Ultrastructural Analysis Reveals Mitochondrial Placement Independent of Synapse Placement in Fine Caliber C. elegans Neurons.

Neurons rely on mitochondria for an efficient supply of ATP and other metabolites. However, while neurons are highly elongated, mitochondria are discrete and limited in number. Due to the slow rates of metabolite diffusion over long distances, it follows that neurons would benefit from an ability to control the distribution of mitochondria to sites of high metabolic activity such as synapses.

Bichromatic exon-reporters reveal voltage-gated Ca -channel splice-isoform diversity across neurons .

Every neuron contains the same genomic information but its complement of proteins is the product of countless neuron-specific steps including pre-mRNA splicing. Despite advances in RNA sequencing techniques, pre-mRNA splicing biases that favor one isoform over another are largely inscrutable in live neurons . Here, in , we developed bichromatic fluorescent reporters to investigate alternate splicing of - a gene that codes the pore-forming α -subunit of the primary neuronal voltage-gated Ca channel (VGCC).

Mitochondrial phosphagen kinases support the volatile power demands of motor nerve terminals.

Motor neurons are the longest neurons in the body, with axon terminals separated from the soma by as much as a meter. These terminals are largely autonomous with regard to their bioenergetic metabolism and must burn energy at a high rate to sustain muscle contraction. Here, through computer simulation and drawing on previously published empirical data, we determined that motor neuron terminals in Drosophila larvae experience highly volatile power demands.

Ultrastructural analysis reveals mitochondrial placement independent of synapse placement in fine caliber C. elegans neurons.

Neurons rely on mitochondria for an efficient supply of ATP and other metabolites. However, while neurons are highly elongated, mitochondria are discrete and limited in number. Due to the slow rates of diffusion over long distances it follows that neurons would benefit from an ability to control the distribution of mitochondria to sites of high metabolic activity, such as synapses.

Physiologic and Nanoscale Distinctions Define Glutamatergic Synapses in Tonic vs Phasic Neurons.

Neurons exhibit a striking degree of functional diversity, each one tuned to the needs of the circuitry in which it is embedded. A fundamental functional dichotomy occurs in activity patterns, with some neurons firing at a relatively constant "tonic" rate, while others fire in bursts, a "phasic" pattern. Synapses formed by tonic versus phasic neurons are also functionally differentiated, yet the bases of their distinctive properties remain enigmatic.

Presynaptic Mitochondrial Volume and Packing Density Scale with Presynaptic Power Demand.

Stable neural function requires an energy supply that can meet the intense episodic power demands of neuronal activity. Neurons have presumably optimized the volume of their bioenergetic machinery to ensure these power demands are met, but the relationship between presynaptic power demands and the volume available to the bioenergetic machinery has never been quantified. Here, we estimated the power demands of six motor nerve terminals in female larvae through direct measurements of neurotransmitter release and Ca entry, and via theoretical estimates of Na entry and power demands at rest.

Computational modeling predicts ephemeral acidic microdomains in the glutamatergic synaptic cleft.

At chemical synapses, synaptic vesicles release their acidic contents into the cleft, leading to the expectation that the cleft should acidify. However, fluorescent pH probes targeted to the cleft of conventional glutamatergic synapses in both fruit flies and mice reveal cleft alkalinization rather than acidification. Here, using a reaction-diffusion scheme, we modeled pH dynamics at the Drosophila neuromuscular junction as glutamate, ATP, and protons (H) were released into the cleft.

Neto-α Controls Synapse Organization and Homeostasis at the Drosophila Neuromuscular Junction.

Glutamate receptor auxiliary proteins control receptor distribution and function, ultimately controlling synapse assembly, maturation, and plasticity. At the Drosophila neuromuscular junction (NMJ), a synapse with both pre- and postsynaptic kainate-type glutamate receptors (KARs), we show that the auxiliary protein Neto evolved functionally distinct isoforms to modulate synapse development and homeostasis. Using genetics, cell biology, and electrophysiology, we demonstrate that Neto-α functions on both sides of the NMJ.

Neuronal Glutamatergic Synaptic Clefts Alkalinize Rather Than Acidify during Neurotransmission.

