In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells

Targeting of axons and dendrites to particular synaptic laminae is an important mechanism by which precise patterns of neuronal connectivity are established. Although axons target specific laminae during development, dendritic lamination has been thought to occur largely by pruning of inappropriately placed arbors. We discovered by in vivo time-lapse imaging that retinal ganglion cell (RGC) dendrites in zebrafish show growth patterns implicating dendritic targeting as a mechanism for contacting appropriate synaptic partners. Populations of RGCs labeled in transgenic animals establish distinct dendritic strata sequentially, predominantly from the inner to outer retina. Imaging individual cells over successive days confirmed that multistratified RGCs generate strata sequentially, each arbor elaborating within a specific lamina. Simultaneous imaging of RGCs and subpopulations of presynaptic amacrine interneurons revealed that RGC dendrites appear to target amacrine plexuses that had already laminated. Dendritic targeting of prepatterned afferents may thus be a novel mechanism for establishing proper synaptic connectivity.     

ALS‐linked protein disulfide isomerase variants cause motor dysfunction

Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) are ER foldases identified as possible ALS biomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized four ALS‐linked mutations recently identified in two major PDI genes, PDIA1 and PDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of these PDI variants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutant PDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of these PDI mutants. Finally, targeting ERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifies ER proteostasis imbalance as a risk factor for ALS, driving initial stages of the disease.     

Dendritic connectivity shapes spatial patterns of genetic diversity: a simulation‐based study

Landscape features notoriously affect spatial patterns of biodiversity. For instance, in dendritic ecological networks (such as river basins), dendritic connectivity has been proposed to create unique spatial patterns of biodiversity. Here, we compared genetic datasets simulated under a lattice‐like, a dendritic and a circular landscape to test the influence of dendritic connectivity on neutral genetic diversity. The circular landscape had a level of connectivity similar to that of the dendritic landscape, so as to isolate the influence of dendricity on genetic diversity. We found that genetic diversity and differentiation varied strikingly among the three landscapes. For instance, the dendritic landscape generated higher total number of alleles and higher global F_st than the lattice‐like landscape, and these indices also varied between the dendritic and the circular landscapes, suggesting an effect of dendricity. Furthermore, in the dendritic landscape, allelic richness was higher in highly connected demes (e.g. confluences in rivers) than in low‐connected demes (e.g. upstream and downstream populations), which was not the case in the circular landscape, hence confirming the major role of dendricity. This led to bell‐shaped distributions of allelic richness along an upstream–downstream gradient. Conversely, genetic differentiation (F_st) was lower in highly than in low‐connected demes (which was not observed in circular landscape), and significant patterns of isolation by distance (IBD) were also observed in the dendritic landscape. We conclude that in dendritic networks, the combined influence of dendricity and connectivity generates unique spatial patterns of neutral genetic diversity, which has implications for population geneticists and conservationists.     

Neuroprotective Effects on the Morphology of Somatic Motoneurons Following the Death of Neighboring Motoneurons: A Role for Microglia?

Partial depletion of spinal motoneuron populations induces dendritic atrophy in neighboring motoneurons, and treatment with testosterone protects motoneurons from induced dendritic atrophy. We explored a potential mechanism for this induced atrophy and protection by testosterone, examining the microglial response to partial depletion of motoneurons. Motoneurons innervating the vastus medialis muscles of adult male rats were killed by intramuscular injection of cholera toxin‐conjugated saporin; some saporin‐injected rats were treated with testosterone. Microglia were later visualized via immunohistochemical staining, classified as monitoring or activated, and counted stereologically. Partial motoneuron depletion increased the number of activated microglia in the quadriceps motor pool, and this increase was attenuated with testosterone treatment. The attenuation in microglial response could reflect an effect of testosterone on suppressing microglia activation, potentially sparing motoneuron dendrites. Alternatively, testosterone could be neuroprotective, sparing motoneuron dendrites, secondarily resulting in reduced microglial activation. To discriminate between these hypotheses, following partial motoneuron depletion, rats were treated with minocycline to inhibit microglial activation. Motoneurons innervating the ipsilateral vastus lateralis muscle were later labeled with cholera toxin‐conjugated horseradish peroxidase, and dendritic arbors were reconstructed. Reduction of microglial activation by minocycline did not prevent induced dendritic atrophy following partial motoneuron depletion. Further, reduction of microglial activation by minocycline treatment resulted in dendritic atrophy in intact animals. Together, these findings indicate that the neuroprotective effect of testosterone on dendrites following motoneuron death is not due to inhibiting microglial activation, and that microglial activity contributes to the normal maintenance of dendritic arbors.     

