Biology subjects

Zarin, A. A.

Publications and source records attributed to Zarin, A. A..

8 recordsLinked to original sources

bioRxiv2026

Three-dimensional object-based quantification of nicotinic acetylcholine receptor subunit colocalization using Imaris

Neural circuit function depends not only on synaptic connectivity but also on the molecular composition and spatial organization of neurotransmitter receptors. Determining whether receptor subunits occupy shared or distinct domains requires spatial measurements capable of resolving sparse receptor puncta across neural tissue and, when needed, restricting analysis to defined cellular compartments. Conventional intensity-based colocalization metrics can be difficult to interpret for sparse, punctate signals and do not inherently provide object-level classification or cell-specific analysis. Here, we describe a three-dimensional object-based colocalization workflow for endogenously tagged nicotinic acetylcholine receptor (nAChR) subunits imaged by Airyscan super-resolution confocal microscopy and analyzed in Imaris. Individual receptor puncta are segmented as 3D surface objects and classified as colocalized or non-colocalized using a control-defined spatial overlap criterion. The workflow supports analysis within a defined region of the ventral nerve cord neuropil or within genetically labeled motor-neuron dendritic compartments. For dendrite-restricted analysis, receptor puncta are first selected by their overlap with the dendritic surface before receptor-receptor overlap is quantified. The overlap criterion is empirically calibrated using positive and negative biological controls processed under the same imaging and analysis conditions, providing a reproducible alternative to arbitrary threshold selection. This approach enables receptor organization to be quantified within the neuropil and genetically labeled motor-neuron dendritic compartments and can be adapted to other epitope-tagged receptors, ion channels, and synaptic proteins.

neuroscience↗

bioRxiv2025

A Drosophila Tonic Motor Neuron Reinnervates Ectopic Muscles Fully Deprived of Native Tonic and Phasic Inputs

Motor neurons (MNs) form precise neuromuscular junctions (NMJs) during development, but the extent to which individual MNs can reinnervate fully denervated muscles in vivo remains poorly understood. In the Drosophila larva, each muscle is co- innervated by a tonic and a phasic glutamatergic MN. Here, we show that the tonic MN1 undergoes robust heterosynaptic sprouting and forms ectopic NMJs when neighboring muscles are deprived of both their native tonic and phasic inputs. This structural plasticity is not induced by silencing, but instead requires the physical ablation of adjacent MNs. Live imaging of the same MN1 axons in individual animals reveals that sprouting initiates early and expands progressively across larval stages. In contrast, phasic MNs show minimal remodeling, indicating that tonic MNs possess a greater intrinsic capacity for neuroplasticity. Notably, as MN1 establishes new synapses on targets it does not normally innervate, it redistributes pre-synaptic territory across both native and ectopic muscles. These findings identify a subtype-specific, injury-induced rewiring program in an intact motor circuit.

neuroscience↗

bioRxiv2025

Motor Neurons Decode Cholinergic Inputs via Spatially Distinct nAChR Subunits to Drive Locomotion in Drosophila larvae

Neural circuits consist of neurons that differ not only in their neurotransmitter identities but also in the types and subcellular localization of neurotransmitter receptors (NRs) they express. This receptor diversity enables distinct responses to the same neurotransmitter, highlighting the need to understand NR distribution and function to fully interpret circuit logic. Here, we focus on nicotinic acetylcholine receptors (nAChRs), the primary mediators of fast excitatory transmission in the Drosophila central nervous system (CNS). Functional nAChRs are pentamers assembled from a pool of 10 subunits (1-7, {beta}1-{beta}3), yet their in vivo expression and function remain poorly defined. We used T2A-Gal4 lines and endogenous protein tagging to examine nAChR expression in larval motor neurons (MNs) and identified eight subunits (1-3, 5-7, {beta}1, {beta}2) expressed in these cells. MN-specific knockdown of individual subunits caused distinct locomotor defects, indicating their functional importance. Co-localization analysis revealed some subunit pairs are frequently co-expressed at the same synapses, while others localize to distinct subcellular domains. Supporting this, double knockdown of co-localized subunits did not worsen locomotor phenotypes compared to single knockdowns, whereas knockdown of non-co-localized subunit pairs produced additive defects. These results suggest that different nAChR subtypes are strategically positioned in discrete synaptic domains within single MNs, where they serve non-redundant roles. Our findings provide new insight into the spatial organization and functional diversity of nAChRs in motor circuits that drive locomotion. Significance StatementMotor circuits rely on precise neurotransmitter signaling, yet the diversity and subcellular organization of neurotransmitter receptors remain poorly understood. Using Drosophila larvae, we show that motor neurons express multiple nicotinic acetylcholine receptor (nAChR) subunits, which localize to distinct synaptic domains and play non-redundant roles in locomotion. These findings reveal a previously underappreciated level of receptor compartmentalization within single neurons and demonstrate that spatially organized nAChRs are essential for coordinated movement. By integrating genetic, imaging, and behavioral approaches, our work provides a new framework for understanding how receptor diversity shapes motor output and highlights the importance of mapping receptor localization to decode circuit function in both health and disease.

