Drosophila Motor Neurons Research Articles

Modelling transport and mean age of dense core vesicles in large axonal arbours.

A model simulating the transport of dense core vesicles (DCVs) in type II axonal terminals of Drosophila motoneurons has been developed. The morphology of type II terminals is characterized by the large number of en passant boutons. The lack of both scaled-up DCV transport and scaled-down DCV capture in boutons results in a less efficient supply of DCVs to distal boutons. Furthermore, the large number of boutons that DCVs pass as they move anterogradely until they reach the most distal bouton may lead to the capture of a majority of DCVs before they turn around in the most distal bouton to move in the retrograde direction. This may lead to a reduced retrograde flux of DCVs and a lack of DCV circulation in type II terminals. The developed model simulates DCV concentrations in boutons, DCV fluxes between the boutons, age density distributions of DCVs and the mean age of DCVs in various boutons. Unlike published experimental observations, our model predicts DCV circulation in type II terminals after these terminals are filled to saturation. This disagreement is likely because experimentally observed terminals were not at steady state, but rather were accumulating DCVs for later release. Our estimates show that the number of DCVs in the transiting state is much smaller than that in the resident state. DCVs travelling in the axon, rather than DCVs transiting in the terminal, may provide a reserve of DCVs for replenishing boutons after a release. The techniques for modelling transport of DCVs developed in our paper can be used to model the transport of other organelles in axons.

Mining the Drosophila Gustatory Receptor Family for New Thermosensitive Proteins

Gustatory receptors (Gr) are a family of transmembrane proteins that have been extensively studied in the context of insect taste and odor sensory systems. Drosophila Gr28b(D) is the first gustatory receptor shown to be involved in thermosensation. In a previous study, we showed that Gr28b(D) exhibits a non-inactivating, temperature dependent, cation non-specific current, when expressed in Xenopus laevis oocytes. More recently, we explored the temperature sensitivity of Gr28b(D) orthologs from five Drosophila species (D. yakuba, simulans, pseudoobscura, willistoni, and mojavensis) that inhabit different environments. When expressed in Xenopus oocytes, Gr28b(D) orthologs exhibit various temperature sensitivities and current densities. Additionally, when expressed in Drosophila motor neurons, along with a genetically-encoded calcium sensor (GCaMP6f), Gr28b(D) orthologs exhibit unique changes in the firing pattern of individual cells, in a temperature-dependent fashion. Finally, flies over-expressing GR28b(D) orthologs exhibit different temperature-dependent behavior. By comparing their primary structure and through mutagenesis experiments, we identified functional domains within the Gr proteins and obtained mutants with various properties. These results identify novel thermosensitive gustatory receptors in Drosophila and expand the pool of potential candidates for thermogenetics tools. They also provide clues for further structure/function studies aimed at elucidating the molecular biophysics of thermosensation.

A De Novo RAPGEF2 Variant Identified in a Sporadic Amyotrophic Lateral Sclerosis Patient Impairs Microtubule Stability and Axonal Mitochondria Distribution.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is frequently linked to microtubule abnormalities and mitochondrial trafficking defects. Whole exome sequencing (WES) of patient-parent trios has proven to be an efficient strategy for identifying rare de novo genetic variants responsible for sporadic ALS (sALS). Using a trio-WES approach, we identified a de novo RAPGEF2 variant (c.4069G>A, p.E1357K) in a patient with early-onset sALS. To assess the pathogenic effects of this variant, we have used patient-derived skin fibroblasts and motor neuron-specific overexpression of the RAPGEF2-E1357K mutant protein in Drosophila. Patient fibroblasts display reduced microtubule stability and defective microtubule network morphology. The intracellular distribution, ultrastructure, and function of mitochondria are also impaired in patient cells. Overexpression of the RAPGEF2 mutant in Drosophila motor neurons reduces the stability of axonal microtubules and disrupts the distribution of mitochondria to distal axons and neuromuscular junction (NMJ) synapses. We also show that the recruitment of the pro-apoptotic protein BCL2-associated X (BAX) to mitochondria is significantly increased in patient fibroblasts compared with control cells. Finally, increasing microtubule stability through pharmacological inhibition of histone deacetylase 6 (HDAC6) rescues defects in the intracellular distribution of mitochondria and BAX. Overall, our data suggest that the RAPGEF2 variant identified in this study can drive ALS-related pathogenic effects through microtubule dysregulation.