Neuroprotective effects of testosterone metabolites and dependency on receptor action on the morphology of somatic motoneurons following the death of neighboring motoneurons

Partial depletion of spinal motoneuron populations induces dendritic atrophy in neighboring motoneurons, and treatment with testosterone is neuroprotective, attenuating induced dendritic atrophy. In this study we examined whether the protective effects of testosterone could be mediated via its androgenic or estrogenic metabolites. Furthermore, to assess whether these neuroprotective effects were mediated through steroid hormone receptors, we used receptor antagonists to attempt to prevent the neuroprotective effects of hormones after partial motoneuron depletion. Motoneurons innervating the vastus medialis muscles of adult male rats were selectively killed by intramuscular injection of cholera toxin‐conjugated saporin. Simultaneously, some saporin‐injected rats were treated with either dihydrotestosterone or estradiol, alone or in combination with their respective receptor antagonists, or left untreated. Four weeks later, motoneurons innervating the ipsilateral vastus lateralis muscle were labeled with cholera toxin‐conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Compared with intact normal animals, partial motoneuron depletion resulted in decreased dendritic length in remaining quadriceps motoneurons. Dendritic atrophy was attenuated with both dihydrotestosterone and estradiol treatment to a degree similar to that seen with testosterone, and attenuation of atrophy was prevented by receptor blockade. Together, these findings suggest that neuroprotective effects on motoneurons can be mediated by either androgenic or estrogenic hormones and require action via steroid hormone receptors, further supporting a role for hormones as neurotherapeutic agents in the injured nervous system. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 691–707, 2017     

Regeneration studies on a crayfish neuromuscular system. I. Connectivity changes after intersegmental nerve transplants

The superficial flexor muscles of the crayfish are a neuromuscular system of a few muscle cells innervated by six neurons in a precise position-dependent pattern. The neurons are capable of regenerating their normal connectivity patterns within a short span of time when conditions are favorable. The superficial flexor muscles of the second and third segments, despite their similarities in neuronal and muscle cell size and number, have distinctive connectivity patterns; some homologous neurons form similar patterns but other homologous neurons form patterns that are reversed between segments. We transplanted each segment's nerve into each other's muscle in order to observe regeneration of the nerves into a target area that differed in connectivity patterns from their original muscle. During the first weeks of regeneration all neurons formed a connectivity pattern with more connections medially and declining connections laterally, a pattern determined by the medial location of the nerve transplant. This pattern is maintained for most of the neurons, but for some there is an eventual reduction in medial connections as maximum synapse formation shifts to the lateral muscle fibers. Three of the eight neurons studied were able to regenerate connectivity patterns that corresponded to their segment of origin and not to their host muscle. This suggests that intersegmental muscle differences are not influencing the formation of these connectivity patterns, so the neurons will follow their inherent synaptogenesis program.     

Theoretical analysis of a synaptotropic dendrite growth mechanism

It is generally believed that the genome cannot encode explicit instructions to form each synaptic connection in the nervous system, but may provide general neurite growth mechanisms which will result in proper connectivity. Recent in vivo imaging has provided evidence for a synaptotropic growth mechanism, wherein synapses could influence dendrite growth by selectively stabilizing filopodia upon which they form. We undertook a theoretical investigation into the consequences of such a growth process. Discrete stochastic simulations demonstrate that the synaptotropic mechanism can result in decreased dendritic wiring length, is capable of searching for regions of high density pre-synaptic partners, and can recapitulate specific patterns of dendrite growth and connectivity. A mean-field analysis shows that growth by selective stabilization of filopodia can be approximated as a reaction-diffusion system, with a spatially varying diffusion constant that depends on the probability of synapse formation. Thus, growth will occur faster in regions of appropriate synaptic connections, and the net growth can be shown to climb a gradient of synaptic partner density. Synaptotropic growth thus presents a mechanism for the emergent development of connectivity based on local properties of the circuit elements, rather than explicit dependence on global guidance molecules or innate predetermined branching programs.     