neuroscience↗

bioRxiv2024

Heterogeneous responses to embryonic critical period perturbations among different components of the Drosophila larval locomotor circuit

As developing neural circuits become functional, they undergo a phase of heightened plasticity that facilitates network tuning in response to intrinsic and/or extrinsic stimuli. These developmental windows are termed critical periods (CPs), because perturbations during, but not outside, the CP can lead to lasting changes, such as the formation of sub-optimal or unstable networks. How separate, but connected elements, within a network might respond differently to a CP perturbation is not well understood. To study this, we used the locomotor network of the Drosophila larva as a model and heat stress as a CP stimulus that has ecological relevance. When embryos experienced heat stress the subsequent development of their locomotor network is changed, creating larvae with reduced crawling speed and decreased network stability. Developing body wall muscles and central neurons are sensitive to heat stress perturbations during distinct, consecutive phases of embryogenesis. Within the CNS, transient embryonic CP perturbation leads to increased synaptic drive from premotor interneurons to motoneurons, which in turn adopt reduced excitability. In contrast, the peripheral neuromuscular junction maintains normal synaptic transmission, despite significant structural changes. Thus, connected elements respond differentially to a CP perturbation, suggesting a sequence, or hierarchy, of network adjustment during the CP.

neuroscience↗

bioRxiv2023

Neuromuscular Basis of Drosophila larval escape behavior

When threatened by dangerous or harmful stimuli, animals engage in diverse forms of rapid escape behaviors. In Drosophila larvae, one type of escape response involves C-shaped bending and lateral rolling followed by rapid forward crawling. The sensory circuitry that promotes larval escape has been extensively characterized; however, the motor programs underlying rolling are unknown. Here, we characterize the neuromuscular basis of rolling escape behavior. We used high-speed, volumetric, Swept Confocally-Aligned Planar Excitation (SCAPE) microscopy to image muscle activity during larval rolling. Unlike sequential peristaltic muscle contractions that progress from segment to segment during forward and backward crawling, the muscle activity progresses circumferentially during bending and rolling escape behavior. We propose that progression of muscular contraction around the larvas circumference results in a transient misalignment between weight and the ground support forces, which generates a torque that induces stabilizing body rotation. Therefore, successive cycles of slight misalignment followed by reactive aligning rotation lead to continuous rolling motion. Supporting our biomechanical model, we found that disrupting the activity of muscle groups undergoing circumferential contraction progression lead to rolling defects. We use EM connectome data to identify premotor to motor connectivity patterns that could drive rolling behavior, and perform neural silencing approaches to demonstrate the crucial role of a group of glutamatergic premotor neurons in rolling. Our data reveal body-wide muscle activity patterns and putative premotor circuit organization for execution of the rolling escape response. Significance StatementTo escape from dangerous stimuli, animals execute escape behaviors that are fundamentally different from normal locomotion. The rolling escape behavior of Drosophila larvae consists of C-shaped bending and rolling. However, the muscle contraction patterns that lead to rolling are poorly understood. We find that following the initial body bending, muscles contract in a circumferential wave around the larva as they enter the bend, maintaining unidirectional rolling that resembles cylinder rolling on a surface. We study the structure of motor circuits for rolling, inhibit different motor neurons to determine which muscles are essential for rolling, and propose circuit and biomechanical models for roll generation. Our findings provide insights into how motor circuits produce diverse motor behaviors.

