Biology subjects

Liewald, J. F.

Publications and source records attributed to Liewald, J. F..

8 recordsLinked to original sources

bioRxiv2026

The copine protein NRA-1 regulates activity-dependent cholinergic signalling and sensory integration in C. elegans

Membrane-associated proteins regulate the localisation and function of ligand-gated ion channels, yet how they shape synaptic efficacy and behaviour remains poorly understood. Here, we identify the copine family protein NRA-1 as an activity-dependent regulator of cholinergic signalling and sensory circuit function. Electrophysiological recordings at the neuromuscular junction of C. elegans revealed that loss of nra-1 does not alter responses to acute agonist application or the initial response to synaptic stimulation but selectively impairs sustained and repetitive cholinergic transmission during ongoing activity. Single-channel recordings further demonstrated that NRA-1 does not affect the unitary conductance of levamisole-sensitive acetylcholine receptors (L-AChRs) but instead regulates receptor gating by increasing channel closed times and reducing opening frequency, resulting in an overall decrease in receptor activity. Despite these synaptic defects, nra-1 mutants displayed normal baseline locomotion but exhibited impaired chemotaxis, abnormal food localisation and defective egg-laying behaviour. Together, our findings identify NRA-1 as an activity-dependent regulator of postsynaptic receptor function that sustains cholinergic signalling during repeated activity and links receptor dynamics to sensory behaviour. These results establish copine proteins as important modulators of synaptic efficacy and suggest that activity-dependent control of receptor function represents a conserved mechanism for tuning neural circuit performance.

neuroscience↗

bioRxiv2024

Loss of Flower/FLWR-1 induces an increase in neuronal excitability and causes defective recycling of synaptic vesicles

The Flower protein was suggested to couple the fusion of synaptic vesicles (SVs) to their recycling in different model organisms. It is supposed to trigger activity-dependent bulk endocytosis by conducting Ca2+ at endocytic sites. However, this mode of action is debated. Here, we investigated the role of the Caenorhabditis elegans homolog FLWR-1 in neurotransmission. Our results confirm that FLWR-1 facilitates the recycling of SVs at the neuromuscular junction (NMJ). Ultrastructural analysis of synaptic boutons after hyperstimulation revealed an accumulation of large endocytic structures in flwr-1 mutants. These findings do not support a role of FLWR-1 in the formation of bulk endosomes but rather a function in their breakdown. Unexpectedly, loss of FLWR-1 led to increased neuronal Ca2+ levels in axon terminals during stimulation, particularly in GABAergic motor neurons, causing excitation-inhibition imbalance. We found that this increased NMJ transmission might be caused by deregulation of MCA-3, the nematode orthologue of the plasma membrane Ca2+ ATPase (PMCA). In vivo molecular interactions indicated that FLWR-1 may be a positive regulator of the PMCA and might influence its recycling through modification of plasma membrane levels of PI(4,5)P2.

neuroscience↗

bioRxiv2024

Neuropeptidergic regulation of neuromuscular signaling in larval zebrafish alters swimming behavior and synaptic transmission

The regulation of synaptic transmission is crucial for plasticity, homeostasis and learning. Chemical synaptic transmission is thus modulated to accommodate different activity levels, which also enables homeostatic scaling in pre- and postsynaptic compartments. In nematodes, cAMP signaling enhances cholinergic neuron output, and these neurons use neuropeptide signaling to modulate synaptic vesicle content. To explore if this mechanism is conserved in vertebrates, we studied the involvement of neuropeptides in cholinergic transmission at the neuromuscular junction of larval zebrafish. Optogenetic stimulation by photoactivated adenylyl cyclase (bPAC) resulted in elevated locomotion as measured in behavioural assays. Furthermore, post-synaptic patch-clamp recordings revealed that in bPAC transgenics, the frequency of miniature excitatory postsynaptic currents (mEPSCs) was increased after photostimulation. These results suggested that cAMP-mediated activation of ZF motor neurons leads to increased fusion of SVs, consequently resulting in enhanced neuromuscular activity. We generated mutants lacking the neuropeptide processing enzyme carboxypeptidase E (cpe), and the most abundant neuropeptide precursor in motor neurons, tachykinin (tac1). Both mutants showed exaggerated locomotion after photostimulation. cpe mutants exhibit lower mEPSC frequency during photostimulation and less large-amplitude mEPSCs. In tac1 mutants mEPSC frequency was not affected but amplitudes were significantly smaller. Exaggerated locomotion in the mutants thus reflected upscaling of postsynaptic excitability. cpe and tac1 mutant muscles expressed more nicotinic acetylcholine receptors (nAChR) on their surface. Thus, neuropeptide signaling regulates synaptic transmitter output in zebrafish motor neurons, and muscle cells homeostatically regulate nAChR surface expression, compensating reduced presynaptic input. This mechanism may be widely conserved in the animal kingdom.