Transcriptomics of aged Drosophila motor neurons reveals a matrix metalloproteinase that impairs motor function.

SummaryThe neuromuscular junction (NMJ) is responsible for transforming nervous system signals into motor behavior and locomotion. In the fruit fly Drosophila melanogaster, an age‐dependent decline in motor function occurs, analogous to the decline experienced in mice, humans, and other mammals. The molecular and cellular underpinnings of this decline are still poorly understood. By specifically profiling the transcriptome of Drosophila motor neurons across age using custom microarrays, we found that the expression of the matrix metalloproteinase 1 (dMMP1) gene reproducibly increased in motor neurons in an age‐dependent manner. Modulation of physiological aging also altered the rate of dMMP1 expression, validating dMMP1 expression as a bona fide aging biomarker for motor neurons. Temporally controlled overexpression of dMMP1 specifically in motor neurons was sufficient to induce deficits in climbing behavior and cause a decrease in neurotransmitter release at neuromuscular synapses. These deficits were reversible if the dMMP1 expression was shut off again immediately after the onset of motor dysfunction. Additionally, repression of dMMP1 enzymatic activity via overexpression of a tissue inhibitor of metalloproteinases delayed the onset of age‐dependent motor dysfunction. MMPs are required for proper tissue architecture during development. Our results support the idea that matrix metalloproteinase 1 is acting as a downstream effector of antagonistic pleiotropy in motor neurons and is necessary for proper development, but deleterious when reactivated at an advanced age.

Issue Cover (February 2018)

Front cover: Background: Alcohol abuse disorders are associated with adverse neurological symptoms and neurotoxicity. However, despite being one of the most commonly abused substances, the physiological effects of ethanol are not well defined. Main finding: By combining Drosophila, an ideal system for modelling ethanol disorders given their strong homology with mammals and natural response to ethanol, with high spatiotemporal resolution live-imaging of vesicle motility, we found that acute, low doses of ethanol result in increased flux of fluorescently-labeled neuropeptide vesicles toward neuromuscular junctions. Pharmacological treatments to increase (wortmannin) or decrease (LiCl) active GSK-3 levels revealed that GSK-3 is critical in mediating this stimulatory effect of ethanol. Implications: These results yield novel insight into the neurophysiological effects of ethanol and provide a possible link between behavioral responses to ethanol and neuropeptide transport. Image content: Time (y axis, 30 seconds) versus distance (x axis, 90 μm) graph of real-time movement of GFP-tagged neuropeptide vesicles in living Drosophila larvae. The reduced vesicle motility by wortmannin feeding is partially rescued by acute treatment with low dose ethanol. Read the full article ‘Ethanol stimulates the in vivo axonal movement of neuropeptide dense-core vesicles in Drosophila motor neurons’ by G. J. Iacobucci and S. Gunawardena (J. Neurochem. 2018, vol. 144(4), pp. 466–482) on doi: 10.1111/jnc.14230

Differing Strategies Despite Shared Lineages of Motor Neurons and Glia to Achieve Robust Development of an Adult Neuropil in Drosophila

In vertebrates and invertebrates, neurons and glia are generated in a stereotyped manner from neural stem cells, but the purpose of invariant lineages isnot understood. We show that two stem cells thatproduce leg motor neurons in Drosophila also generate neuropil glia, which wrap and send processes into the neuropil where motor neuron dendrites arborize. The development of the neuropil glia and leg motor neurons is highly coordinated. However, although motor neurons have a stereotyped birth order and transcription factor code, the number and individual morphologies of the glia born from these lineages are highly plastic, yet the final structure they contribute to is highly stereotyped. We suggest that the shared lineages of these two cell types facilitate the assembly of complex neural circuits and that the two birth order strategies-hardwired for motor neurons and flexible for glia-are important for robust nervous system development, homeostasis, and evolution.