The dogma that the synaptic cleft acidifies during neurotransmission is based on the corelease of neurotransmitters and protons from synaptic vesicles, and is supported by direct data from sensory ribbon-type synapses. However, it is unclear whether acidification occurs at non-ribbon-type synapses. Here we used genetically encoded fluorescent pH indicators to examine cleft pH at conventional neuronal synapses.

Endogenous Tagging Reveals Differential Regulation of Ca Channels at Single Active Zones during Presynaptic Homeostatic Potentiation and Depression.

Neurons communicate through Ca-dependent neurotransmitter release at presynaptic active zones (AZs). Neurotransmitter release properties play a key role in defining information flow in circuits and are tuned during multiple forms of plasticity. Despite their central role in determining neurotransmitter release properties, little is known about how Ca channel levels are modulated to calibrate synaptic function.

The application of 'kisser' probes for resolving the distribution and microenvironment of membrane proteins in situ.

Membrane proteins play a lead role in the formation and function of synapses, but, despite revolutions in immunology and molecular genetics, limitations persist in our ability to investigate membrane proteins in the context of an intact synapse. Here, we introduce a simple but novel approach to resolving the distribution of endogenous membrane proteins in either live or fixed tissues. The technique involves transgenic expression of a protein with an extracellular tag, a generic transmembrane domain, and an intracellular terminus that mimics the intracellular anchoring motifs of the endogenous protein of interest.

The Krebs Cycle Enzyme Isocitrate Dehydrogenase 3A Couples Mitochondrial Metabolism to Synaptic Transmission.

Neurotransmission is a tightly regulated Ca-dependent process. Upon Ca influx, Synaptotagmin1 (Syt1) promotes fusion of synaptic vesicles (SVs) with the plasma membrane. This requires regulation at multiple levels, but the role of metabolites in SV release is unclear.

Na /H exchange via the Drosophila vesicular glutamate transporter mediates activity-induced acid efflux from presynaptic terminals.

Key Points: Intracellular pH regulation is vital to neurons as nerve activity produces large and rapid acid loads in presynaptic terminals. Rapid clearance of acid loads is necessary to maintain control of neurotransmission, but neuronal acid clearance mechanisms remain poorly understood. Glutamate is loaded into synaptic vesicles via the vesicular glutamate transporter (VGLUT), a mechanism conserved across phyla, and this study reports a previously unknown role for VGLUT as an acid-extruding protein when deposited in the plasmamembrane during exocytosis.

High-Probability Neurotransmitter Release Sites Represent an Energy-Efficient Design.

Nerve terminals contain multiple sites specialized for the release of neurotransmitters. Release usually occurs with low probability, a design thought to confer many advantages. High-probability release sites are not uncommon, but their advantages are not well understood.

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.

The lack of CuZnSOD leads to impaired neurotransmitter release, neuromuscular junction destabilization and reduced muscle strength in mice.

Elevated reactive oxygen species (ROS) production and ROS-dependent protein damage is a common observation in the pathogenesis of many muscle wasting disorders, including sarcopenia. However, the contribution of elevated ROS levels to -a breakdown in neuromuscular communication and muscle atrophy remains unknown. In this study, we examined a copper zinc superoxide dismutase [CuZnSOD (Sod1)] knockout mouse (Sod1-/-), a mouse model of elevated oxidative stress that exhibits accelerated loss of muscle mass, which recapitulates many phenotypes of sarcopenia as early as 5 months of age.

Expression of multiple transgenes from a single construct using viral 2A peptides in Drosophila.

Expression of multiple reporter or effector transgenes in the same cell from a single construct is increasingly necessary in various experimental paradigms. The discovery of short, virus-derived peptide sequences that mediate a ribosome-skipping event enables generation of multiple separate peptide products from one mRNA. Here we describe methods and vectors to facilitate easy production of polycistronic-like sequences utilizing these 2A peptides tailored for expression in Drosophila both in vitro and in vivo.

The redistribution of Drosophila vesicular monoamine transporter mutants from synaptic vesicles to large dense-core vesicles impairs amine-dependent behaviors.

