Rewarding Capacity of Optogenetically Activating a Giant GABAergic Central-Brain Interneuron in Larval .

Larvae of the fruit fly are a powerful study case for understanding the neural circuits underlying behavior. Indeed, the numerical simplicity of the larval brain has permitted the reconstruction of its synaptic connectome, and genetic tools for manipulating single, identified neurons allow neural circuit function to be investigated with relative ease and precision. We focus on one of the most complex neurons in the brain of the larva (of either sex), the GABAergic anterior paired lateral neuron (APL).

Centrosome-dependent microtubule modifications set the conditions for axon formation.

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.

Separation of presynaptic Ca2 and Ca1 channel function in synaptic vesicle exo- and endocytosis by the membrane anchored Ca pump PMCA.

Synaptic vesicle (SV) release, recycling, and plastic changes of release probability co-occur side by side within nerve terminals and rely on local Ca signals with different temporal and spatial profiles. The mechanisms that guarantee separate regulation of these vital presynaptic functions during action potential (AP)-triggered presynaptic Ca entry remain unclear. Combining genetics with electrophysiology and imaging reveals the localization of two different voltage-gated calcium channels at the presynaptic terminals of glutamatergic neuromuscular synapses (the Ca2 homolog, Dmca1A or cacophony, and the Ca1 homolog, Dmca1D) but with spatial and functional separation.

A quick and versatile protocol for the 3D visualization of transgene expression across the whole body of larval .

Larval are used as a genetically accessible study case in many areas of biological research. Here we report a fast, robust and user-friendly procedure for the whole-body multi-fluorescence imaging of larvae; the protocol has been optimized specifically for larvae by systematically tackling the pitfalls associated with clearing this small but cuticularized organism. Tests on various fluorescent proteins reveal that the recently introduced monomeric infrared fluorescent protein (mIFP) is particularly suitable for our approach.

Radial somatic F-actin organization affects growth cone dynamics during early neuronal development.

The centrosome is thought to be the major neuronal microtubule-organizing center (MTOC) in early neuronal development, producing microtubules with a radial organization. In addition, albeit in vitro, recent work showed that isolated centrosomes could serve as an actin-organizing center, raising the possibility that neuronal development may, in addition, require a centrosome-based actin radial organization. Here, we report, using super-resolution microscopy and live-cell imaging of cultured rodent neurons, F-actin organization around the centrosome with dynamic F-actin aster-like structures with F-actin fibers extending and retracting actively.

F-actin patches associated with glutamatergic synapses control positioning of dendritic lysosomes.

Organelle positioning within neurites is required for proper neuronal function. In dendrites, with their complex cytoskeletal organization, transport of organelles is guided by local specializations of the microtubule and actin cytoskeleton, and by coordinated activity of different motor proteins. Here, we focus on the actin cytoskeleton in the dendritic shaft and describe dense structures consisting of longitudinal and branched actin filaments.

Prominent Postsynaptic and Dendritic Exocytosis of Endogenous BDNF Vesicles in BDNF-GFP Knock-in Mice.

Brain-derived neurotrophic factor (BDNF) is a secreted messenger molecule that is crucial for neuronal function and induction of synaptic plasticity. Although altered availability of BDNF underlies many neurological deficits and neurodegenerative disorders, secretion dynamics of endogenous BDNF are unexplored. We generated a BDNF-GFP knock-in (KiBE) mouse, in which GFP-labeled BDNF is expressed under the control of the unaltered endogenous mouse BDNF gene regulatory elements.

Click Chemistry (CuAAC) and Detection of Tagged Synthesized Proteins in .

Copper-catalyzed azide-alkyne-cycloaddition (CuAAC), also known as 'click chemistry' serves as a technique for bio-orthogonal, that is, bio-compatible labeling of macromolecules including proteins or lipids. Click chemistry has been widely used to covalently, selectively, and efficiently attach probes such as fluorophores or biotin to small bio-orthogonal chemical reporter groups introduced into macromolecules. In bio-orthogonal non-canonical amino acid tagging (BONCAT) and fluorescent non-canonical amino acid tagging (FUNCAT) proteins are metabolically labeled with a non-canonical, azide-bearing amino acid and subsequently CuAAC-clicked either to an alkyne-bearing biotin (BONCAT) for protein purification, Western blot, or mass spectrometry analyses or to an alkyne-bearing fluorophore (FUNCAT) for immunohistochemistry.

Functional architecture of reward learning in mushroom body extrinsic neurons of larval Drosophila.

The brain adaptively integrates present sensory input, past experience, and options for future action. The insect mushroom body exemplifies how a central brain structure brings about such integration. Here we use a combination of systematic single-cell labeling, connectomics, transgenic silencing, and activation experiments to study the mushroom body at single-cell resolution, focusing on the behavioral architecture of its input and output neurons (MBINs and MBONs), and of the mushroom body intrinsic APL neuron.

