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Neuromodulatory signalingviaG protein-coupled receptor (GPCRs) plays a pivotal role in regulating neural network function and animal behavior. Recent efforts have led to the development of optogenetic tools to induce G protein-mediated signaling, with the promise of acute and cell type-specific manipulation of neuromodulatory signals. However, designing and deploying optogenetically functionalized GPCRs (optoXRs) with accurate specificity and activity to mimic endogenous signalingin vivoremains challenging. Here we optimized the design of optoXRs by considering evolutionary conserved GPCR-G protein interactions and demonstrate the feasibility of this approach using twoDrosophilaDopamine receptors (optoDopRs). We validated these optoDopRs showing that they exhibit high signaling specificity and light sensitivityin vitro.In vivowe detected receptor and cell type-specific effects of dopaminergic signaling in various behaviors including the ability of optoDopRs to rescue loss of the endogenous receptors. This work demonstrates that OptoXRs can enable optical control of neuromodulatory receptor specific signaling in functional and behavioral studies.

Optogenetic silencing allows to reveal the necessity of selected neuronal populations for various neurophysiological functions ranging from specific behaviors to neuronal network activity. An ideal optogenetic silencing tool should be switchable between active and inactive states with precise timing while preserving its activity in the absence of light until switched to an inactive state. Although bistable anion-conducting channelrhodopsins (ACRs) were previously engineered to reach this goal, their conducting state life time was limited to only a few minutes and some ACRs were not fully switchable. Here we report Aion, a bistable ACR displaying a long-lasting open state with a spontaneous closing time constant close to 15 min. Moreover, Aion can be switched between the open and closed state with millisecond precision using blue and orange light, respectively. The long conducting state enables overnight silencing of neurons with minimal light exposure. We further generated trafficking-optimized versions of Aion, which show enhanced membrane localization and allow precisely timed, long-lasting all-optical control of nociceptive responses in larvae of Drosophila melanogaster.

Summary Animals display selective escape behaviors when faced with environmental threats. Selection of the appropriate response by the underlying neuronal network is key to maximizing chances of survival, yet the underlying network mechanisms are so far not fully understood. Using synapse-level reconstruction of the Drosophila larval network paired with physiological and behavioral readouts, we uncovered a circuit that gates selective escape behavior for noxious light through acute and input-specific neuropeptide action. Sensory neurons required for avoidance of noxious light and escape in response to harsh touch, each converge on discrete domains of neuromodulatory hub neurons. We show that acute release of hub neuron-derived insulin-like peptide 7 (Ilp7) and cognate relaxin family receptor (Lgr4) signaling in downstream neurons are required for noxious light avoidance, but not harsh touch responses. Our work highlights a role for compartmentalized circuit organization and neuropeptide release from regulatory hubs, acting as central circuit elements gating escape responses.

Dendritic morphogenesis requires dynamic microtubules (MTs) to form a coordinated cytoskeletal network during development. Dynamic MTs are characterized by their number, polarity and speed of polymerization. Previous studies described a correlation between anterograde MT growth and terminal branch extension in Drosophila dendritic arborization (da) neurons, suggesting a model that anterograde MT polymerization provides a driving force for dendritic branching. We recently found that the Ste20-like kinase Tao specifically regulates dendritic branching by controlling the number of dynamic MTs in a kinase activity-dependent fashion, without affecting MT polarity or speed. This finding raises the interesting question of how MT dynamics affects dendritic morphogenesis, and if Tao kinase activity is developmentally regulated to coordinate MT dynamics and dendritic morphogenesis. We explored the possible correlation between MT dynamics and dendritic morphogenesis together with the activity changes of Tao kinase in C1da and C4da neurons during larval development. Our data show that spatiotemporal changes in the number of dynamic MTs, but not polarity or polymerization speed, correlate with dendritic branching and Tao kinase activity. Our findings suggest that Tao kinase limits dendritic branching by controlling the abundance of dynamic MTs and we propose a novel model on how regulation of MT dynamics might influence dendritic morphogenesis.

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets., Currently, bidirectional control of activity in the same neurons in the same experiment is difficult. Here the authors report a Bidirectional Pair of Opsins for Light-induced Excitation and Silencing, BiPOLES, which they use in a range of organisms including worms, fruit flies, mice and ferrets.

Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson’s disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair., Author summary The death of physiologically important cells and tissues underlies of a wide range of diseases, including the neurodegenerative disorder Parkinson’s disease. Currently, the two major strategies to counter cell degeneration are the injection of soluble growth factor peptides and growth factor gene therapy. Importantly, both strategies can lead to the undesired activation of healthy bystander cells or the non-natural permanent modification of cells and their internal signals. Here, we developed a light-based method to overcome these limitations. The use of optogenetics allowed delivering cell type-specific pro-survival signals in a genetic model of Parkinson’s disease. In Drosophila and human cells that exhibit loss of the PINK1 gene, akin to autosomal recessive Parkinson’s disease, we efficiently suppressed disease phenotypes using a light-activated tyrosine kinase receptor. This work demonstrates a ‘remote controlled’ and thus spatio-temporally precise strategy to interfere with degeneration and may open new avenues towards tissue repair in a variety of disease models, including but not limited to diseases of the brain.

Information is carried between brain regions through neurotransmitter release from axonal presynaptic terminals. Understanding the functional roles of defined neuronal projection pathways in cognitive and behavioral processes requires temporally precise manipulation of their activity in vivo. However, existing optogenetic tools have low efficacy and off-target effects when applied to presynaptic terminals, while chemogenetic tools are difficult to control in space and time. Here, we show that a targeting-enhanced mosquito homologue of the vertebrate encephalopsin (eOPN3) can effectively suppress synaptic transmission through the Gi/o signaling pathway. Brief illumination of presynaptic terminals expressing eOPN3 triggers a lasting suppression of synaptic output that recovers spontaneously within minutes in vitro as well as in vivo. In freely moving mice, eOPN3-mediated suppression of dopaminergic nigrostriatal afferents leads to an ipsiversive rotational bias. We conclude that eOPN3 can be used to selectively suppress neurotransmitter release at synaptic terminals with high spatiotemporal precision, opening new avenues for functional interrogation of long-range neuronal circuits in vivo.

Microtubule (MT) modifications are critical during axon development, with stable MTs populating the axon. How these modifications are spatially coordinated is unclear. Here, via high-resolution microscopy, we show that early developing neurons have fewer somatic acetylated MTs restricted near the centrosome. At later stages, however, acetylated MTs spread out in soma and concentrate in growing axon. Live imaging in early plated neurons of the MT plus-end protein, EB3, show increased displacement and growth rate near the MTOC, suggesting local differences that might support axon selection. Moreover, F-actin disruption in early developing neurons, which show fewer somatic acetylated MTs, does not induce multiple axons, unlike later stages. Overexpression of centrosomal protein 120 (Cep120), which promotes MT acetylation/stabilization, induces multiple axons, while its knockdown downregulates proteins modulating MT dynamics and stability, hampering axon formation. Collectively, we show how centrosome-dependent MT modifications contribute to axon formation.

Animals display a plethora of escape behaviors when faced with environmental threats. Selection of the appropriate response by the underlying neuronal network is key to maximize chances of survival. We uncovered a somatosensory network in Drosophila larvae that encodes two escape behaviors through input-specific neuropeptide action. Sensory neurons required for avoidance of noxious light and escape in response to harsh touch, each converge on discrete domains of the same neuromodulatory hub neurons. These gate harsh touch responses via short Neuropeptide F, but noxious light avoidance via compartmentalized, acute Insulin-like peptide 7 action and cognate Relaxin-family receptor signaling in connected downstream neurons. Peptidergic hub neurons can thus act as central circuit elements for first order processing of converging sensory inputs to gate specific escape responses.One Sentence SummaryCompartment-specific neuropeptide action regulates sensory information processing to elicit discrete escape behavior in Drosophila larvae.

Optogenetics has been harnessed to shed new mechanistic light on current therapies and to develop future treatment strategies. This has been to date achieved by the correction of electrical signals in neuronal cells and neural circuits that are affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and thereby may modify progression of degenerative disorders, has never been demonstrated in an animal disease model. Here, we reengineered the human andDrosophila melanogasterREarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson’s disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRETviathe PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to overcome limitations of current strategies towards a spatio-temporal regulation of tissue repair.Significance StatementThe death of physiologically important cell populations underlies of a wide range of degenerative disorders, including Parkinson’s disease (PD). Two major strategies to counter cell degeneration, soluble growth factor injection and growth factor gene therapy, can lead to the undesired activation of bystander cells and non-natural permanent signaling responses. Here, we employed optogenetics to deliver cell type-specific pro-survival signals in a genetic model of PD. InDrosophilaand human cells exhibiting loss of the PINK1 kinase, akin to autosomal recessive PD, we efficiently suppressed disease phenotypes using a light-activated tyrosine kinase receptor. This work demonstrates a spatio-temporally precise strategy to interfere with degeneration and may open new avenues towards tissue repair in disease models.

