Biology subjects

Macleod, G. T.

Publications and source records attributed to Macleod, G. T..

8 recordsLinked to original sources

bioRxiv2025

An optogenetic assay of Drosophila larval motor neuron performance in vivo

BackgroundOver fifty million people worldwide currently live with neurodegenerative diseases, many of which are the result of pathogenic gene variants. Genetically malleable model organisms provide an avenue for research into the genetic bases of these diseases, and the large motor neurons of fruit fly larvae provide a test bed for investigating neuronal mechanisms impacted by pathogenic gene variants. However, it is challenging to collect information from these neurons under physiological conditions as they terminate on muscle fibers that are perpetually contracting - driven by motor neuron burst-firing. New MethodAs a test of in vivo neuronal performance, we expressed light-activated opsins in motor-neurons of unrestrained intact Drosophila larva and used light pulses to drive cyclical body-wall contractions that were captured on camera and analyzed offline. ResultsWe describe the assembly of an apparatus to systematically activate motor-neurons in Drosophila larvae and an image acquisition system to capture the resulting body-wall contractions. To test the utility of the assay we performed a motor-neuron specific knock-down of dMiro, an adaptor for mitochondrial transport into motor-neuron terminals. As predicted, contractions were poorly sustained in larvae with impaired axonal transport of mitochondria. Comparison with Existing MethodsThis in vivo assay allows for a test of sustained neuronal performance while sidestepping the shortcomings of electrophysiological assays of neurotransmission in situ where hemolymph-like solutions may not recapitulate hemolymph properties, axons are severed and where recordings are mechanically disrupted at endogenous firing rates. Secondly, unlike adult climbing assays and larval locomotion assays, performance is assayed independently of the organisms motivation to perform or ability to detect stimuli. ConclusionsWe demonstrated that Drosophila 3rd instar larvae cannot sustain body-wall contractions if mitochondria are not delivered to motor nerve terminals - validating a motor neuron performance assay in a model organism suited for molecular genetic analysis.

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bioRxiv2025

Optimal Neuromuscular Performance Requires Motor Neuron Phosphagen Kinases

Phosphagen systems are crucial for muscle bioenergetics - rapidly regenerating ATP to support the high metabolic demands of intense musculoskeletal activity. However, their roles in motor neurons that drive muscle contraction have received little attention. Here, we knocked down expression of the primary phosphagen kinase [Arginine Kinase 1; ArgK1] in Drosophila larval motor neurons and assessed the impact on presynaptic energy metabolism and neurotransmission in situ. Fluorescent metabolic probes showed a deficit in presynaptic energy metabolism and some glycolytic compensation. Glycolytic compensation was revealed through a faster elevation in lactate at high firing frequencies, and the accumulation of pyruvate subsequent to firing. Our performance assays included two tests of endurance: enforced cycles of presynaptic calcium pumping, and, separately, enforced body-wall contractions for extended periods. Neither test of endurance revealed deficits when ArgK1 was knocked down. The only performance deficits were detected at firing frequencies that approached, or exceeded, twice the firing frequencies recorded during fictive locomotion, where both electrophysiology and SynaptopHluorin imaging showed an inability to sustain neurotransmitter release. Our computational modeling of presynaptic bioenergetics indicates that the phosphagen systems contribution to motor neuron performance is likely through the removal of ADP in microdomains close to sites of ATP hydrolysis, rather than the provision of a deeper reservoir of ATP. Taken together, these data demonstrate that, as in muscle fibers, motor neurons rely on phosphagen systems during activity that imposes intense energetic demands.

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bioRxiv2024

Bichromatic exon-reporters reveal voltage-gated Ca2+-channel splice-isoform diversity across Drosophila neurons in vivo

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 in situ. Here, in Drosophila, we developed bichromatic fluorescent reporters to investigate alternate splicing of cacophony - a gene that codes the pore-forming 1-subunit of the primary neuronal voltage-gated Ca2+ channel (VGCC). These reporters reveal a neuron-specific pattern of exon biases, including stereotypical differences between neurons of the same neurotransmitter type and ostensibly the same function. Information about exon splicing biases of individual neurons in vivo provides clues to the role of VGCC motifs and the role of those neurons in the context of local circuits. The application of this technology to a large gene such as cacophony provides a precedence for effective exon-reporter design for other Drosophila genes.

