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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.

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.

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.

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.

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.

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

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.

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.

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

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.

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.