Biology subjects

Muraro, N. I.

Publications and source records attributed to Muraro, N. I..

4 recordsLinked to original sources

bioRxiv2026

Huntingtin is a cell-autonomous regulator of neuropeptide trafficking and clock output

Huntingtons disease is a severe neurodegenerative condition arising from an abnormal CAG repeat expansion in the HTT gene, which leads to the production of a mutant Huntingtin protein carrying an extended polyglutamine stretch. Although the field has largely centred on the toxic effects gained by this mutant protein, growing evidence points to the loss of normal wild-type Huntingtin function as an additional contributor to disease progression. Despite this, the cell-intrinsic roles of wild-type Huntingtin in neuronal biology remain poorly defined, in part because disentangling its specific contributions from broader network-level effects has proven technically challenging. To address this knowledge gap, we took advantage of the Drosophila huntingtin homolog (htt) and selectively manipulated its expression in the small lateral ventral neurons (sLNvs), a discrete cluster of just eight circadian pacemaker neurons that govern behavioral rhythmicity and sleep. Through targeted genetic knock-down, we show that reducing htt levels in sLNvs weakens the robustness of free-running circadian rhythms and substantially increases sleep in female flies. These behavioral changes are not rooted in developmental abnormalities, as restricting htt knock-down to adult flies reproduces the sleep phenotype across both beam-crossing and video-based locomotion assays. At the cellular level, htt loss disrupts dense core vesicle trafficking along sLNv axons, altering the fraction of motile vesicles and their velocity, and abolishing the time-of-day-dependent fluctuations in vesicle dynamics observed in these neurons. Complementary electrophysiological recordings using whole-cell patch-clamp further reveal that htt knock-down lowers action potential firing rates without perturbing resting membrane potential. Together, these results identify huntingtin as a cell-autonomous regulator of neuropeptide trafficking, neuronal excitability and circadian output. Beyond advancing our understanding of wild-type huntingtin physiology, this work carries direct relevance for HD therapeutic strategies, particularly those involving huntingtin-lowering approaches, by highlighting functions that may be unintentionally compromised.

neuroscience↗

bioRxiv2024

Neuronal synchronization in Drosophila

Rhythms are intrinsic to biological processes across temporal and spatial scales. In the brain, the synchronized oscillatory activity of neurons creates collective rhythms that are essential for complex functions. While this is a recognized phenomenon in the mammalian brain, information about insect neuronal synchrony and its underlying mechanisms is scarce. In the fly brain, neuronal oscillations were reported in individual lateral ventral neurons (LNvs), which play a key role in circadian and sleep behaviors. However, it is still unclear whether and how these participate in a collective rhythm. In this work, we perform thorough whole-cell patch clamp recordings of LNvs, and demonstrate consistent membrane potential oscillations. We show that oscillations degrade over time, and disappear upon exposure to an acetylcholine receptor blocker. Together with a flat phase response curve, these results suggest that oscillations are exogenously produced. Prompted by these results, we propose a generic forced oscillator theory that can account for the experimental phase response. The theory further predicts that neurons with similar properties should oscillate in synchrony with zero lags, while neurons with different properties may show coherent oscillations with non-zero lags. We confirm this prediction through simultaneous patch clamp recordings of neuronal pairs, revealing that large LNvs are consistently advanced relative to small LNvs. Additionally, we find that other neurons in the accessory medulla also exhibit coherent membrane potential oscillations, with diverse lags. Our findings suggest the intriguing possibility that brain waves may arise from collective neuronal activity within this region of the fly brain.

neuroscience↗

bioRxiv2020

Dopamine signaling in wake promoting clock neurons is not required for the normal regulation of sleep in Drosophila

Dopamine is a wakefulness promoting neuromodulator in mammals and fruit flies. In D. melanogaster, the network of clock neurons that drives sleep/activity cycles comprises both wake and sleep promoting cell types, indicating that the sleep-wake circuitry is intimately linked to the circadian clock. The large and small ventrolateral neurons (l-LNvs and s-LNvs) have been identified as wake-promoting neurons within the clock neuron network. The l-LNvs are innervated by dopaminergic neurons, and earlier work proposed that dopamine signaling raises cAMP levels in the l-LNvs and thus induces excitatory electrical activity (action potential firing), which results in wakefulness and inhibits sleep. Here, we test this hypothesis by combining cAMP imaging and patch-clamp recordings in isolated brains. We find that dopamine application indeed increases cAMP levels and depolarizes the l-LNvs, but surprisingly, it does not result in increased firing rates. Down-regulation of the excitatory dopamine receptor, Dop1R1 in the l-and s-LNvs, but not of Dop1R2, abolished the depolarization of l-LNvs in response to dopamine. This indicates that dopamine signals via Dop1R1 to the l-LNvs. Down-regulation of Dop1R1 or Dop1R2 receptors in the l- and s-LNvs does not affect sleep. Unexpectedly, we find a moderate decrease of daytime sleep with down-regulation of Dop1R1 and of nighttime sleep with down-regulation of Dop1R2. Since the l-LNvs do not utilize Dop1R2 receptors and the s-LNvs respond also to dopamine, we conclude that the s-LNvs are responsible for the observed decrease in nighttime sleep. In summary, dopamine signaling in the wake-promoting LNvs is not required for daytime arousal, but likely promotes nighttime sleep via the s-LNvs. Significance statementIn insect and mammalian brains, sleep promoting networks are intimately linked to the circadian clock, and the mechanisms underlying sleep and circadian timekeeping are evolutionarily ancient and highly conserved. Here we show that dopamine, one important sleep modulator in flies and mammals, plays surprisingly complex roles in the regulation of sleep by clock containing neurons. Dopamine inhibits neurons in a central brain sleep center to promote sleep and excites wake-promoting circadian clock neurons. It is therefore predicted to promote wakefulness through both of these networks. Nevertheless, our results reveal that dopamine acting on wake promoting clock neurons promotes sleep, revealing a previously unappreciated complexity in the dopaminergic control of sleep.

neuroscience↗

bioRxiv2020

High frequency neuronal bursting is essential for circadian and sleep behaviors in Drosophila

Circadian rhythms have been extensively studied in Drosophila, however, still little is known about how the electrical properties of clock neurons are specified. We have performed a behavioral genetic screen through the downregulation of candidate ion channels in the lateral ventral neurons (LNvs) and show that the hyperpolarization-activated cation current Ih is important for the behaviors that the LNvs command: temporal organization of locomotor activity and sleep. Using whole-cell patch clamp electrophysiology we demonstrate that small LNvs are bursting neurons, and that Ih is necessary to achieve the high frequency bursting firing pattern characteristic of both types of LNvs. Since firing in bursts has been associated to neuropeptide release, we hypothesized that Ih would be important for LNvs communication. Indeed, herein we demonstrate that Ih is fundamental for the recruitment of PDF filled dense core vesicles to the terminals at the dorsal protocerebrum and for their timed release, and hence for the temporal coordination of circadian behaviors.

neuroscience↗