Dendritic arborization is highly regulated and requires tight control of dendritic growth, branching, cytoskeletal dynamics, and ion channel expression to ensure proper function. Abnormal dendritic development can result in altered network connectivity, which has been linked to neurodevelopmental disorders, including autism spectrum disorders (ASDs). How neuronal growth control programs tune dendritic arborization to ensure function is still not fully understood. UsingDrosophiladendritic arborization (da) neurons as a model, we identified the conserved Ste20-like kinase Tao as a negative regulator of dendritic arborization. We show that Tao kinase activity regulates cytoskeletal dynamics and sensory channel localization required for proper sensory function in both male and female flies. We further provide evidence for functional conservation of Tao kinase, showing that its ASD-linked human ortholog, Tao kinase 2 (Taok2), could replaceDrosophilaTao and rescue dendritic branching, dynamic microtubule alterations, and behavioral defects. However, several ASD-linked Taok2 variants displayed impaired rescue activity, suggesting that Tao/Taok2 mutations can disrupt sensory neuron development and function. Consistently, we show that Tao kinase activity is required in developing and as well as adult stages for maintaining normal dendritic arborization and sensory function to regulate escape and social behavior. Our data suggest an important role for Tao kinase signaling in cytoskeletal organization to maintain proper dendritic arborization and sensory function, providing a strong link between developmental sensory aberrations and behavioral abnormalities relevant for Taok2-dependent ASDs.SIGNIFICANCE STATEMENTAutism spectrum disorders (ASDs) are linked to abnormal dendritic arbors. However, the mechanisms of how dendritic arbors develop to promote functional and proper behavior are unclear. We identifiedDrosophilaTao kinase, the ortholog of the ASD risk gene Taok2, as a regulator of dendritic arborization in sensory neurons. We show that Tao kinase regulates cytoskeletal dynamics, controls sensory ion channel localization, and is required to maintain somatosensory functionin vivo. Interestingly, ASD-linked human Taok2 mutations rendered it nonfunctional, whereas its WT form could restore neuronal morphology and function inDrosophilalacking endogenous Tao. Our findings provide evidence for a conserved role of Tao kinase in dendritic development and function of sensory neurons, suggesting that aberrant sensory function might be a common feature of ASDs.

Adhesion-type G protein-coupled receptors (aGPCRs) participate in a vast range of physiological processes. Correspondingly, these receptors are associated with diverse human diseases, such as developmental disorders, defects of the nervous system, allergies and cancer. Several aGPCRs have recently been linked to mechanosensitive functions suggesting that processing of mechanical stimuli may be a common feature of this receptor family. CIRL (ADGRL/Latrophilin, LPHN), one of the oldest members of the aGPCR family, sensitizes sensory responses of larvalDrosophilato gentle touch and sound by amplifying mechanosensory signal transduction in low-threshold mechanoreceptors (Scholz et al., 2015; 2017). In the present study, we show thatCirlis also expressed in high-threshold mechanical nociceptors where it adjusts nocifensive behaviour under physiological and pathophysiological conditions. Optogeneticin vivoexperiments indicate that CIRL quenches cAMP levels in both mechanosensory submodalities. However, contrasting its effect in touch sensitive neurons, CIRL dampens the response of nociceptors to mechanical stimulation. Consistent with this finding, rat nociceptors display a drop inCirl1expression during allodynia. Taken together, these results demonstrate that CIRL exerts opposing modulatory functions in low-threshold mechanosensors and high-threshold nociceptors. This intriguing bipolar action likely facilitates the separation of mechanosensory signals carrying different physiological information.

Nociception is an evolutionarily conserved mechanism to encode and process harmful environmental stimuli. Like most animals, Drosophila melanogaster larvae respond to a variety of nociceptive stimuli, including noxious touch and temperature, with stereotyped escape responses through activation of multimodal nociceptors. How behavioral responses to these different modalities are processed and integrated by the downstream network remains poorly understood. By combining trans-synaptic labeling, ultrastructural analysis, calcium imaging, optogenetics and behavioral analyses, we uncovered a circuit specific for mechanonociception but not thermonociception. Notably, integration of mechanosensory input from innocuous and nociceptive sensory neurons is required for robust mechanonociceptive responses. We further show that neurons integrating mechanosensory input facilitate primary nociceptive output by releasing short neuropeptide F, the Drosophila neuropeptide Y homolog. Our findings unveil how integration of somatosensory input and neuropeptide-mediated modulation can produce robust modality-specific escape behavior.