The Drosophila Gr28bD product is a non-specific cation channel that can be used as a novel thermogenetic tool

Extrinsic control of single neurons and neuronal populations is a powerful approach for understanding how neural circuits function. Adding new thermogenetic tools to existing optogenetic and other forms of intervention will increase the complexity of questions that can be addressed. A good candidate for developing new thermogenetic tools is the Drosophila gustatory receptor family, which has been implicated in high-temperature avoidance behavior. We examined the five members of the Gr28b gene cluster for temperature-dependent properties via three approaches: biophysical characterization in Xenopus oocytes, functional calcium imaging in Drosophila motor neurons, and behavioral assays in adult Drosophila. Our results show that Gr28bD expression in Xenopus oocytes produces a non-specific cationic current that is activated by elevated temperatures. This current is non-inactivating and non-voltage dependent. When expressed in Drosophila motor neurons, Gr28bD can be used to change the firing pattern of individual cells in a temperature-dependent fashion. Finally, we show that pan-neuronal or motor neuron expression of Gr28bD can be used to alter fruit fly behavior with elevated temperatures. Together, these results validate the potential of the Gr28bD gene as a founding member of a new class of thermogenetic tools.

A simple microfluidic platform for the partial treatment of insuspendable tissue samples with orientation control.

Microfluidic devices have extensively been applied to study biological samples, including single cells. Exploiting laminar flows on a small scale, microfluidics allow for the selective and partial exposure of samples to various chemical treatments. Traditionally, suspendable samples are first flowed into formed microchannels and are allowed to adhere to the channel floor randomly with no control over sample placement or orientation, before being subjected to partial treatment. This severely limits the choice of samples and the extent of sample preparations. Here, we overcame this limit by reversing the sequence. We prepared the samples first on glass substrates. A patterned silicone slab was then placed on the substrate to form channels at an appropriate orientation with respect to the sample. We used liquid silicone rubber (LSR) as the base material. Its compliance (low elastic modulus) and its adhesion to glass offer the necessary seal to form the microchannels naturally. The applicability of the device was demonstrated by testing single axons of embryonic Drosophila motor neurons in vivo. A segment of the axons was subjected to drugs that inhibit myosin activities or block voltage-gated sodium ion channels. In response, the axons reduced the clustering of neuro-transmitter vesicles at the presynaptic terminal of neuromuscular junctions, or increased the calcium intake and underwent membrane hyperpolarization, respectively. Such fundamental studies cannot be carried out using conventional microfluidics.

Islet Coordinately Regulates Motor Axon Guidance and Dendrite Targeting through the Frazzled/DCC Receptor

Motor neuron axon targeting in the periphery is correlated with the positions of motor neuron inputs in the CNS, but how these processes are coordinated to form a myotopic map remains poorly understood. We show that the LIM homeodomain factor Islet (Isl) controls targeting of both axons and dendrites in Drosophila motor neurons through regulation of the Frazzled (Fra)/DCC receptor. Isl is required for fra expression in ventrally projecting motor neurons, and isl and fra mutants have similar axon guidance defects. Single-cell labeling indicates that isl and fra are also required for dendrite targeting in a subset of motor neurons. Finally, overexpression of Fra rescues axon and dendrite targeting defects in isl mutants. These results indicate that Fra acts downstream of Isl in both the periphery and the CNS, demonstrating how a single regulatory relationship is used in multiple cellular compartments to coordinate neural circuit wiring.

Differential effects of phytotherapic preparations in the hSOD1 Drosophila melanogaster model of ALS

The present study was aimed at characterizing the effects of Withania somnifera (Wse) and Mucuna pruriens (Mpe) on a Drosophila melanogaster model for Amyotrophic Lateral Sclerosis (ALS). In particular, the effects of Wse and Mpe were assessed following feeding the flies selectively overexpressing the wild human copper, zinc-superoxide dismutase (hSOD1-gain-of-function) in Drosophila motoneurons. Although ALS-hSOD1 mutants showed no impairment in life span, with respect to GAL4 controls, the results revealed impairment of climbing behaviour, muscle electrophysiological parameters (latency and amplitude of ePSPs) as well as thoracic ganglia mitochondrial functions. Interestingly, Wse treatment significantly increased lifespan of hSDO1 while Mpe had not effect. Conversely, both Wse and Mpe significantly rescued climbing impairment, and also latency and amplitude of ePSPs as well as failure responses to high frequency DLM stimulation. Finally, mitochondrial alterations were any more present in Wse- but not in Mpe-treated hSOD1 mutants. Hence, given the role of inflammation in the development of ALS, the high translational impact of the model, the known anti-inflammatory properties of these extracts, and the viability of their clinical use, these results suggest that the application of Wse and Mpe might represent a valuable pharmacological strategy to counteract the progression of ALS and related symptoms.