Caldendrin Directly Couples Postsynaptic Calcium Signals to Actin Remodeling in Dendritic Spines.

Compartmentalization of calcium-dependent plasticity allows for rapid actin remodeling in dendritic spines. However, molecular mechanisms for the spatio-temporal regulation of filamentous actin (F-actin) dynamics by spinous Ca-transients are still poorly defined. We show that the postsynaptic Ca sensor caldendrin orchestrates nano-domain actin dynamics that are essential for actin remodeling in the early phase of long-term potentiation (LTP).

Microtubules Modulate F-actin Dynamics during Neuronal Polarization.

Neuronal polarization is reflected by different dynamics of microtubule and filamentous actin (F-actin). Axonal microtubules are more stable than those in the remaining neurites, while dynamics of F-actin in axonal growth cones clearly exceed those in their dendritic counterparts. However, whether a functional interplay exists between the microtubule network and F-actin dynamics in growing axons and whether this interplay is instrumental for breaking cellular symmetry is currently unknown.

Cell Type-specific Metabolic Labeling of Proteins with Azidonorleucine in .

Advanced mass spectrometry technology has pushed proteomic analyses to the forefront of biological and biomedical research. Limitations of proteomic approaches now often remain with sample preparations rather than with the sensitivity of protein detection. However, deciphering proteomes and their context-dependent dynamics in subgroups of tissue-embedded cells still poses a challenge, which we meet with a detailed version of our recently established protocol for cell-selective and temporally controllable metabolic labeling of proteins in .

Periodic F-actin structures shape the neck of dendritic spines.

Most of the excitatory synapses on principal neurons of the forebrain are located on specialized structures called dendritic spines. Their morphology, comprising a spine head connected to the dendritic branch via a thin neck, provides biochemical and electrical compartmentalization during signal transmission. Spine shape is defined and tightly controlled by the organization of the actin cytoskeleton.

A Dendritic Golgi Satellite between ERGIC and Retromer.

The local synthesis of transmembrane proteins underlies functional specialization of dendritic microdomains in neuronal plasticity. It is unclear whether these proteins have access to the complete machinery of the secretory pathway following local synthesis. In this study, we describe a probe called pGolt that allows visualization of Golgi-related organelles for live imaging in neurons.

Cell-selective labelling of proteomes in Drosophila melanogaster.

The specification and adaptability of cells rely on changes in protein composition. Nonetheless, uncovering proteome dynamics with cell-type-specific resolution remains challenging. Here we introduce a strategy for cell-specific analysis of newly synthesized proteomes by combining targeted expression of a mutated methionyl-tRNA synthetase (MetRS) with bioorthogonal or fluorescent non-canonical amino-acid-tagging techniques (BONCAT or FUNCAT).

KCNQ5 K(+) channels control hippocampal synaptic inhibition and fast network oscillations.

KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) K(+) channels dampen neuronal excitability and their functional impairment may lead to epilepsy.

Terminal axonal arborization and synaptic bouton formation critically rely on abp1 and the arp2/3 complex.

Neuronal network formation depends on properly timed and localized generation of presynaptic as well as postsynaptic structures. Although of utmost importance for understanding development and plasticity of the nervous system and neurodegenerative diseases, the molecular mechanisms that ensure the fine-control needed for coordinated establishment of pre- and postsynapses are still largely unknown. We show that the F-actin-binding protein Abp1 is prominently expressed in the Drosophila nervous system and reveal that Abp1 is an important regulator in shaping glutamatergic neuromuscular junctions (NMJs) of flies.

Fast neurotransmitter release regulated by the endocytic scaffold intersectin.

Sustained fast neurotransmission requires the rapid replenishment of release-ready synaptic vesicles (SVs) at presynaptic active zones. Although the machineries for exocytic fusion and for subsequent endocytic membrane retrieval have been well characterized, little is known about the mechanisms underlying the rapid recruitment of SVs to release sites. Here we show that the Down syndrome-associated endocytic scaffold protein intersectin 1 is a crucial factor for the recruitment of release-ready SVs.

The Drosophila larval neuromuscular junction as a model for scaffold complexes at glutamatergic synapses: benefits and limitations.

Based on unbeatable genetic accessibility and relative simplicity, the Drosophila larval neuromuscular junction has become a widely used model system for studying functional and structural aspects of excitatory glutamatergic synapses. Membrane-associated guanylate kinase-like proteins (MAGUKs) are first-order scaffolding molecules enriched at many cellular junctions, including synapses, where they coordinate multiple binding partners, including cell adhesion molecules and ion channels. The enrichment of the prototypic MAGUK Discs-Large at larval NMJs apparently parallels the high abundance of its homologs at excitatory synapses in the mammalian central nervous system.

A perisynaptic ménage à trois between Dlg, DLin-7, and Metro controls proper organization of Drosophila synaptic junctions.

Structural plasticity of synaptic junctions is a prerequisite to achieve and modulate connectivity within nervous systems, e.g., during learning and memory formation.