific areas in the spinal cord and in the brain. Sensory afferent fibers are heterogeneous in many aspects. For example, sensory nerves are classified as Aa, -b, -d and C-fibers according to their diameter and degree of myelination. It is widely accepted that small sensory fibers tend to respond to vigorous or noxious stimuli and related to nociception. Thus these fibers are specifically called nociceptors. Most of nociceptors respond to noxious mechanical stimuli and heat. In addition, these sensory fibers also respond to chemical stimuli [Davis et al. (1993)] such as capsaicin. Thus, nociceptors are considered polymodal. Recent advance in research on ion channels in sensory neurons reveals molecular mechanisms underlying how various types of stimuli can be transduced to neural signals transmitted to the brain for pain perception. In particular, electrophysiological studies on ion channels characterize biophysical properties of ion channels in sensory neurons. Furthermore, molecular biology leads to identification of genetic structures as well as molecular properties of ion channels in sensory neurons. These ion channels are expressed in axon terminals as well as in cell soma. When these channels are activated, inward currents or outward currents are generated, which will lead to depolarization or hyperpolarization of the membrane causing increased or decreased excitability of sensory neurons. In order to depolarize the membrane of nerve terminals, either inward currents should be generated or outward currents should be inhibited. So far, many cationic channels that are responsible for the excitation of sensory neurons are introduced recently. Activation of these channels in sensory neurons is evidently critical to the generation of nociceptive signals. The main channels responsible for inward membrane currents in nociceptors are voltage-activated sodium and calcium channels, while outward current is carried mainly by potassium ions. In addition, activation of non-selective cation channels is also responsible for the excitation of sensory neurons. Thus, excitability of neurons can be controlled by regulating expression or by modulating activity of these channels.     

NGF signaling in sensory neurons: evidence that early endosomes carry NGF retrograde signals

Target-derived NGF promotes the phenotypic maintenance of mature dorsal root ganglion (DRG) nociceptive neurons. Here, we provide in vivo and in vitro evidence for the presence within DRG neurons of endosomes containing NGF, activated TrkA, and signaling proteins of the Rap1/Erk1/2, p38MAPK, and PI3K/Akt pathways. Signaling endosomes were shown to be retrogradely transported in the isolated sciatic nerve in vitro. NGF injection in the peripheral target of DRG neurons increased the retrograde transport of p-Erk1/2, p-p38, and pAkt in these membranes. Conversely, NGF antibody injections decreased the retrograde transport of p-Erk1/2 and p-p38. Our results are evidence that signaling endosomes, with the characteristics of early endosomes, convey NGF signals from the target of nociceptive neurons to their cell bodies.     

Multiple morphogens and rapid elongation promote segmental patterning during development

The vertebrate hindbrain is segmented into rhombomeres (r) initially defined by distinct domains of gene expression. Previous studies have shown that noise-induced gene regulation and cell sorting are critical for the sharpening of rhombomere boundaries, which start out rough in the forming neural plate (NP) and sharpen over time. However, the mechanisms controlling simultaneous formation of multiple rhombomeres and accuracy in their sizes are unclear. We have developed a stochastic multiscale cell-based model that explicitly incorporates dynamic morphogenetic changes (i.e. convergent-extension of the NP), multiple morphogens, and gene regulatory networks to investigate the formation of rhombomeres and their corresponding boundaries in the zebrafish hindbrain. During pattern initiation, the short-range signal, fibroblast growth factor (FGF), works together with the longer-range morphogen, retinoic acid (RA), to specify all of these boundaries and maintain accurately sized segments with sharp boundaries. At later stages of patterning, we show a nonlinear change in the shape of rhombomeres with rapid left-right narrowing of the NP followed by slower dynamics. Rapid initial convergence improves boundary sharpness and segment size by regulating cell sorting and cell fate both independently and coordinately. Overall, multiple morphogens and tissue dynamics synergize to regulate the sizes and boundaries of multiple segments during development.     

Muscle-derived factors that support survival and promote fiber outgrowth from embryonic chick spinal motor neurons in culture

The purposes of the experiments reported is to provide an unambiguous demonstration that embryonic skeletal muscle contains factors that act directly on embryonic spinal motor neurons both to support their survival and to stimulate the outgrowth of neurites. Cells of lumbar and brachial ventral spinal cords from 6-day-old chick embryos were separated by centrifugation in a two-step metrizamide gradient, and a motor neuron enriched fraction was obtained. Motor neurons were identified by retrogradely labeling with rhodamine isothiocyanate, and were enriched fourfold in the motor neuron fraction relative to unfractionated cells. In culture, the isolated motor neurons died within 3-4 days unless they were supplemented with embryonic chick skeletal muscle extract. Two functionally distinct entities separable by ammonium sulfate precipitation were responsible for the effects of muscle extracts on motor neurons. The 0-25% ammonium sulfate precipitate contained molecules that alone had no effect on neuronal survival but when bound to polyornithine-coated culture substrata, stimulated neurite outgrowth and potentiated the survival activity present in muscle. Most of this activity was due to a laminin-like molecule being immunoprecipitated with antisera against laminin, and immunoblotting demonstrated the presence of both the A and B chains of laminin. A long-term survival activity resided in the 25-70% ammonium sulfate fraction, and its apparent total and specific activities were strongly dependent on the culture substrate. In contrast to the motor neurons, the cells from the other metrizamide fraction (including neuronal cells) could be kept in culture for a prolonged time without addition of exogenous factor(s).     

Cellular mechanisms of dendrite pruning in Drosophila: insights from in vivo time-lapse of remodeling dendritic arborizing sensory neurons

Regressive events that refine exuberant or inaccurate connections are critical in neuronal development. We used multi-photon, time-lapse imaging to examine how dendrites of Drosophila dendritic arborizing (da) sensory neurons are eliminated during early metamorphosis, and how intrinsic and extrinsic cellular mechanisms control this deconstruction. Removal of the larval dendritic arbor involves two mechanisms: local degeneration and branch retraction. In local degeneration, major branch severing events entail focal disruption of the microtubule cytoskeleton, followed by thinning of the disrupted region, severing and fragmentation. Retraction was observed at distal tips of branches and in proximal stumps after severing events. The pruning program of da neuron dendrites is steroid induced; cell-autonomous dominant-negative inhibition of steroid action blocks local degeneration, although retraction events still occur. Our data suggest that steroid-induced changes in the epidermis may contribute to dendritic retraction. Finally, we find that phagocytic blood cells not only engulf neuronal debris but also attack and sever intact branches that show signs of destabilization.