Alexander Gottschalk

Gap junctions (GJs), formed by connexins in vertebrates and innexins in invertebrates, enable direct ion flow between adjacent cells. GJ coupling is typically analyzed by electrophysiological methods, which, however, are invasive and cannot be conducted in intact animals. Here, we used all-optical methods to non-invasively investigate electrical coupling in body wall muscle (BWM) cells of intact Caenorhabditis elegans. We analyzed the roles of specific innexins (unc-9, inx-11, inx-16) in BWMs, using the genetically encoded fluorescent voltage indicator QuasAr2 to assess spontaneous muscle activity. unc-9 mutants showed strongly reduced coupling and asynchronous activity, and this was accompanied by severe locomotion defects. Overexpression of the murine connexin Cx36 increased synchronization but disrupted locomotion. inx-16 mutants exhibited increased excitability of individual muscle cells, likely due to reduced leak currents, while inx-11 mutants displayed only moderate defects. Patch-clamp recordings confirmed altered action potentials and increased input resistance in inx-16 mutants. Additionally, we established a cell-specific optogenetic voltage clamp (cOVC) method, directly revealing changes in junctional conductance in live, non-dissected animals. In sum, our findings demonstrate that a balanced level of GJ coupling is essential for coordinated muscle activity and proper motor behavior.

Gap junctions (GJs), formed by connexins in vertebrates and innexins in invertebrates, enable direct ion flow between adjacent cells. GJ coupling is typically analyzed by electrophysiological methods, which, however, are invasive and cannot be conducted in intact animals. Here, we used all-optical methods to non-invasively investigate electrical coupling in body wall muscle (BWM) cells of intact Caenorhabditis elegans . We analyzed the roles of specific innexins ( unc-9, inx-11, inx-16 ) in BWMs, using the genetically encoded fluorescent voltage indicator QuasAr2 to assess spontaneous muscle activity. unc-9 mutants showed strongly reduced coupling and asynchronous activity, and this was accompanied by severe locomotion defects. Overexpression of the murine connexin Cx36 increased synchronization but disrupted locomotion. inx-16 mutants exhibited increased excitability of individual muscle cells, likely due to reduced leak currents, while inx-11 mutants displayed only moderate defects. Patch-clamp recordings confirmed altered action potentials and increased input resistance in inx-16 mutants. Additionally, we established a cell-specific optogenetic voltage clamp (cOVC) method, directly revealing changes in junctional conductance in live, non-dissected animals. In sum, our findings demonstrate that a balanced level of GJ coupling is essential for coordinated muscle activity and proper motor behavior. Significance statement: Coordination of cells in muscular organs requires electrical coupling via intercellular channels termed gap junctions (GJs). How this coupling contributes to coordinated activity like locomotion in an intact animal is difficult to assess, since commonly used electrophysiological methods require dissection, and are incompatible with behavior. Here, we developed all-optical electrophysiology methods to study GJ coupling in live C. elegans . Different innexin mutants, as well as overexpression of mammalian connexin Cx36, induced different locomotion defects and, compared to wild type, were accompanied by more or less electrical coupling and synchronicity of individual muscle cells. Loss of some innexins caused the ‘insulated’ muscle cells to be more excitable. Thus, GJ coupling is required for fine-tuning locomotion, and can be studied by voltage imaging.

Gap junctions (GJs) are intercellular channels that mediate electrical signals and transfer of small molecules. They are crucial for brain, heart, and other organ functions. While molecular structures of purified homomeric GJs are available, information of in situ structures is lacking. In vivo, GJs can form heteromers with different functionalities and may associate with other proteins. Here, we analyzed Caenorhabditis elegans GJs by cryo–electron tomography and subtomogram averaging. We observed hexagonal arrays of GJs at cellular junctions in primary embryonal cells that displayed distinct wide and narrow conformations. Moreover, we found a cap-like, cytosolic protein assembly enclosing the channel pore. We propose that the cap is formed by the stomatin UNC-1, known to interact with UNC-9 innexins. This is corroborated by matching AlphaFold3 models of UNC-1 multimers with our subtomogram average structure; by expressing GFP-tagged UNC-1, leading to cap structures with additional density; and by coarse-grained MD simulations. UNC-1/stomatin rings may affect GJ formation or functions, possibly beyond nematodes.

Effective optogenetic inhibition of neuronal activity requires tools that can reliably silence neurons across diverse conditions and cell types. Potassium-selective channelrhodopsins (KCRs) have emerged as promising alternatives to chloride-conducting channels for optogenetic inhibition of cellular excitability, but they exhibit dynamic ion selectivity shift under prolonged or intense illumination, causing activation. This inhibition to activation transition limits their utility for silencing circuits. Through behavioral and electrophysiological analyses in Drosophila , and C. elegans , we found that the C29D variant of KCR1 maintains the most stable potassium selectivity among tested variants. While other KCR variants showed excitation defects, KCR1-C29D consistently provided robust in vivo inhibition across cell types, illumination conditions, and species. Electrophysiological recordings revealed that while most KCRs show declining PK/PNa ratios during illumination, KCR1-C29D maintains stable ion selectivity, consistent with its robust silencing capability. This work resolves a key limitation of KCR optogenetics and shows that KCR1-C29D is a superior tool for reliable inhibition. Our findings offer valuable mechanistic and practical insights for the design of next generation optogenetic inhibitors.

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 homologue 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, the 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 phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂).