Structure and function of neuronal dendrites

Neurons represent the cellular substrate for information processing in the nervous system. Already around 1900 the Spanish neuroanatomist Ramón y Cajal proposed that neurons possess two discrete functional domains, the axonal and the somatodendritic compartment. Cajal established the foundation for the neuron doctrine by suggesting dendrites to be the synaptic input regions of neurons, and that information processing travels from dendritic regions towards axon terminals and output synapses (“the theory of dynamic polarization”, Shepherd, 1991). Despite a number of exceptions, for most neurons this rule prevails to the present. Therefore, dendritic architecture has two fundamental functions in the nervous system. First dendrites expand the receptive surface of neurons, and their shape dictates how many and which presynaptic neurons can contact a postsynaptic dendritic arbor. Thus, dendritic structure influences the number of synapses as well as the wiring logic within neuronal networks. Second dendritic structure impacts the temporal and spatial integration of postsynaptic potentials. Accordingly, in different types of neurons with different functions dendritic gestalt differs significantly, and dendritic architecture often serves to classify neuron types. In most cases, however, the specific function of dendritic architecture remains largely elusive. Dendritic structure analysis is further bedeviled by dendrites exhibiting voltage-gated ion channels which themselves vastly modify function and computing power. Although a multitude of neurodevelopmental and neurodegenerative disorders coincides with dendritic defects, it often remains unclear whether these structural defects are the cause or a consequence of the dysfunction. Therefore, on the one hand it is important to determine the contribution of dendritic structure to the function of different types of healthy neurons. On the other hand the question arises whether dendritic defects impact neuronal function qualitatively and to what degree of dendritic defect neuronal function can be maintained. This article will first summarize basic functions of passive dendritic architecture which applies for most neurons but confers variable characteristics to different types of neurons. It will be discussed how the location of input synapses in a passive electrical structure affects the integration of postsynaptic potentials. Then principles will be introduced how this localization-dependence of synaptic inputs into dendrites can be compensated for. And finally, an identified Drosophila motoneuron will serve as an example that at least in specific types of neurons basic function can be maintained with a minimum number of dendrites and input synapses. By contrast, in this example dendritic structure is imperative for fine tuning of adaptive behavioral functions which are essential for survival and reproduction. These findings will then be discussed in the context of other neuronal functions.

Mechanism of Axonal Contractility in Embryonic Drosophila Motor Neurons In Vivo

Several in vitro and limited in vivo experiments have shown that neurons maintain a rest tension along their axons intrinsically. They grow in response to stretch but contract in response to loss of tension. This contraction eventually leads to the restoration of the rest tension in axons. However, the mechanism by which axons maintain tension in vivo remains elusive. The objective of this work is to elucidate the key cytoskeletal components responsible for generating tension in axons. Toward this goal, in vivo experiments were conducted on single axons of embryonic Drosophila motor neurons in the presence of various drugs. Each axon was slackened mechanically by bringing the neuromuscular junction toward the central nervous system multiple times. In the absence of any drug, axons shortened and restored the straight configuration within 2–4 min of slackening. The total shortening was ∼40% of the original length. The recovery rate in each cycle, but not the recovery magnitude, was dependent on the axon’s prior contraction history. For example, the contraction time of a previously slackened axon may be twice its first-time contraction. This recovery was significantly hampered with the depletion of ATP, inhibition of myosin motors, and disruption of actin filaments. The disruption of microtubules did not affect the recovery magnitude, but, on the contrary, led to an enhanced recovery rate compared to control cases. These results suggest that the actomyosin machinery is the major active element in axonal contraction, whereas microtubules contribute as resistive/dissipative elements.

