Drosophila enabled promotes synapse morphogenesis and regulates active zone form and function.

Background: Recent studies of synapse form and function highlight the importance of the actin cytoskeleton in regulating multiple aspects of morphogenesis, neurotransmission, and neural plasticity. The conserved actin-associated protein Enabled (Ena) is known to regulate development of the Drosophila larval neuromuscular junction through a postsynaptic mechanism. However, the functions and regulation of Ena within the presynaptic terminal has not been determined.

Target-dependent retrograde signaling mediates synaptic plasticity at the Drosophila neuromuscular junction.

Neurons that innervate multiple targets often establish synapses with target-specific strengths, and local forms of synaptic plasticity. We have examined the molecular-genetic mechanisms that allow a single Drosophila motoneuron, the ventral Common Exciter (vCE), to establish connections with target-specific properties at its various synaptic partners. By driving transgenes in a subset of vCE's targets, we found that individual target cells are able to independently control the properties of vCE's innervating branch and synapses.

Activity-Dependent Synaptic Refinement: New Insights from .

During development, neurons establish inappropriate connections as they seek out their synaptic partners, resulting in supernumerary synapses that must be pruned away. The removal of miswired synapses usually involves electrical activity, often through a Hebbian spike-timing mechanism. A novel form of activity-dependent refinement is used by that may be non-Hebbian, and is critical for generating the precise connectivity observed in that system.

Calcium Signaling during Synaptic Refinement at the Neuromuscular Junction.

Neural activity plays a key role in pruning aberrant synapses in various neural systems, including the mammalian cortex, where low-frequency (0.01 Hz) calcium oscillations refine topographic maps. However, the activity-dependent molecular mechanisms remain incompletely understood.

Cyclic nucleotide signaling is required during synaptic refinement at the Drosophila neuromuscular junction.

The removal of miswired synapses is a fundamental prerequisite for normal circuit development, leading to clinical problems when aberrant. However, the underlying activity-dependent molecular mechanisms involved in synaptic pruning remain incompletely resolved. Here the dynamic properties of intracellular calcium oscillations and a role for cAMP signaling during synaptic refinement in intact Drosophila embryos were examined using optogenetic tools.

Mutations in KATNB1 cause complex cerebral malformations by disrupting asymmetrically dividing neural progenitors.

Exome sequencing analysis of over 2,000 children with complex malformations of cortical development identified five independent (four homozygous and one compound heterozygous) deleterious mutations in KATNB1, encoding the regulatory subunit of the microtubule-severing enzyme Katanin. Mitotic spindle formation is defective in patient-derived fibroblasts, a consequence of disrupted interactions of mutant KATNB1 with KATNA1, the catalytic subunit of Katanin, and other microtubule-associated proteins. Loss of KATNB1 orthologs in zebrafish (katnb1) and flies (kat80) results in microcephaly, recapitulating the human phenotype.

A systematic nomenclature for the insect brain.

Despite the importance of the insect nervous system for functional and developmental neuroscience, descriptions of insect brains have suffered from a lack of uniform nomenclature. Ambiguous definitions of brain regions and fiber bundles have contributed to the variation of names used to describe the same structure. The lack of clearly determined neuropil boundaries has made it difficult to document precise locations of neuronal projections for connectomics study.

Retrograde BMP signaling at the synapse: a permissive signal for synapse maturation and activity-dependent plasticity.

At the Drosophila neuromuscular junction (NMJ), the loss of retrograde, trans-synaptic BMP signaling causes motoneuron terminals to have fewer synaptic boutons, whereas increased neuronal activity results in a larger synapse with more boutons. Here, we show that an early and transient BMP signal is necessary and sufficient for NMJ growth as well as for activity-dependent synaptic plasticity. This early critical period was revealed by the temporally controlled suppression of Mad, the SMAD1 transcriptional regulator.

TUNEL-antibody double-labeling method for Drosophila embryos.

The terminal deoxynucleotide transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method for monitoring targeted cell ablation is based on the in situ labeling of DNA fragmentation sites in nuclei of intact fixed cells. Unlike other methods of detecting dying cells, the use of fixed material allows antigen expression to be monitored at the same time that apoptosis is confirmed in the targeted cells. Double-labeling of Drosophila embryos using the TUNEL reaction and fluorescently tagged antibodies can be adapted to the selected antigen.

Genetic systems for functional cell ablation in Drosophila.

The selective removal of cells by ablation is a powerful tool in the study of eukaryotic developmental biology, providing much information about the origin, fate, or function of these cells in the developing organism. In Drosophila, three main methods have been used to ablate cells: chemical, genetic, and laser ablation. Each method has its own applicability with regard to developmental stage and the cells to be ablated, and its own limitations.

Embryonic cell ablation in Drosophila using lasers.

Cell ablation is a powerful tool in the study of eukaryotic developmental biology. The selective removal of cells by ablation may provide much information about their origin, fate, or function in the developing organism. Laser-based techniques have an advantage over genetic or chemical ablation methods in that the operations can be performed in essentially any cell pattern and at any time in development.

Setup for functional cell ablation with lasers: coupling of a laser to a microscope.

The selective removal of cells by ablation is a powerful tool in the study of eukaryotic developmental biology, providing much information about their origin, fate, or function in the developing organism. In Drosophila, three main methods have been used to ablate cells: chemical, genetic, and laser ablation. Each method has its own applicability with regard to developmental stage and the cells to be ablated, and its own limitations.