Chemical synaptic transmission at the neuromuscular junction (NMJ) is regulated by electrical activity of the motor circuit, but may also be affected by neuromodulation. Here, we assessed the role of neuropeptide signaling in the plasticity of NMJ function in Caenorhabditis elegans. We show that the CAPS (Ca²⁺-dependent activator protein for secretion) ortholog UNC-31, which regulates exocytosis of dense core vesicles, affects both pre- and post-synaptic functional properties, as well as NMJ-mediated locomotion. Despite reduced evoked acetylcholine (ACh) transmission, the loss of unc-31 results in a more vigorous response to presynaptic stimulation, i.e., enhanced muscle contraction and Ca²⁺ transients. Based on expression profiles, we identified neuropeptides involved in both cholinergic (FLP-6, FLP-15, NLP-9, NLP-15, NLP-21, and NLP-38) and GABAergic motor neurons (FLP-15, NLP-15), that mediate normal transmission at the NMJ. In the absence of these peptides, neurons fail to upregulate their ACh output in response to increased cAMP signaling; for flp-15; nlp-15 double mutants, we observed overall increased postsynaptic currents, indicating that these neuropeptides may be inhibitory. We also identified proprotein convertases encoded by aex-5/kpc-3 and egl-3/kpc-2 that act synergistically to generate these neuropeptides. We propose that postsynaptic homeostatic scaling, mediated by increased muscle activation, likely through excitability, might compensate for the reduced cholinergic transmission in mutants affected for neuropeptide signaling, thus maintaining net synaptic strength. We show that in the absence of UNC-31 muscle excitability is modulated by upregulating the expression of the muscular L-type voltage-gated Ca²⁺ channel EGL-19. Our results unveil a role for neuropeptidergic regulation in synaptic plasticity, linking changes in presynaptic transmission to compensatory changes in muscle excitability.

Among the most frequent applications of optogenetic tools are those for depolarization of excitable cells such as neurons and muscles. Equally important are inhibitory tools that act through cellular hyperpolarization, which often rely on chloride conductance. Yet, in vivo, re- and hyperpolarization is typically mediated by potassium. Recently, light-gated ion channels with high potassium selectivity were identified (Kalium channelrhodopsins, KCRs), and their use described in different organisms. Here, we characterized HcKCR1 and WiChR in cholinergic neurons and muscles of the nematode Caenorhabditis elegans. Hyperpolarization of both cell types induces muscle relaxation and elongation of the animals. We analyzed body length changes during illumination, 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 maintained inhibition for the duration of the stimulus in some transgenic strains. Electrophysiological characterization verified that initial hyperpolarization quickly changed to depolarization, and current-voltage relationships indicate that the initial channel selectivity for potassium became progressively tolerant for sodium during the light pulse, scaling with light intensity. For animals expressing WiChR, we again observed brief hyperpolarization during continuous illumination; however, still during the stimulus, this changed to depolarization. This effect was long-lasting, requiring 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 expression and illumination parameters.

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.

Ultraviolet (UV) light is dangerous to unpigmented organisms, inducing photodamage of cells and DNA. The transparent nematode Caenorhabditis elegans detects light and exhibits negative phototaxis in order to evade sunlight. UV absorption is detected by the photosensor protein LITE-1 that also responds to reactive oxygen species. We investigated which neurons express LITE-1 and act as noxious photosensors and how they transmit this sensation to the nervous system to evoke escape behavior. We identified the interneuron AVG as a main focus of LITE-1 function in mediating the noxious light-evoked escape behavior, with minor roles of the interneuron PVT, the sensory ASK neurons, and touch receptor neurons. AVG is activated by blue light, and also its optogenetic stimulation causes escape behavior, while its optogenetic inhibition reduced escape. Signaling from AVG involves chemical neurotransmission, likely directly to premotor interneurons, and to other cells, by extrasynaptic signaling through the neuropeptide NLP-10. NLP-10 signaling is somewhat required for the acute response, yet is more important for maintaining responsiveness to repeated noxious stimuli. The source of NLP-10 in this context is largely AVG, yet also other cells contribute, possibly ASK. We uncover entry points of sensory information to neuronal circuits mediating noxious UV/blue light responses.

Gap junctions (GJ) are intercellular channels that mediate electrical signals and the transfer of small molecules. GJs are crucial for the functions of the brain, heart and other organs. While structures of purified homomeric GJs are available, we lack in situ structures. In vivo , GJs can form heteromers with different functionalities, and may associate with other proteins. Here, we analyzed Caenorhabditis elegans GJs by cryo-electron tomography and sub-tomogram averaging. We observed hexagonal arrays of GJs at cellular junctions in primary embryonal cell culture that displayed distinct wide and narrow conformations. Moreover, in about 20% of the observed channels, we found a cap-like, cytosolic protein assembly enclosing the channel pore. We propose that the cap-structure is formed by the stomatin UNC-1, which is known to interact with C. elegans GJs, and strengthen this hypothesis by matching AlphaFold3 models of UNC-1 multimers with our GJ average. Furthermore, expressing UNC-1 and the C. elegans innexin UNC-9 in HEK cells resulted in similar structures at cell-cell contacts. UNC-1/stomatin ring assemblies may affect GJ formation or functions like rectification, that might be evolutionarily conserved. Significance Statement: Gap junction (GJ) channels connect neighboring cells. Structures of (purified) GJs have been studied in vitro , but not in situ . We identified GJ channels in primary Caenorhabditis elegans cells by cryo-electron tomography, and analyzed their structure by sub-tomogram averaging. The channels transverse the membranes of connected cells, and AlphaFold3 (AF3) models of the GJ subunit UNC-9, assuming dodecamers, fit the experimentally obtained surface map well. We observed a cytosolic ‘cap’ structure on the GJ channels. The stomatin protein UNC-1 is known to physically interact with UNC-9 GJs. AF3 models of UNC-1 hexadecamers fit the cap structure, indicating that it may be formed by UNC-1, providing a first idea how UNC-1 interacts with, and may functionally influence, GJ channels.