HIGHLIGHTS: eOPN3 is a mosquito-derived rhodopsin that inhibits neurotransmission in neurons. Activation of eOPN3 activates the G(i/o) pathway and reduces Ca(2+) channel activity; eOPN3 can suppress neurotransmission in a variety of cell types in vitro and in vivo. Activation of eOPN3 in nigrostriatal dopamine axons modulates locomotor behavior. SUMMARY: Information is carried between brain regions through neurotransmitter release from axonal presynaptic terminals. Understanding the functional roles of defined neuronal projection pathways requires temporally precise manipulation of their activity. However, existing inhibitory optogenetic tools have low efficacy and off-target effects when applied to presynaptic terminals, while chemogenetic tools are difficult to control in space and time. Here, we show that a targeting-enhanced mosquito homolog of the vertebrate encephalopsin (eOPN3) can effectively suppress synaptic transmission through the G(i/o) signaling pathway. Brief illumination of presynaptic terminals expressing eOPN3 triggers a lasting suppression of synaptic output that recovers spontaneously within minutes in vitro and in vivo. In freely moving mice, eOPN3-mediated suppression of dopaminergic nigrostriatal afferents induces a reversible ipsiversive rotational bias. We conclude that eOPN3 can be used to selectively suppress neurotransmitter release at presynaptic terminals with high spatiotemporal precision, opening new avenues for functional interrogation of long-range neuronal circuits in vivo.

Sensory circuits are typically established during early development, yet how circuit specificity and function are maintained during organismal growth has not been elucidated. To gain insight we quantitatively investigated synaptic growth and connectivity in the Drosophila nociceptive network during larval development. We show that connectivity between primary nociceptors and their downstream neurons scales with animal size. We further identified the conserved Ste20-like kinase Tao as a negative regulator of synaptic growth required for maintenance of circuit specificity and connectivity. Loss of Tao kinase resulted in exuberant postsynaptic specializations and aberrant connectivity during larval growth. Using functional imaging and behavioral analysis we show that loss of Tao-induced ectopic synapses with inappropriate partner neurons are functional and alter behavioral responses in a connection-specific manner. Our data show that fine-tuning of synaptic growth by Tao kinase is required for maintaining specificity and behavioral output of the neuronal network during animal growth., It is unclear how circuit specificity and function are maintained during organismal growth. In this study, authors show that connectivity between primary nociceptors and their downstream neurons scales with animal size and that Ste20-like kinase Tao acts as a negative regulator of synaptic growth required for maintenance of circuit specificity and connectivity.

Developmental pruning of axons and dendrites is critical for the formation of precise neuronal connections, but the mechanisms underlying developmental pruning are not fully understood. Here we investigated the function of JNK signaling in dendrite pruning using Drosophila class IV dendritic arborization (c4da) neurons as a model. We found that loss of JNK or its canonical downstream effectors d-Jun or d-Fos led to dendrite pruning defects in c4da neurons. Interestingly, our data showed that JNK activity in c4da neurons remained constant from larval to pupal stages but the expression of d-Fos was specifically activated by ecdysone receptor B1 (EcRB1) at early pupal stages, suggesting that ecdysone signaling provides a temporal control for the regulation of dendrite pruning by JNK signaling. Thus, our work not only identified a novel pathway involved in dendrite pruning and a new downstream target of EcRB1 in c4da neurons, but also revealed that JNK and Ecdysone signaling coordinate to promote dendrite pruning.

Multiple sclerosis (MS) is characterized by inflammatory insults that drive neuroaxonal injury. However, knowledge about neuron-intrinsic responses to inflammation is limited. By leveraging neuron-specific messenger RNA profiling, we found that neuroinflammation leads to induction and toxic accumulation of the synaptic protein bassoon (Bsn) in the neuronal somata of mice and patients with MS. Neuronal overexpression of Bsn in flies resulted in reduction of lifespan, while genetic disruption of Bsn protected mice from inflammation-induced neuroaxonal injury. Notably, pharmacological proteasome activation boosted the clearance of accumulated Bsn and enhanced neuronal survival. Our study demonstrates that neuroinflammation initiates toxic protein accumulation in neuronal somata and advocates proteasome activation as a potential remedy.