Monitoring membrane excitability in Drosophila expressing modified shaker constructs.

The Drosophila neuromuscular junction (NMJ) ranks as one of the preeminent model systems for studying synaptic development, function, and plasticity. This protocol describes the use of the two-electrode voltage clamp (TEVC) to examine potassium (K(+)) currents mediated by voltage-gated ion channels, and gives several genetic and pharmacological methods that are used to study the currents. Drosophila larval muscle fibers possess three major K(+) currents.

Experimental methods for examining synaptic plasticity in Drosophila.

The Drosophila neuromuscular junction (NMJ) ranks as one of the preeminent model systems for studying synaptic development, function, and plasticity. In this article, we review the experimental genetic methods that include the use of mutated or reengineered ion channels to manipulate the synaptic connections made by motor neurons onto larval body-wall muscles. We also provide a consideration of environmental and rearing conditions that phenocopy some of the genetic manipulations.

Hydroxyurea ablation of mushroom bodies in Drosophila.

Chemical ablation is an effective tool for studying nervous system development and function in Drosophila. Hydroxyurea (HU) inhibits ribonucleotide reductase, blocking DNA synthesis, and killing dividing cells. The specificity of HU ablation is thus dependent on developmental events.

X-gal staining of the central nervous system in adult Drosophila.

The Drosophila nervous system provides a valuable model for studying various aspects of brain development and function. The postembryonic Drosophila brain is especially useful, because specific neuron types derive from specific progenitors at specific times. Elucidating the means by which diverse neuron types derive from a limited number of progenitors can contribute significantly to our understanding of the genetic and molecular mechanisms involved in developmental neurobiology.

Antibody staining of the central nervous system in adult Drosophila.

The Drosophila nervous system provides a valuable model for studying various aspects of brain development and function. The postembryonic Drosophila brain is especially useful, because specific neuron types derive from specific progenitors at specific times. Elucidating the means by which diverse neuron types derive from a limited number of progenitors can contribute significantly to our understanding of the genetic and molecular mechanisms involved in developmental neurobiology.

Dissection of adult Drosophila brains.

The Drosophila nervous system provides a valuable model for studying various aspects of brain development and function. The postembryonic Drosophila brain is especially useful, because specific neuron types derive from specific progenitors at particular times. Elucidating the means by which diverse neuron types derive from a limited number of progenitors can contribute significantly to our understanding of the genetic and molecular mechanisms involved in developmental neurobiology.

Cracking the combinatorial semaphorin code.

In this issue of Neuron, Wu et al. describe a combinatorial code of repulsive Sema-2a and attractive Sema-2b signaling that mediates mechanosensory axonal guidance, fasciculation, and synaptic target selection within the CNS of Drosophila. Their work exemplifies how a detailed, multilevel molecular-genetic analysis (from molecules to behavior) provides fundamental insights into neural circuit development.

Maintenance and regulation of extracellular volume and the ion environment in Drosophila larval nerves.

In mammals and insects, paracellular blood barriers isolate the nervous system from the rest of the animal. Glia and accessory cells of the nervous system use pumps, channels, cotransporters, and exchangers collectively to maintain the extracellular ion environment and osmotic balance in the nervous system. At present, the molecular mechanisms that regulate this process remain unclear.

Drosophila glia use a conserved cotransporter mechanism to regulate extracellular volume.

The nervous system is protected by blood barriers that use multiple systems to control extracellular solute composition, osmotic pressure, and fluid volume. In the human nervous system, misregulation of the extracellular volume poses serious health threats. Here, we show that the glial cells that form the Drosophila blood-nerve barrier have a conserved molecular mechanism that regulates extracellular volume: the Serine/Threonine kinase Fray, which we previously showed is an ortholog of mammalian PASK/SPAK; and the Na-K-Cl cotransporter Ncc69, which we show is an ortholog of human NKCC1.

Presynaptic activity and CaMKII modulate retrograde semaphorin signaling and synaptic refinement.

Establishing synaptic connections often involves the activity-dependent withdrawal of off-target contacts. We describe an in vivo role for temporally patterned electrical activity, voltage-gated calcium channels, and CaMKII in modulating the response of Drosophila motoneurons to the chemorepellent Sema-2a during synaptic refinement. Mutations affecting the Sema-2a ligand, the plexin B receptor (plexB), the voltage-gated Ca(v)2.

Spatial and temporal control of gene expression in Drosophila using the inducible GeneSwitch GAL4 system. I. Screen for larval nervous system drivers.

There is a critical need for genetic methods for the inducible expression of transgenes in specific cells during development. A promising approach for this is the GeneSwitch GAL4 system of Drosophila. With GeneSwitch GAL4 the expression of upstream activating sequence (UAS) effector lines is controlled by a chimeric GAL4 protein that becomes active in the presence of the steroid RU486 (mifepristone).

Lim kinase regulates the development of olfactory and neuromuscular synapses.

Lim Kinase (Limk) belongs to a phylogenetically conserved family of serine/threonine kinases, which have been shown to be potent regulators of the actin cytoskeleton. Despite accumulating evidence of its biochemical actions, its in vivo function has remained poorly understood. The association of the Limk1 gene with Williams Syndrome indicates that proteins of this family play a role in the nervous system.