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bioRxiv2022

A premotor microcircuit to generate behavior-specific muscle activation patterns in Drosophila larvae

Animals can use a common set of muscles and motor neurons (MNs) to generate diverse locomotor behaviors, but how this is accomplished remains poorly understood. Previously, we characterized the muscle activity patterns for Drosophila larval forward and backward locomotion and found that ventral oblique (VO) muscles become active earlier in backward than in forward locomotion (Zarin et al. 2019). Here, we describe how premotor circuits generate differential activation timing of VO muscles. We identify inhibitory (A06c) and excitatory (A27h) premotor neurons (PMNs) with the greatest number of synapses with VO MNs. Strikingly, A06c is a bi-modal PMN that fires before and after VO MNs in forward locomotion but fires only after MNs in backward locomotion. Further, A27h is a forward-dedicated PMN active only in forward locomotion. These two PMNs interconnect with another forward-dedicated excitatory PMN (A18b3), to create feedforward inhibitory microcircuits that define the activity window for VO MNs/muscles, producing precise VO muscle patterns underlying forward locomotion. Silencing A06c, A27h, or A18b3 PMN results in premature VO muscle activation in forward locomotion, resembling early VO activation in backward locomotion. Our results identify PMN micro-circuits that produce unique MN/muscle activity patterns to create behavior-specific motor output.

neuroscience↗

bioRxiv2020

Electrophysiological validation of premotor interneurons monosynaptically connected to the aCC motoneuron in the Drosophila larval CNS.

Mapping the wired connectivity of a nervous system is a prerequisite for full understanding of function. In this respect, such endeavours can be likened to genome sequencing projects. These projects similarly produce impressive amounts of data which, whilst a technical tour-de-force, remain under-utilised without validation. Validation of neuron synaptic connectivity requires electrophysiology which has the necessary temporal and spatial resolution to map synaptic connectivity. However, this technique is not common and requires extensive equipment and training to master, particularly when applied to the small CNS of the Drosophila larva. Thus, validation of connectivity in this CNS has been more reliant on behavioural analyses and, in particular, activity imaging using the calcium-sensor GCaMP. Whilst both techniques are powerful, they each have significant limitations for this purpose. Here we use electrophysiology to validate an array of driver lines reported to label specific premotor interneurons that the Drosophila connectome project suggests are monosynaptically connected to an identified motoneuron termed the anterior corner cell (aCC). Our results validate this proposition for four selected lines. Thus, in addition to validating the connectome with respect to these four premotor interneurons, our study highlights the need to functionally validate driver lines prior to use.

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bioRxiv2018

A command-like descending neuron that coordinately activates backward and inhibits forward locomotion

Command-like descending neurons can induce many behaviors, such as backward locomotion, escape, feeding, courtship, egg-laying, or grooming. In most animals it remains unknown how neural circuits switch between these antagonistic behaviors: via top-down activation/inhibition of antagonistic circuits or via reciprocal inhibition between antagonistic circuits. Here we use genetic screens, intersectional genetics, circuit reconstruction by electron microscopy, and functional optogenetics to identify a bilateral pair of larval \"mooncrawler descending neurons\" (MDNs) with command-like ability to coordinately induce backward locomotion and block forward locomotion; the former by activating a backward-specific premotor neuron, and the latter by disynaptic inhibition of a forward-specific premotor neuron. In contrast, direct reciprocal inhibition between forward and backward circuits was not observed. Thus, MDNs coordinate a transition between antagonistic larval locomotor behaviors. Interestingly, larval MDNs persist into adulthood, where they can trigger backward walking. Thus, MDNs induce backward locomotion in both limbless and limbed animals.\n\nHighlightsO_LIMDN command-like descending neuron induces backward larval locomotion\nC_LIO_LIMDN neurons coordinately regulate antagonistic behaviors (forward/backward locomotion)\nC_LIO_LIMDN-motor circuit validated at structural (TEM) and functional (optogenetic) levels\nC_LIO_LIMDN neurons induce backward locomotion in both limbless larva and limbed adult\nC_LI

neuroscience↗