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bioRxiv2023

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. It is assumed that neurons possess this capacity, but ultrastructural data over substantial portions of a neurons extent that would allow for tests of such hypotheses are scarce. Here, we mined the Caenorhabditis elegans electron micrographs of John White and Sydney Brenner and found systematic differences in average mitochondrial length (ranging from 1.3 to 2.4 m), volume density (3.7% to 6.5%) and diameter (0.18 to 0.24 m) between neurons of different neurotransmitter type and function, but found limited differences in mitochondrial morphometrics between axons and dendrites of the same neurons. Analyses of distance intervals found mitochondria to be distributed randomly with respect to presynaptic specializations, and an indication that mitochondria were displaced from postsynaptic specializations. Presynaptic specializations were primarily localized to varicosities, but mitochondria were no more likely to be found in synaptic varicosities than non-synaptic varicosities. Consistently, mitochondrial volume density was no greater in varicosities with synapses. Therefore, beyond the capacity to disperse mitochondria throughout their length, at least in C. elegans, fine caliber neurons manifest limited sub-cellular control of mitochondrial size and distribution. SIGNIFICANCEBrain function is unequivocally reliant on mitochondrial function for its energy needs, and the mechanisms that cells use to control these organelles is an active field of enquiry. WormImage, a decades old electron microscopy database in the public domain, contains information about the ultrastructural disposition of mitochondria within the nervous system of C elegans over previously unexamined extents. In a largely remote format, a team of students mined this database over the course of the pandemic. They found differences in mitochondrial size and density between neurons, but limited differences between different compartments of the same neurons. Also, while neurons are clearly able to disperse mitochondria throughout their extent, they found little evidence that they "install" mitochondria at synaptic varicosities.

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bioRxiv2022

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 vs phasic neurons are also functionally differentiated, yet the bases of their distinctive properties remain enigmatic. A major challenge towards illuminating the synaptic differences between tonic and phasic neurons is the difficulty in isolating their physiological properties. At the Drosophila neuromuscular junction (NMJ), most muscle fibers are co-innervated by two motor neurons, the tonic "MN-Ib" and phasic "MN-Is". Here, we employed selective expression of a newly developed botulinum neurotoxin (BoNT-C) transgene to silence tonic or phasic motor neurons. This approach revealed major differences in their neurotransmitter release properties, including probability, short-term plasticity, and vesicle pools. Furthermore, Ca2+ imaging demonstrated ~two-fold greater Ca2+ influx at phasic neuron release sites relative to tonic, along with enhanced synaptic vesicle coupling. Finally, confocal and super resolution imaging revealed that phasic neuron release sites are organized in a more compact arrangement, with enhanced stoichiometry of voltage-gated Ca2+ channels relative to other active zone scaffolds. These data suggest that distinctions in active zone nano-architecture and Ca2+ influx collaborate to differentially tune glutamate release at synapses of tonic vs phasic neuronal subtypes.

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bioRxiv2022

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

Neural function relies on cellular energy supplies meeting the episodic demands of synaptic activity, but little is known about the extent to which power demands (energy demands per unit time) fluctuate, or the mechanisms that match supply with demand. Here, in individually-identified glutamatergic motor neuron terminals of Drosophila larvae, we leveraged prior macroscopic estimates of energy demand to generate profiles of power demand from one action potential to the next. These profiles show that signaling demands can exceed non-signaling demands 10-fold within milliseconds, and terminals with the greatest fluctuation (volatility) in power demand have the greatest mitochondrial volume and packing density. We elaborated on this quantitative approach to simulate adenosine triphosphate (ATP) levels during activity and drove ATP production as a function of the reciprocal of the energy state, but this canonical feedback mechanism appeared to be unable to prevent ATP depletion during locomotion. Muscle cells possess a phosphagen system to buffer ATP levels but phosphagen systems have not been described for motor nerve terminals. We examined these terminals for evidence of a phosphagen system and found the mitochondria to be heavily decorated with an arginine kinase, the key element of invertebrate phosphagen systems. Similarly, an examination of mouse cholinergic motor nerve terminals found mitochondrial creatine kinases, the vertebrate analogues of arginine kinases. Knock down of arginine kinase in Drosophila resulted in rapid depletion of presynaptic ATP during activity, indicating that, in motor nerve terminals, as in muscle, phosphagen systems play a critical role in matching power supply with demand. SIGNIFICANCEFailure of metabolic processes to supply neurons with energy at an adequate rate can lead to synaptic dysfunction and cell death under pathological conditions. Using a quantitative approach at fruit fly motor nerve terminals we generated the first temporal profiles of presynaptic power demand during locomotor activity. This approach revealed challenges for the known mechanisms that match cellular power supply to demand. However, we discovered that motor nerve terminals in fruit flies and mice alike are supported by phosphagen systems, more commonly seen in muscles where they store energy and buffer mismatch between power supply and demand. This study highlights an understudied aspect of neuronal bioenergetics which may represent a bulwark against the progression of some neuropathologies.