Thermo-nociception, the detection and behavioral response to noxious temperatures, is a highly conserved action to avoid injury and ensure survival. Basic molecular mechanisms of thermal responses have been elucidated in several model organisms and are of clinical relevance as thermal hypersensitivity (thermos-allodynia) is common in neuropathic pain syndromes. Drosophila larvae show stereotyped escape behavior upon noxious heat stimulation, which can be easily quantified and coupled with molecular genetic approaches. It has been successfully used to elucidate key molecular components and circuits involved in thermo-nociceptive responses. We provide a detailed and updated protocol of this previously described method ( Tracey et al., 2003 ) to apply a defined local heat stimulus to larvae using a fast-regulating hot probe.

Mapping brain function to brain structure is a fundamental task for neuroscience. For such an endeavour, the Drosophila larva is simple enough to be tractable, yet complex enough to be interesting. It features about 10,000 neurons and is capable of various taxes, kineses and Pavlovian conditioning. All its neurons are currently being mapped into a light-microscopical atlas, and Gal4 strains are being generated to experimentally access neurons one at a time. In addition, an electron microscopic reconstruction of its nervous system seems within reach. Notably, this electron microscope-based connectome is being drafted for a stage 1 larva - because stage 1 larvae are much smaller than stage 3 larvae. However, most behaviour analyses have been performed for stage 3 larvae because their larger size makes them easier to handle and observe. It is therefore warranted to either redo the electron microscopic reconstruction for a stage 3 larva or to survey the behavioural faculties of stage 1 larvae. We provide the latter. In a community-based approach we called the Ol1mpiad, we probed stage 1 Drosophila larvae for free locomotion, feeding, responsiveness to substrate vibration, gentle and nociceptive touch, burrowing, olfactory preference and thermotaxis, light avoidance, gustatory choice of various tastants plus odour-taste associative learning, as well as light/dark-electric shock associative learning. Quantitatively, stage 1 larvae show lower scores in most tasks, arguably because of their smaller size and lower speed. Qualitatively, however, stage 1 larvae perform strikingly similar to stage 3 larvae in almost all cases. These results bolster confidence in mapping brain structure and behaviour across developmental stages. The Gulbenkian/Melbourne group was supported by the Fundação para a Ciência e a Tecnologia (FCT; post-doctoral fellowship to M.J.A.-C.: SFRH/BPD/75993/2011; exploratory grant to M.J.A.-C. and C.K.M.: EXPL/BEX-BID/0497/2013). The Münster group was supported by the Cluster of Excellence Cells in Motion (CiM) and the CiM International Max Planck research school (CiM-IMPRS). The Barcelona group was supported by the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013-2017' (SEV-2012-0208), the CERCA Programme/Generalitat de Catalunya, the 'la Caixa' International PhD Programme, and the Spanish Ministry of Science and Innovation (BFU2011-26208). The LIN-GoLM group received support from the Wissenschaftsgemeinschaft Gottfried Wilhelm Leibniz, the State of Sachsen-Anhalt, the Center for Behavioral Brain Sciences Magdeburg and the Otto von Guericke Universität Magdeburg, as well as from the Deutsche Forschungsgemeinschaft (CRC 779 Motivated behaviour: B11; GE1091/4-1) and the European Commission (FP7-ICT project Miniature Insect Model for Active Learning MINIMAL). The Janelia group received support from the Howard Hughes Medical Institute. The Fribourg group was supported by a starter grant from the European Research Council (ERC-2012-StG 309832-PhotoNaviNet) and the Swiss National Science Foundation (31003A_169993). The Hamburg group was supported by the Deutsche Forschungsgemeinschaft (SPP 1926, Next generation optogenetics, SO1337/2-1), and the Landesforschungsförderung Hamburg (LFF-FV27). The LIN-MolSysBiol group received support from the Wissenschaftsgemeinschaft Gottfried Wilhelm Leibniz, the State of Sachsen-Anhalt, the Center for Behavioral Brain Sciences Magdeburg and the Deutsche Forschungsgemeinschaft (CRC 779 Motivated behaviour: B15; YA272/2-1). The Bonn group was supported by the Deutsche Forschungsgemeinschaft (PA 787/7-1) and Cluster of Excellence ImmunoSensation. The Konstanz group was supported by Deutsche Forschungsgemeinschaft (TH1584/1-1, TH1584/3-1), the Baden-Württemberg Stiftung and the Zukunftskolleg of the University of Konstanz.