neuroscience↗

bioRxiv2022

All-optical closed-loop voltage clamp for precise control of muscles and neurons in live animals

Excitable cells can be stimulated or inhibited by optogenetics. Since optogenetic actuation regimes are often static, neurons and circuits can quickly adapt, allowing perturbation, but not true control. Hence, we established an optogenetic voltage-clamp (OVC). The voltage-indicator QuasAr2 provides information for fast, closed-loop optical feedback to the bidirectional optogenetic actuator BiPOLES. Voltage-dependent fluorescence is held within tight margins, thus clamping the cell to distinct potentials. We established the OVC in muscles and neurons of Caenorhabditis elegans, and transferred it to rat hippocampal neurons in slice culture. Fluorescence signals were calibrated to electrically measured potentials, and wavelengths to currents, enabling to determine optical I/V-relationships. The OVC reports on homeostatically altered cellular physiology in mutants and on Ca2+-channel properties, and can dynamically clamp spiking. Combining non-invasive imaging with control capabilities of electrophysiology, the OVC facilitates high-throughput, contact-less electrophysiology in individual cells and paves the way for true optogenetic control in behaving animals.

neuroscience↗

bioRxiv2021

RIM and RIM-binding protein localize synaptic Cav2 channels in a differential manner to regulate transmission in neuronal circuits

At chemical synapses, voltage-gated Ca2+-channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca2+ microdomains they elicit must be located precisely to primed SVs, to evoke rapid transmitter release. Localization is mediated by Rab3 interacting molecule (RIM) and RIM-binding proteins (RIM-BPs), which interact and bind to the C-terminus of the CaV2 VGCC -subunit. We studied this machinery at the mixed cholinergic/GABAergic neuromuscular junction (NMJ) of Caenorhabditis elegans. rimb-1 mutants had mild synaptic defects, through loosening the anchoring of UNC-2/CaV2 and delaying the onset of SV fusion. UNC-10/RIM deletion much more severely affected transmission. Even though postsynaptic depolarization was reduced, rimb-1 mutants had increased cholinergic (but reduced GABAergic) transmission, to compensate for the delayed release. This did not occur when the excitation-inhibition balance was altered by removing GABA transmission. RIMB-1 thus may differentially regulate transmission in mixed circuits. Untethering the UNC-2/CaV2 channel by removing its C-terminal PDZ ligand exacerbated the rimb-1 defects, and similar phenotypes resulted from acute degradation of the CaV2 {beta}-subunit CCB-1. Therefore, untethering of the CaV2 complex is as severe as its elimination, yet does not abolish transmission, likely due to compensation by CaV1. Thus, robustness and flexibility of synaptic transmission emerges from VGCC regulation.

neuroscience↗

bioRxiv2019

Synapsin is required for dense core vesicle capture and cAMP-dependent neuropeptide release

Release of neuropeptides from dense core vesicles (DCVs) is important for neuromodulation. By optogenetics, behavioral analysis, electrophysiology, and electron microscopy, we show that synapsin SNN-1 is required for cAMP-dependent neuropeptide release in Caenorhabditis elegans cholinergic motor neurons. In synapsin mutants, behaviors induced by the photoactivated adenylyl cyclase bPAC, which we previously showed to depend on acetylcholine and neuropeptides, are altered like in animals with reduced cAMP. While synapsin mutants have slight alterations in synaptic vesicle distribution, DCVs were affected much more: DCVs were ~30% reduced in synaptic terminals, and not released following bPAC stimulation. Imaging axonal DCV trafficking, also in genome-engineered mutants in the serine-9 protein kinase A phosphorylation site, showed that synapsin captures DCVs at synapses, making them available for release. In non-phosphorylatable SNN-1B(S9A) mutants, DCVs traffic less and accumulate, likely by enhanced tethering to the actin cytoskeleton. Our work establishes synapsin as a key mediator of neuropeptide release.