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bioRxiv2021

Computational modeling predicts 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 (NMJ) as glutamate, adenosine triphosphate (ATP) and protons (H+) are released into the cleft. The model incorporates bicarbonate and phosphate buffering systems as well as plasma membrane calcium-ATPase (PMCA) activity and predicts substantial cleft acidification but only for fractions of a millisecond following neurotransmitter release. Thereafter, the cleft rapidly alkalinizes and remains alkaline for over 100 milliseconds, as the PMCA removes H+ from the cleft in exchange for calcium ions (Ca2+) from adjacent pre- and post-synaptic compartments; thus recapitulating the empirical data. The extent of synaptic vesicle loading and time course of exocytosis has little influence on the magnitude of acidification. Phosphate, but not bicarbonate buffering is effective at ameliorating the magnitude and time course of the acid spike, while both buffering systems are effective at ameliorating cleft alkalinization. The small volume of the cleft levies a powerful influence on the magnitude of alkalinization and its time course. Structural features that open the cleft to adjacent spaces appear to be essential for alleviating the extent of pH disturbances accompanying neurotransmission. SIGNIFICANCE STATEMENTAcid-base imbalances have surprisingly potent neurological effects highlighting the acute pH sensitivity of many neural mechanisms. Acid-Sensing Ion-Channels (ASICs), which open in response to acid shifts in extracellular pH, are an example of such a mechanism. However, while ASICs open during neurotransmission at conventional glutamatergic synapses, pH-sensitive electrodes and fluorophores show no signs of acidification at these synapses, only alkalinization. To resolve this paradox, we built a computational model which allows a glimpse beyond the experimental limitations of pH-sensitive electrodes and fluorophores. Our model reveals a highly dynamic pH landscape within the synaptic cleft, harboring deep but exceedingly rapid acid transients that give way to a prolonged period of alkalinization, thus reconciling ASIC activation with direct measurements of extracellular pH.

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bioRxiv2021

Presynaptic Mitochondrial Volume and Density Scale with Presynaptic Power Demand

Stable neural function requires an energy supply that can meet the intense episodic power demands of neuronal activity. The bioenergetic machinery of glycolysis and oxidative phosphorylation is highly responsive to such demands, but it must occupy a minimum volume if it is to accommodate these demands. We examined the trade-off between presynaptic power demands and the volume available to the bioenergetic machinery. We quantified the energy demands of six Drosophila motor nerve terminals through direct measurements of neurotransmitter release and Ca2+ entry, and via theoretical estimates of Na+ entry and power demands at rest. Electron microscopy revealed that terminals with the highest power demands contained the greatest volume of mitochondria, indicating that mitochondria are allocated according to presynaptic power demands. In addition, terminals with the greatest power demand-to-volume ratio ([~]66 nmol{middle dot}min-1{middle dot}L-1) harbor the largest mitochondria packed at the greatest density. If we assume sequential and complete oxidation of glucose by glycolysis and oxidative phosphorylation, then these mitochondria are required to produce ATP at a rate of 52 nmol{middle dot}min-1{middle dot}L-1 at rest, rising to 963 during activity. Glycolysis would contribute ATP at 0.24 nmol{middle dot}min-1{middle dot}L-1 of cytosol at rest, rising to 4.36. These data provide a quantitative framework for presynaptic bioenergetics in situ, and reveal that, beyond an immediate capacity to accelerate ATP output from glycolysis and oxidative phosphorylation, over longer time periods presynaptic terminals optimize mitochondrial volume and density to meet power demand. Significance StatementThe remarkable energy demands of the brain are supported by the complete oxidation of its fuel but debate continues regarding a division of labor between glycolysis and oxidative phosphorylation across different cell types. Here we leverage the neuromuscular synapse, a model for studying neurophysiology, to elucidate fundamental aspects of neuronal energy metabolism that ultimately constrain rates of neural processing. We quantified energy production rates required to sustain activity at individual nerve terminals and compared these data with the volume capable of oxidative phosphorylation (mitochondria) and glycolysis (cytosol). We find strong support for oxidative phosphorylation playing a primary role in presynaptic terminals and provide the first in vivo estimates of energy production rates per unit volume of presynaptic mitochondria and cytosol.

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