Active colonization dynamics and diversity patterns are influenced by dendritic network connectivity and species interactions

Habitat network connectivity influences colonization dynamics, species invasions, and biodiversity patterns. Recent theoretical work suggests dendritic networks, such as those found in rivers, alter expectations regarding colonization and dispersal dynamics compared with other network types. As many native and non‐native species are spreading along river networks, this may have important ecological implications. However, experimental studies testing the effects of network structure on colonization and diversity patterns are scarce. Up to now, experimental studies have only considered networks where sites are connected with small corridors, or dispersal was experimentally controlled, which eliminates possible effects of species interactions on colonization dynamics. Here, we tested the effect of network connectivity and species interactions on colonization dynamics using continuous linear and dendritic (i.e., river‐like) networks, which allow for active dispersal. We used a set of six protist species and one rotifer species in linear and dendritic microcosm networks. At the start of the experiment, we introduced species, either singularly or as a community within the networks. Species subsequently actively colonized the networks. We periodically measured densities of species throughout the networks over 2 weeks to track community dynamics, colonization, and diversity patterns. We found that colonization of dendritic networks was faster compared with colonization of linear networks, which resulted in higher local mean species richness in dendritic networks. Initially, community similarity was also greater in dendritic networks compared with linear networks, but this effect vanished over time. The presence of species interactions increased community evenness over time, compared with extrapolations from single‐species setups. Our experimental findings confirm previous theoretical work and show that network connectivity, species‐specific dispersal ability, and species interactions greatly influence the dispersal and colonization of dendritic networks. We argue that these factors need to be considered in empirical studies, where effects of network connectivity on colonization patterns have been largely underestimated.     

Linking dendritic network structures to population demogenetics: The downside of connectivity

Spatial structures strongly influence ecological processes. Connectivity is known to positively influence metapopulation demography and genetics by increasing the rescue effect and thus favoring individual and gene flow between populations. This result has not been tested in the special case of dendritic networks, which encompass stream and cave ecosystem for instance. We propose a first approach using an individual based model to explore the population demography and genetics in various dendritic networks. To do so, we first generate a large number of different networks, and we analyze the relationship between their hydrographical characteristics and connectivity. We show that connectivity mean and variance of connectivity are strongly correlated in dendritic networks. Connectivity segregates two types of networks: Hortonian and non‐Hortonian networks. We then simulate the population dynamics for a simple life cycle in each of the generated networks, and we analyze persistence time as well as populations structure at quasi‐stationary state. Our main results show that connectivity in dendritic networks can promote local extinction and genetic isolation by distance at low dispersal and diminish the size of the metapopulation at high dispersal. We discuss these unexpected findings in the light of connectivity spatial distribution in dendritic networks in the case of our model.     

PTX-induced hyperexcitability affects dendritic shape and GABAergic synapse density but not synapse distribution during <Emphasis Type="Italic">Manduca</Emphasis> postembryonic motoneuron development

During the metamorphosis of the holometabolous insect, Manduca sexta, the postembryonic acquisition of adult specific motor behaviors is accompanied by changes in dendritic architecture, membrane currents, and input synapses of identified motoneurons. This study aims to test whether increased activity affects dendritic architecture and sub-dendritic input synapse distribution of the identified flight motoneuron 5 (MN5). Systemic injections of the chloride channel blocker, picrotoxin (PTX), during early pupal stages increase pupal reflex responsiveness, but overall development is not impaired. MN5 input resistance, resting membrane potential, and spiking threshold are not affected. Bath application of PTX to isolated ventral nerve cords evokes spiking in pupal and adult flight motoneurons. Quantitative three-dimensional reconstructions of the dendritic tree of the adult MN5 show that systemic PTX injections into early pupae cause dendritic overgrowth and reduce the density of GABAergic inputs. In contrast, the distribution patterns of GABAergic terminals throughout the dendritic tree remain unaltered. This indicates that increased overall excitability might cause dendritic overgrowth and decreased inhibitory input during postembryonic motoneuron remodeling, whereas sub-dendritic synapse targeting might be controlled by activity-independent signals. Behavioral testing reveals that these neuronal changes do not impede the animal’s ability to fly, but impair maximum flight performance.     