neuroscience↗

bioRxiv2019

Rhodopsin-based voltage imaging tools for use in excitable cells of Caenorhabditis elegans

Genetically encoded voltage indicators (GEVIs) based on microbial rhodopsins utilize the voltage-sensitive fluorescence of the all-trans retinal (ATR) cofactor, while in electrochromic (eFRET) sensors, donor fluorescence drops when the rhodopsin acts as depolarization-sensitive acceptor. We systematically assessed Arch(D95N), Archon, and QuasAr, as well as the eFRET sensors MacQ-mCitrine and QuasAr-mOrange, in C. elegans. ATR-bearing rhodopsins reported on voltage changes in body wall muscles (BWMs) and the pharynx, the feeding organ, where Arch(D95N) showed ca. 125 % {Delta}F/F increase per 100 mV. The ATR fluorescence is very dim, however, using the retinal analog dimethylaminoretinal (DMAR), it was boosted 250-fold. eFRET sensors provided sensitivities of 45 % to 78 % {Delta}F/F per 100 mV, induced by BWM action potentials (APs). All sensors reported differences in muscle depolarization induced by a voltage-gated Ca2+-channel mutant. Optogenetically evoked de-or hyperpolarization of motor neurons increased or eliminated AP activity and caused a rise or drop in BWM sensor fluorescence. Last, we could analyze voltage dynamics across the entire pharynx, showing uniform depolarization but compartmentalized repolarization of anterior and posterior parts. Our work establishes all-optical, non-invasive electrophysiology in intact C. elegans.

neuroscience↗

bioRxiv2018

Functionally asymmetric motor neurons coordinate locomotion of Caenorhabditis elegans

Invertebrate nervous systems are valuable models for fundamental principles of the control of behavior. Ventral nerve cord (VNC) motor neurons in Caenorhabditis elegans represent one of the best studied locomotor circuits, with known connectivity and functional information about most of the involved neuron classes. However, for one of those, the AS motor neurons (AS MNs), no physiological data is available. Combining specific expression and selective illumination, we precisely targeted AS MNs by optogenetics and addressed their role in the locomotion circuit. After photostimulation, AS MNs induce currents in post-synaptic body wall muscles (BWMs), exhibiting an initial asymmetry of excitatory output. This may facilitate complex regulatory motifs for adjusting direction during navigation. By behavioral and photo-inhibition experiments, we show that AS MNs contribute to propagation of the antero-posterior body wave during locomotion. By Ca2+-imaging in AS MNs and in their synaptic partners, we also reveal that AS MNs play a role in mediating forward and backward locomotion by integrating activity of premotor interneurons (PINs), as well as in coordination of the dorso-ventral body wave. AS MNs do not exhibit pacemaker properties, but potentially gate VNC central pattern generators (CPGs), as indicated by ceasing of locomotion when AS MNs are hyperpolarized. AS MNs provide positive feedback to the PIN AVA via gap junctions, a feature found also in other locomotion circuits. In sum, AS MNs have essential roles in coordinating locomotion, combining several functions, and emphasizing the compressed nature of the C. elegans nervous system in comparison to higher animals.\n\nHighlightsA class of motor neurons with unidentified function - AS cholinergic motor neurons - was characterized in C. elegans.\n\nAS neurons show asymmetry in both input and output and are specialized in coordination of dorso-ventral undulation bends.\n\nAS neurons mediate antero-posterior propagation of the undulatory body wave during locomotion.\n\nAS neurons integrate signals for forward and reverse locomotion from premotor interneurons and may gate ventral nerve cord central pattern generators (CPGs) via gap junctions.

neuroscience↗