Compartmentalized Regulation of Parkin-Mediated Mitochondrial Quality Control in the Drosophila Nervous System In Vivo.

In neurons, the normal distribution and selective removal of mitochondria are considered essential for maintaining the functions of the large asymmetric cell and its diverse compartments. Parkin, a E3 ubiquitin ligase associated with familial Parkinson's disease, has been implicated in mitochondrial dynamics and removal in cells including neurons. However, it is not clear how Parkin functions in mitochondrial turnover in vivo, or whether Parkin-dependent events of the mitochondrial life cycle occur in all neuronal compartments. Here, using the live Drosophila nervous system, we investigated the involvement of Parkin in mitochondrial dynamics, distribution, morphology, and removal. Contrary to our expectations, we found that Parkin-deficient animals do not accumulate senescent mitochondria in their motor axons or neuromuscular junctions; instead, they contain far fewer axonal mitochondria, and these displayed normal motility behavior, morphology, and metabolic state. However, the loss of Parkin did produce abnormal tubular and reticular mitochondria restricted to the motor cell bodies. In addition, in contrast to drug-treated, immortalized cells in vitro, mature motor neurons rarely displayed Parkin-dependent mitophagy. These data indicate that the cell body is the focus of Parkin-dependent mitochondrial quality control in neurons, and argue that a selection process allows only healthy mitochondria to pass from cell bodies to axons, perhaps to limit the impact of mitochondrial dysfunction. Parkin has been proposed to police mitochondrial fidelity by binding to dysfunctional mitochondria via PTEN (phosphatase and tensin homolog)-induced putative kinase 1 (PINK1) and targeting them for autophagic degradation. However, it is unknown whether and how the PINK1/Parkin pathway regulates the mitochondrial life cycle in neurons in vivo Using Drosophila motor neurons, we show that parkin disruption generates an abnormal mitochondrial network in cell bodies in vivo and reduces the number of axonal mitochondria without producing any defects in their axonal transport, morphology, or metabolic state. Furthermore, while cultured neurons display Parkin-dependent axonal mitophagy, we find this is vanishingly rare in vivo under normal physiological conditions. Thus, both the spatial distribution and mechanism of mitochondrial quality control in vivo differ substantially from those observed in vitro.

Mechanism of Axonal Contractility in Embryonic Drosophila Motor Neurons in Vivo

Several in vitro and limited in vivo experiments have shown that neurons maintain a rest tension along their axons intrinsically. They grow in response to stretch but contract in response to loss of tension. This contraction eventually leads to the restoration of the rest tension in axons. However, the mechanism by which axons maintain tension in vivo remains elusive. The objective of this work is to elucidate the key cytoskeletal components responsible for generating tension in axons. Towards this goal, in vivo experiments were conducted on single axons of embryonic drosophila motor neurons in the presence of various drugs. Each axon was slackened mechanically by bringing the neuromuscular junction (NMJ) towards the central nervous system (CNS) multiple times. In the absence of any drug, axons always shortened and restored the straight configuration each time within 2-4 minutes of slackening. The total shortening was about 40% of the original length. The recovery rate in each cycle, but not the recovery magnitude, was dependent on the number of times the axon had previously been slackened. This recovery was significantly hampered with the depletion of ATP, inhibition of myosin motors, and disruption of actin filaments, but not with the disruption of microtubules. These results suggest that the actomyosin-machinery is the major active element in axonal contraction while microtubules contribute passively as resistive elements.

Distal spike initiation zone location estimation by morphological simulation of ionic current filtering demonstrated in a novel model of an identified Drosophila motoneuron.