As animals grow, many early born structures grow by cell expansion rather than cell addition; thus growth of distinct structures must be coordinated to maintain proportionality. This phenomenon is particularly widespread in the nervous system, with dendrite arbors of many neurons expanding in concert with their substrate to sustain connectivity and maintain receptive field coverage as animals grow. After rapidly growing to establish body wall coverage, dendrites of Drosophila class IV dendrite arborization (C4da) neurons grow synchronously with their substrate, the body wall epithelium, providing a system to study how proportionality is maintained during animal growth. Here, we show that the microRNA bantam (ban) ensures coordinated growth of C4da dendrites and the epithelium through regulation of epithelial endoreplication, a modified cell cycle that entails genome amplification without cell division. In Drosophila larvae, epithelial endoreplication leads to progressive changes in dendrite-extracellular matrix (ECM) and dendrite-epithelium contacts, coupling dendrite/substrate expansion and restricting dendrite growth beyond established boundaries. Moreover, changes in epithelial expression of cell adhesion molecules, including the beta-integrin myospheroid (mys), accompany this developmental transition. Finally, endoreplication and the accompanying changes in epithelial mys expression are required to constrain late-stage dendrite growth and structural plasticity. Hence, modulating epithelium-ECM attachment probably influences substrate permissivity for dendrite growth and contributes to the dendrite-substrate coupling that ensures proportional expansion of the two cell types.

Mapping brain function to brain structure is a fundamental task for neuroscience. For such an endeavour, the

Summary: During differentiation, neurons require a high lipid supply for membrane formation as they elaborate complex dendritic morphologies. While glia-derived lipids support neuronal growth during development, the importance of cell-autonomous lipid production for dendrite formation has been unclear. Using Drosophila larva dendritic arborization (da) neurons, we show that dendrite expansion relies on cell-autonomous fatty acid production. The nociceptive class four (CIV) da neurons form particularly large space-filling dendrites. We show that dendrite formation in these CIVda neurons additionally requires functional sterol regulatory element binding protein (SREBP), a crucial regulator of fatty acid production. The dendrite simplification in srebp mutant CIVda neurons is accompanied by hypersensitivity of srebp mutant larvae to noxious stimuli. Taken together, our work reveals that cell-autonomous fatty acid production is required for proper dendritic development and establishes the role of SREBP in complex neurons for dendrite elaboration and function. : Ziegler et al. highlight the endogenous role of fatty acid synthesis for proper neuronal dendrite growth during development. Using Drosophila da neurons, they show that large CIVda neurons cell-autonomously rely on fatty acid synthesis through the lipid synthesis master regulator SREBP. Keywords: Drosophila, dendrite differentiation, fatty acids, lipids, SREBP, metabolism, brain, nociception

Precise patterning of dendritic arbors is critical for the wiring and function of neural circuits. Dendrite-extracellular matrix (ECM) adhesion ensures that the dendrites of Drosophila dendritic arborization (da) sensory neurons are properly restricted in a 2D space, and thereby facilitates contact-mediated dendritic self-avoidance and tiling. However, the mechanisms regulating dendrite-ECM adhesion in vivo are poorly understood. Here, we show that mutations in the semaphorin ligand sema-2b lead to a dramatic increase in self-crossing of dendrites due to defects in dendrite-ECM adhesion, resulting in a failure to confine dendrites to a 2D plane. Furthermore, we find that Sema-2b is secreted from the epidermis and signals through the Plexin B receptor in neighboring neurons. Importantly, we find that Sema-2b/PlexB genetically and physically interacts with TORC2 complex, Tricornered (Trc) kinase, and integrins. These results reveal a novel role for semaphorins in dendrite patterning and illustrate how epidermal-derived cues regulate neural circuit assembly.

Genetic engineering of natural light-gated ion channels has proven a powerful way to generate optogenetic tools for a wide variety of applications. In recent years, blue-light activated engineered anion-conducting channelrhodopsins (eACRs) have been developed, improved, and were successfully applied in vivo. We asked whether the approaches used to create eACRs can be transferred to other well-characterized cation-conducting channelrhodopsins (CCRs) to obtain eACRs with a broad spectrum of biophysical properties. We generated 22 variants using two conversion strategies applied to 11 CCRs and screened them for membrane expression, photocurrents and anion selectivity. We obtained two novel eACRs, Phobos and Aurora, with blue- and red-shifted action spectra and photocurrents similar to existing eACRs. Furthermore, step-function mutations greatly enhanced the cellular operational light sensitivity due to a slowed-down photocycle. These bi-stable eACRs can be reversibly toggled between open and closed states with brief light pulses of different wavelengths. All new eACRs reliably inhibited action potential firing in pyramidal CA1 neurons. In Drosophila larvae, eACRs conveyed robust and specific light-dependent inhibition of locomotion and nociception.