Neuronal adaptation accompanying metamorphosis in the flatfish

Flatfish provide a natural paradigm to investigate adaptive changes in the central nervous system of vertebrates. During their metamorphosis, the animals undergo a 90 degrees tilt to one side or the other to become the bottom-adapted adult flatfish. The eye on the down side is pushed over to the up side. Thus, vestibular and oculomotor coordinate systems rotate 90 degrees relative to each other. As a result, during swimming movements different types of compensatory eye movements are produced before and after metamorphosis by the same vestibular stimulation. Intracellular staining of central neurons with horseradish peroxidase revealed that in postmetamorphic flatfish second-order horizontal canal neurons contact vertical eye muscle motoneuron pools on both sides of the brain via pathways that are absent in all other vertebrates studied. These unique connections provide the necessary and sufficient connectivity to adapt the flatfish's eye movement system to the animals' postmetamorphic existence. Although the adult fish has a bilaterally asymmetric appearance, the central nervous connectivity reestablishes symmetry in the vestibulo-oculomotor system.     

Exogenous testosterone prevents motoneuron atrophy induced by contralateral motoneuron depletion

Gonadal steroids exhibit neuroprotective and neurotherapeutic effects. The lumbar spinal cord of male rats contains a highly androgen-sensitive population of motoneurons, the spinal nucleus of the bulbocavernosus (SNB), whose morphology and function are dependent on testosterone in adulthood. Unilateral SNB motoneuron depletion induces dendritic atrophy in contralateral SNB motoneurons, but this atrophy is reversed in previously castrated males treated with testosterone. In the present experiment we test the hypothesis that the morphology of SNB motoneurons is protected from atrophy after contralateral motoneuron depletion by exogenous testosterone alone (i.e., with no delay between castration and testosterone replacement). We unilaterally depleted SNB motoneurons by intramuscular injection of cholera toxin conjugated saporin. Simultaneously, some saporin-injected rats were castrated and immediately given replacement testosterone. Four weeks later, contralateral SNB motoneurons were labeled with cholera toxin conjugated HRP, soma sizes were measured, and dendritic arbors were reconstructed. Contralateral SNB motoneuron depletion induced somal atrophy and dendritic retraction, but testosterone treatment prevented both of these effects. Thus, the presence of high-normal levels of testosterone prevents motoneuron atrophy induced by contralateral motoneuron depletion. These data support a therapeutic role for testosterone in preventing atrophy induced by motoneuron injury.     

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.     

Regeneration of an identifiable motoneuron in the crayfish. I. Patterns of reconnection and synaptic strength established in normal and altered target areas

The superficial flexor muscles of the crayfish are innervated in a position-dependent connectivity pattern, which can be reestablished when the nerve to the muscle is cut. This article deals with the regeneration of the largest excitor motoneuron under three different target scenarios: (1) a normal target with all the muscle fibers present, (2) a reduced target lacking the medial or the lateral muscle fiber population, and (3) when the nerve enters the target in the middle of the muscle field. In scenario 1 the neuron is able to regenerate the normal connectivity pattern within 10 weeks after surgery: all the lateral fibers become innervated, with a linear decline in the probability of connections over the medial fibers. The medial fibers become transiently hyperinnervated before the normal pattern of connections is established. In scenario 2 the normal pattern of connections is established only when the lateral fibers were present; with only medial cells as a target, the transient hyperinnervation stage is stable and no decline in connections was observed. Analysis of regenerated junction potential sizes during the stable hyperinnervation stage show abnormal patterns, suggesting that some aspects of the regeneration program of this neuron can be affected when signals from its prime target cells are missing. In scenario 3 growth begins in both directions until the entire muscle becomes innervated. The normal pattern of connectivity finally emerges after continued lateral growth and diminished medial growth, suggesting that the position of the muscle fibers influences connectivity patterns during the final stages of regeneration.     