Monoamine neurotransmitters are stored in both synaptic vesicles (SVs), which are required for release at the synapse, and large dense-core vesicles (LDCVs), which mediate extrasynaptic release. The contributions of each type of vesicular release to specific behaviors are not known. To address this issue, we generated mutations in the C-terminal trafficking domain of the Drosophila vesicular monoamine transporter (DVMAT), which is required for the vesicular storage of monoamines in both SVs and LDCVs.

Neuron-specific expression of CuZnSOD prevents the loss of muscle mass and function that occurs in homozygous CuZnSOD-knockout mice.

Deletion of copper-zinc superoxide dismutase (CuZnSOD) in Sod1(-/-) mice leads to accelerated loss of muscle mass and force during aging, but the losses do not occur with muscle-specific deletion of CuZnSOD. To determine the role of motor neurons in the muscle decline, we generated transgenic Sod1(-/-) mice in which CuZnSOD was expressed under control of the synapsin 1 promoter (SynTgSod1(-/-) mice). SynTgSod1(-/-) mice expressed CuZnSOD in brain, spinal cord, and peripheral nerve, but not in other tissues.

Mitochondrial free Ca²⁺ levels and their effects on energy metabolism in Drosophila motor nerve terminals.

Mitochondrial Ca²⁺ uptake exerts dual effects on mitochondria. Ca²⁺ accumulation in the mitochondrial matrix dissipates membrane potential (ΔΨm), but Ca²⁺ binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca²⁺ ([Ca²⁺]m) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined.

Genetically encoded pH-indicators reveal activity-dependent cytosolic acidification of Drosophila motor nerve termini in vivo.

All biochemical processes, including those underlying synaptic function and plasticity, are pH sensitive. Cytosolic pH (pH(cyto)) shifts are known to accompany nerve activity in situ, but technological limitations have prevented characterization of such shifts in vivo. Genetically encoded pH-indicators (GEpHIs) allow for tissue-specific in vivo measurement of pH.

Forward-filling of dextran-conjugated indicators for calcium imaging at the Drosophila larval neuromuscular junction.

Calcium imaging is a technique in which Ca(2+)-binding molecules are loaded into live cells and as they bind Ca(2+) they "indicate" the concentration of free calcium through a change in either the intensity or the wavelength of light emitted (fluorescence or bioluminescence). There are several possible methods for loading synthetic Ca(2+) indicators into subcellular compartments, including topical application of membrane-permeant Ca(2+) indicators, forward-filling of dextran conjugates, and direct injection. Calcium imaging is a highly informative technique in neurobiology because Ca(2+) is involved in many neuronal signaling pathways and serves as the trigger for neurotransmitter release.

Topical application of indicators for calcium imaging at the Drosophila larval neuromuscular junction.

Calcium imaging is a technique in which Ca(2+)-binding molecules are loaded into live cells and as they bind Ca(2+) they "indicate" the concentration of free calcium through a change in either the intensity or the wavelength of light emitted (fluorescence or bioluminescence). There are several possible methods for loading synthetic Ca(2+) indicators into subcellular compartments, including topical application of membrane-permeant Ca(2+) indicators, forward-filling of dextran conjugates, and direct injection. Calcium imaging is a highly informative technique in neurobiology because Ca(2+) is involved in many neuronal signaling pathways and serves as the trigger for neurotransmitter release.

Calcium imaging at the Drosophila larval neuromuscular junction.

Calcium imaging uses optical imaging techniques to measure the concentration of free calcium [Ca(2+)] in live cells. It is a highly informative technique in neurobiology because Ca(2+) is involved in many neuronal signaling pathways and serves as the trigger for neurotransmitter release. The technique relies on loading Ca(2+) indicators into cells, measuring the quantity and/or wavelength of the photons emitted by the Ca(2+) indicator, and interpreting these data in terms of [Ca(2+)].

Imaging and analysis of nonratiometric calcium indicators at the Drosophila larval neuromuscular junction.

Ca(2+) indicators can be loaded into a Drosophila larval neuromuscular junction (NMJ) preparation using several methods, including topical application of membrane-permeant Ca(2+) indicators, forward-filling of dextran conjugates, and direct injection. This article describes how such an NMJ preparation loaded with Ca(2+) indicator is set up for imaging of the muscle fiber during stimulation of its innervating nerve cell. A simple protocol is provided for collecting and analyzing a set of imaging data, together with the sequence of calculations involved in image analysis.