Self-avoidance allows a neuron’s dendrites to recognize and repel each other. It constitutes one of the basic mechanisms facilitating even patterning of the receptive field. Self-avoidance enables neurons to distinguish self- from nonself-dendrites in complex systems so their receptive fields can coexist in overlapping territories. The phenomenon was first described in leech and later in Drosophila and C. elegans, with recent studies in vertebrate systems revealing self-avoidance as a universally conserved mechanism required to pattern dendrites of many if not all neurons. Molecularly, members of the immunoglobulin superfamily (IgSF), in particular Down’s syndrome cell adhesion molecule (Dscam), and clustered protocadherins (Pcdhs) play a prominent role in mediating self-avoidance. Here, I will summarize the findings and advances in our understanding of the conceptual and molecular nature of dendrite self-avoidance from the cellular to organismal level and discuss its contribution to dendritic patterning.

Alzheimer's disease (AD) is characterized by neurofibrillary tangles and extracellular plaques, which consist mainly of beta-amyloid derived from the beta-amyloid precursor protein (APP). An additional feature of AD is axonopathy, which might contribute to impairment of cognitive functions. Specifically, axonal transport defects have been reported in AD animal models, including mice and flies that overexpress APP and tau. Here we demonstrate that the APP-induced traffic jam of vesicles in peripheral nerves of Drosophila melanogaster larvae depends on the four residues NPTY motif in the APP intracellular domain. Furthermore, heterologous expression of Fe65 and JIP1b, scaffolding proteins interacting with the NPTY motif, also perturb axonal transport. Together, these data indicate that JIP1b or Fe65 may be involved in the APP-induced axonal transport defect. Moreover, we have characterized neurotransmission at the neuromuscular junction in transgenic larvae that express human APP. Consistent with the observation that these larvae do not show any obvious movement deficits, we found no changes in basal synaptic transmission. However, short-term synaptic plasticity was affected by overexpression of APP. Together, our results show that overexpression of APP induces partial stalling of axonal transport vesicles, paralleled by abnormalities in synaptic plasticity, which may provide a functional link to the deterioration of cognitive functions observed in AD.

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease, but its physiological function and that of its mammalian paralogs, the amyloid precursor-like proteins 1 and 2 (APLPs), is still poorly understood. APP has been proposed to form dimers, a process that could promote cell adhesion via trans-dimerization. We investigated the dimerization and cell adhesion properties of APP/APLPs and provide evidence that all three paralogs are capable of forming homo- and heterocomplexes. Moreover, we show that trans-interaction of APP family proteins promotes cell–cell adhesion in a homo- and heterotypic fashion and that endogenous APLP2 is required for cell–cell adhesion in mouse embryonic fibroblasts. We further demonstrate interaction of all the three APP family members in mouse brain, genetic interdependence, and molecular interaction of APP and APLPs in synaptically enriched membrane compartments. Together, our results provide evidence that homo- and heterocomplexes of APP/APLPs promote trans-cellular adhesion in vivo.

Amyloid precursor protein (APP) processing is of major interest in Alzheimer's disease research, since sequential cleavages by β- and γ-secretase lead to the formation of the 4-kDa amyloid Aβ protein peptide that accumulates in Alzheimer's disease brain. The processing of APP involves proteolytic conversion by different secretases leading to α-, β-, γ-, δ-, and ϵ-cleavages. Since modulation of these cleavages represents a rational therapeutic approach to control amyloid formation, its interference with the processing of the members of the APP gene family is of considerable importance. By using C-terminally tagged constructs of APLP-1 and APLP-2 and the untagged proteins, we have characterized their proteolytic C-terminal fragments produced in stably transfected SH-SY5Y cells. Pharmacological manipulation with specific protease inhibitors revealed that both homologues are processed by α- and γ-secretase-like cleavages, and that their intracellular domains can be released by cleavage at ϵ-sites. APLP-2 processing appears to be the most elaborate and to involve alternative cleavage sites. We show that APLP-1 is the only member of the APP gene family for which processing can be influenced by N-glycosylation. Additionally, we were able to detect p3-like fragments of APLP-1 and p3-like and Aβ-like fragments of APLP-2 in the media of stably transfected SH-SY5Y cells.