Studying ion channel currents generated distally from the recording site is difficult because of artifacts caused by poor space clamp and membrane filtering. A computational model can quantify artifact parameters for correction by simulating the currents only if their exact anatomical location is known. We propose that the same artifacts that confound current recordings can help pinpoint the source of those currents by providing a signature of the neuron’s morphology. This method can improve the recording quality of currents initiated at the spike initiation zone (SIZ) that are often distal to the soma in invertebrate neurons. Drosophila being a valuable tool for characterizing ion currents, we estimated the SIZ location and quantified artifacts in an identified motoneuron, aCC/MN1-Ib, by constructing a novel multicompartmental model. Initial simulation of the measured biophysical channel properties in an isopotential Hodgkin-Huxley type neuron model partially replicated firing characteristics. Adding a second distal compartment, which contained spike-generating Na+ and K+ currents, was sufficient to simulate aCC’s in vivo activity signature. Matching this signature using a reconstructed morphology predicted that the SIZ is on aCC’s primary axon, 70 μm after the most distal dendritic branching point. From SIZ to soma, we observed and quantified selective morphological filtering of fast activating currents. Non-inactivating K+ currents are filtered ∼3 times less and despite their large magnitude at the soma they could be as distal as Na+ currents. The peak of transient component (NaT) of the voltage-activated Na+ current is also filtered more than the magnitude of slower persistent component (NaP), which can contribute to seizures. The corrected NaP/NaT ratio explains the previously observed discrepancy when the same channel is expressed in different cells. In summary, we used an in vivo signature to estimate ion channel location and recording artifacts, which can be applied to other neurons.

Loss of the Coffin-Lowry syndrome-associated gene RSK2 alters ERK activity, synaptic function and axonal transport in Drosophila motoneurons.

ABSTRACTPlastic changes in synaptic properties are considered as fundamental for adaptive behaviors. Extracellular-signal-regulated kinase (ERK)-mediated signaling has been implicated in regulation of synaptic plasticity. Ribosomal S6 kinase 2 (RSK2) acts as a regulator and downstream effector of ERK. In the brain, RSK2 is predominantly expressed in regions required for learning and memory. Loss-of-function mutations in human RSK2 cause Coffin-Lowry syndrome, which is characterized by severe mental retardation and low IQ scores in affected males. Knockout of RSK2 in mice or the RSK ortholog in Drosophila results in a variety of learning and memory defects. However, overall brain structure in these animals is not affected, leaving open the question of the pathophysiological consequences. Using the fly neuromuscular system as a model for excitatory glutamatergic synapses, we show that removal of RSK function causes distinct defects in motoneurons and at the neuromuscular junction. Based on histochemical and electrophysiological analyses, we conclude that RSK is required for normal synaptic morphology and function. Furthermore, loss of RSK function interferes with ERK signaling at different levels. Elevated ERK activity was evident in the somata of motoneurons, whereas decreased ERK activity was observed in axons and the presynapse. In addition, we uncovered a novel function of RSK in anterograde axonal transport. Our results emphasize the importance of fine-tuning ERK activity in neuronal processes underlying higher brain functions. In this context, RSK acts as a modulator of ERK signaling.

A TRPV Channel in Drosophila Motor Neurons Regulates Presynaptic Resting Ca2+ Levels, Synapse Growth, and Synaptic Transmission

Presynaptic resting Ca(2+) influences synaptic vesicle (SV) release probability. Here, we report that a TRPV channel, Inactive (Iav), maintains presynaptic resting [Ca(2+)] by promoting Ca(2+) release from the endoplasmic reticulum in Drosophila motor neurons, and is required for both synapse development and neurotransmission. We find that Iav activates the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin, which is essential for presynaptic microtubule stabilization at the neuromuscular junction. Thus, loss of Iav induces destabilization of presynaptic microtubules, resulting in diminished synaptic growth. Interestingly, expression of human TRPV1 in Iav-deficient motor neurons rescues these defects. We also show that the absence of Iav causes lower SV release probability and diminished synaptic transmission, whereas Iav overexpression elevates these synaptic parameters. Together, our findings indicate that Iav acts as a key regulator of synaptic development and function by influencing presynaptic resting [Ca(2+)].

Synapsin regulates activity-dependent outgrowth of synaptic boutons at the Drosophila neuromuscular junction.

