Biology subjects

Seidenthal, M.

Publications and source records attributed to Seidenthal, M..

4 recordsLinked to original sources

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

Potassium-selective channelrhodopsins can exert hyper- or depolarizing effects in excitable cells of Caenorhabditis elegans, depending on experimental condition

One of the most frequent applications of optogenetic tools is for depolarization and stimulation of excitable cells such as neurons and muscles. Equally important, but less frequently used, are inhibitory tools that suppress activity through cellular hyperpolarization. These tools often rely on chloride conductance. Yet, in vivo, re- and hyperpolarization is typically mediated by potassium. In recent years, light-gated ion channels with a high preference for potassium were identified (Kalium channelrhodopsins, KCRs), and their inhibitory potential described in different organisms. Here, we characterized HcKCR1 and WiChR, in cholinergic neurons and muscles of Caenorhabditis elegans. Hyperpolarization of these cell types both induces muscle relaxation and, consequently, an elongation of the animals. Thus, we analyzed body length before, during, and after illumination, to assess KCR effectiveness, and to benchmark stimulation parameters like light intensity and duration. For HcKCR1 in cholinergic neurons, continuous illumination at high light intensities (1-4.5 mW/mm2) evoked only a transient elongation, while stimulation at 0.1 mW/mm2 could maintain inhibition for the duration of the stimulus in some transgenic strains. For animals expressing WiChR in body wall muscle cells or cholinergic neurons, we again observed brief hyperpolarization during continuous illumination, however, still during the stimulus, this changed to body contraction, corresponding to depolarization. This effect was long lasting, and required dozens of seconds for reversion, but could be reduced by pulsed illumination and fully avoided by less efficient channel activation using green or orange light. Hence, KCRs can be applied to hyperpolarize C. elegans cells, but require optimized illumination parameters. Article summaryTo inhibit excitable cells, light-gated, potassium-selective channels (KCRs) can be used. This study explores whether stimulation of KCRs HcKCR1 and WiChR in cholinergic neurons and muscle cells of Caenorhabditis elegans can induce inhibition during illumination. While inhibition could be achieved, depending on light conditions, the authors unexpectedly also observed excitation. These effects may occur due to a combination of high conductivity of KCRs, and partial conductance of other cations. These findings highlight the need for specific experimental conditions in future studies utilizing these tools. The authors also present conditions that can partially or fully avoid the unwanted depolarizing effects.

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

pOpsicle: An all-optical reporter system for synaptic vesicle recycling combining pH-sensitive fluorescent proteins with optogenetic manipulation of neuronal activity

pH-sensitive fluorescent proteins are widely used to study synaptic vesicle (SV) fusion and recycling. When targeted to the lumen of SVs, fluorescence of these proteins is quenched by the acidic pH. Following SV fusion, they are exposed to extracellular neutral pH, resulting in a fluorescence increase. SV fusion, recycling and acidification can thus be tracked by tagging integral SV proteins with pH-sensitive proteins. Neurotransmission is generally stimulated by electrophysiology, which is not feasible in small, intact animals, thus limiting the approach to cell culture regimes. Previous in vivo approaches depended on distinct (sensory) stimuli, thus limiting the addressable neuron types. To overcome these limitations, we established an all-optical approach to stimulate and visualize SV fusion and recycling. We combined distinct pH-sensitive fluorescent proteins (inserted into the SV protein synaptogyrin) and light-gated channelrhodopsins (ChRs) for optical stimulation, overcoming optical crosstalk and thus enabling an all-optical approach. We generated two different variants of the pH-sensitive optogenetic reporter of vesicle recycling (pOpsicle) and tested them in cholinergic neurons of intact Caenorhabditis elegans nematodes. First, we combined the red fluorescent protein pHuji with the blue-light gated ChR2(H134R), and second, the green fluorescent pHluorin combined with the novel red-shifted ChR ChrimsonSA. In both cases, fluorescence increases were observed after optical stimulation. Increase and subsequent decline of fluorescence was affected by mutations of proteins involved in SV fusion and endocytosis. These results establish pOpsicle as a non-invasive, all-optical approach to investigate different steps of the SV cycle.

neuroscience↗