The cleavage of proteins within their transmembrane domain by Presenilin (PS) has an important role in different signalling pathways and in Alzheimer's disease. Nevertheless, not much is known about the regulation of PS activity. It has been suggested that substrate recognition by the PS complex depends only on the size of the extracellular domain independent of the amino-acid sequence and that PS activity is constitutive in all cells that express the minimal components of the complex. We report here the development of an in vivo reporter system that allowed us to analyse the processing of human amyloid precursor protein (APP) and the Notch receptor tissue specifically during Drosophila development in the living organism. Using this system, we demonstrate differences between APP and Notch processing and show that PS-mediated cleavage of APP can be regulated in different cell types independent of the size of the extracellular domain.

New dicarbonylnickel catalysts with a variety of mono- and bidentate phosphine ligands, such as Ph2P(CH2)xPPh(CH2)ySi(OEt)3 (x=2, 3; y=2, 3), have been synthesized and fully characterized, three of them by X-ray analyses. All the catalysts have been immobilized on silica by reaction of the ethoxysilane or hydroxy groups of the phosphine linkers with the surface silanol groups. The clean immobilization was proved by 31P solid-state NMR. It has been demonstrated that the COSi bridge, in contrast to the strong SiOSi bond, is easily cleaved by organic solvents and water. With respect to the cyclotrimerization of phenylacetylene the optimal reaction conditions for all molecular and immobilized catalysts have been determined. Cyclooctane was the best solvent, and the optimal reaction temperature was 100 °C for reactions in homogeneous phase, and 155 °C for surface-bound catalysts. The bite angles and spacer lengths of the chelating ligands did not play a major role for the performance of the homogeneous or immobilized catalysts. The latter could be recycled for 12 times, with final substrate conversions still typically between 30 and 40%, and turnover numbers (TON) around 1500. With an excess of substrate in one run a TON of 4810 was obtained for an immobilized carbonylnickel complex with a dppp-type chelate phosphine (x=3, y=2). Reduction of the surface coverage generally resulted in catalysts with longer lifetimes, for example, reduction to one fourth gave a record TON of 7616 for this catalyst. The selectivities of the catalysts all followed one trend. The main products were the cyclotrimers 1,2,4- and 1,3,5-triphenylbenzene, besides dimers, linear trimers and tetramers as side-products. The fraction of the latter decreased at higher temperatures with homogeneous catalysts, and vanished entirely after a few cycles with the immobilized catalysts. On recycling, the initial ratio of unsymmetrical to symmetrical cyclotrimers of typically 5 : 1 gradually changed to about 1 : 1 for all catalysts.

Prior to the discovery of natural anion conducting channelrhodopsins (nACRs), artificial anion conducting channelrhodopsins (aACRs) have been developed, improved and were successfully applied in vivo. We asked whether the approaches used to create aACRs can be transferred to other well-characterized cation-conducting channelrhodopsins to obtain aACRs with a broader variation of biophysical properties. The screen of 18 different constructs yielded two new aACRs, termed Phobos and Aurora, which express well in neurons and provide sufficient photocurrents for efficient silencing. They can be activated with far blue or far red light, respectively, making them suitable for combination with other spectrally distinct actuators or sensors of the optogenetic toolbox. Furthermore, introduction of step function mutations slowed down the photocycle of the new aACRs up to 10000-fold, thus greatly enhancing light sensitivity. These light-switchable versions of Phobos and Aurora can further be reversibly toggled between open and closed states with brief light pulses of different wavelengths. Both variants and their step function mutants showed perfect selectivity for chloride ions and where tested in hippocampal slice cultures, reliably inhibiting action potential firing. In drosophila larvae, the new aACR variants showed robust and specific light-dependent in vivo inhibition of locomotion and nociception. Ongoing experiments will reveal in vivo aACR functionality in rodents and adult drosophila.

The amyloid precursor protein (APP) plays a central role in Alzheimer’s disease (AD) and has essential synapse promoting functions. Synaptogenic activity as well as cell adhesion properties of APP presumably depend on trans-cellular dimerization via its extracellular domain. Since neuronal APP is extensively processed by secretases, it raises the question if APP shedding affects its cell adhesion and synaptogenic properties. We show that inhibition of APP shedding using cleavage deficient forms of APP or a dominant negative α-secretase strongly enhanced its cell adhesion and synaptogenic activity suggesting that synapse promoting function of APP is tightly regulated by α-secretase mediated processing, similar to other trans-cellular synaptic adhesion molecules., Frontiers in Cellular Neuroscience, 8, ISSN:1662-5102