Independently Outgrowing Neurons and Geometry-Based Synapse Formation Produce Networks with Realistic Synaptic Connectivity

Neuronal signal integration and information processing in cortical networks critically depend on the organization of synaptic connectivity. During development, neurons can form synaptic connections when their axonal and dendritic arborizations come within close proximity of each other. Although many signaling cues are thought to be involved in guiding neuronal extensions, the extent to which accidental appositions between axons and dendrites can already account for synaptic connectivity remains unclear. To investigate this, we generated a local network of cortical L2/3 neurons that grew out independently of each other and that were not guided by any extracellular cues. Synapses were formed when axonal and dendritic branches came by chance within a threshold distance of each other. Despite the absence of guidance cues, we found that the emerging synaptic connectivity showed a good agreement with available experimental data on spatial locations of synapses on dendrites and axons, number of synapses by which neurons are connected, connection probability between neurons, distance between connected neurons, and pattern of synaptic connectivity. The connectivity pattern had a small-world topology but was not scale free. Together, our results suggest that baseline synaptic connectivity in local cortical circuits may largely result from accidentally overlapping axonal and dendritic branches of independently outgrowing neurons.     

Remote Modulation of Current Transfer from Dendritic Glutamatergic Synapses by GABA-ergic Synapses of the Somatic Zone of Motoneurons: a Simulation Study

Until now, information concerning spatial interaction of postsynaptic excitation and inhibition in neuronal dendrites remains rather limited. In model experiments, we studied spatial effects of tonic co-activation of GABA-ergic synapses situated on the soma and axon hillock of a motoneuron and dendritic glutamatergic synapses with receptors sensitive or insensitive to N-methyl-D-aspartate. We analyzed distribution maps of the transmembrane potentials and excitatory currents transferred toward the soma over the reconstructed dendritic arborization of a rat abducens motoneuron (three-dimensional reconstruction). In the motoneuron, isolated tonic excitation of glutamatergic synapses induced two stable states of low (downstate) or high (upstate) spatially heterogeneous dendritic depolarization, which decayed with unequal rates along different dendritic paths. In this case, the local steady-state current-voltage relation of the dendritic membrane became N-shaped due to a limb of the negative slope within a certain voltage range. The upstate corresponding to plateau potentials associated with stereotyped motor activity patterns was analyzed in detail. In this state, most proximal dendritic sites were the main sources of the excitatory current reaching the soma, while the contribution from distal sites was negligible. Co-activation of GABA-synapses located at the soma and axon hillock reduced this depolarization and shifted the main excitatory current source from a perisomatic location to the middle, structurally more complex, region of the dendritic arborization. The more remote dendritic region having a greater membrane area and receiving a greater number of synaptic contacts became directly involved in the supply of the trigger zone by the excitatory current. We suggest that a special, not described earlier, operational mechanism of postsynaptic inhibition is manifested in the above spatial effects of activation of strategically located inhibitory synapses, and that the list of known crucial inhibitory mechanisms (namely hyperpolarization and shunting of the postsynaptic membrane) must be expanded.     

Simulations of the alpha motoneuron pool electromyogram reflex at different preactivation levels in man

The alpha motoneuron pool and the surface electromyogram (EMG) of the human soleus muscle are modelled, respectively, by an alpha motoneuron pool model generating the firing patterns in the motor units of e muscle and by a muscle model using these discharge patterns to simulate the surface EMG. In the alpha motoneuron pool model, we use a population of motoneurons in which cellular properties like cell size and membrane conductance are distributed according to experimentally observed data. By calculating the contribution from each motor unit, the muscle model predicts the EMG. Wave forms of the motor unit action potentials in the surface EMG are obtained from experimental data. Using the model, we are able to give a quantitative prediction of the motoneuron pool activity and the reflex EMG output at different preactivation levels. The simulated data are consistent with experimentally obtained results in healthy humans. During static isometric muscle preactivations, the simulations show that the reflex strength is highly dependent on the intrinsic threshold properties of the alpha motoneuron pool. Received: 27 April 1993/Accepted in revised form: 8 September 1993