Patterned depolarization of Drosophila motor neurons can rapidly induce the outgrowth of new synaptic boutons at the larval neuromuscular junction (NMJ), providing a model system to investigate mechanisms underlying acute structural plasticity. Correlative light and electron microscopy analysis revealed that new boutons typically form near the edge of postsynaptic reticulums of presynaptic boutons. Unlike mature boutons, new varicosities have synaptic vesicles which are distributed uniformly throughout the bouton and undeveloped postsynaptic specializations. To characterize the presynaptic mechanisms mediating new synaptic growth induced by patterned activity, we investigated the formation of new boutons in NMJs lacking synapsin [Syn(-)], a synaptic protein important for vesicle clustering, neurodevelopment, and plasticity. We found that budding of new boutons at Syn(-) NMJs was significantly diminished, and that new boutons in Syn(-) preparations were smaller and had reduced synaptic vesicle density. Since synapsin is a target of protein kinase A (PKA), we assayed whether activity-dependent synaptic growth is regulated via a cAMP/PKA/synapsin pathway. We pretreated preparations with forskolin to raise cAMP levels and found this manipulation significantly enhanced activity-dependent synaptic growth in control but not Syn(-) preparations. To examine the trafficking of synapsin during synaptic growth, we generated transgenic animals expressing fluorescently tagged synapsin. Fluorescence recovery after photobleaching analysis revealed that patterned depolarization promoted synapsin movement between boutons. During new synaptic bouton formation, synapsin redistributed upon stimulation toward the sites of varicosity outgrowth. These findings support a model whereby synapsin accumulates at sites of synaptic growth and facilitates budding of new boutons via a cAMP/PKA-dependent pathway.

Estimation of spike initiation zone and synaptic input parameters of a Drosophila motoneuron using a morphologically reconstructed model

The fruitfly Drosophila melanogaster has recently emerged as a powerful model for studying neuronal circuit development and function through identified neurons. A major obstacle to these studies has been a lack of computational models of these neurons for generating and testing hypotheses with fast-throughput simulations. Here, we constructed a computational model of an identified Drosophila motoneuron, aCC/MN1-Ib, from its recorded biophysical and anatomical properties [1]. Constructing this model required estimating the location and properties of distal spike-generating Na+ and K+ channels. The effects of channel location were simulated by including anatomical details in the model through a morphological reconstruction. By simulating a match to the observed in vivo electrophysiological characteristics, we predict the anatomical location of the spike initiation zone (SIZ) to be distal on aCC’s primary axon [2]. The complex morphology of the neuron effectively filters fast activating ionic currents which occur at the SIZ but are recoded from the soma in vivo. In particular, the transient component of the voltage-activated sodium current is extensively filtered relative to the slower persistent component. Input-output behavior in a broader network context was also predicted [2]: about 20 miniature excitatory potentials are required for initiating an action potential and about 200 individual potentials underlie evoked spontaneous rhythmic currents (SRC) in this neuron. Our model of aCC, which is the first morphologically-realistic active model of an identified Drosophila motoneuron, provides a template for further investigation of neuronal function in health and disease.

Enhanced tethered-flight duration and locomotor activity by overexpression of the human gene SOD1 in Drosophila motorneurons.

Mutation of the human gene superoxide dismutase (hSOD1) is associated with the fatal neurodegenerative disease familial amyotrophic lateral sclerosis (Lou Gehrig’s disease). Selective overexpression of hSOD1 in Drosophila motorneurons increases lifespan to 140% of normal. The current study was designed to determine resistance to lifespan decline and failure of sensorimotor functions by overexpressing hSOD1 in Drosophila‘s motorneurons. First, we measured the ability to maintain continuous flight and wingbeat frequency (WBF) as a function of age (5 to 50 days). Flies overexpressing hSOD1 under the D42-GAL4 activator were able to sustain flight significantly longer than controls, with the largest effect observed in the middle stages of life. The hSOD1-expressed line also had, on average, slower wingbeat frequencies in late, but not early life relative to age-matched controls. Second, we examined locomotor (exploratory walking) behavior in late life when flies had lost the ability to fly (age ≥ 60 d). hSOD1-expressed flies showed significantly more robust walking activity relative to controls. Findings show patterns of functional decline dissimilar to those reported for other life-extended lines, and suggest that the hSOD1 gene not only delays death but enhances sensorimotor abilities critical to survival even in late life.