A genetically encoded tool for reconstituting synthetic modulatory neurotransmission and reconnect neural circuits in vivo

Current chemogenetic and optogenetic tools allow manipulation of existing neural circuits, but lack the capability to rationally rewrite circuits by creating or modifying synaptic connections. This research addresses this gap by introducing HySyn, a system designed to reconnect neural circuits in vivo by building synthetic modulatory neurotransmission. The researchers leverage the unique properties of the Hydra nervous system, a loosely connected nerve net employing neuropeptides for neurovascular neuromodulation. They hypothesize that reconstituting this peptidergic synapse in other systems will enable the examination of adjacent chemical synaptic relationships and the creation of functional connections between non-adjacent neurons. This would also allow for orthogonal neuromodulation of targeted endogenous circuits, mimicking the in vivo function of neuromodulatory systems. The development of HySyn is crucial because it offers a new way to understand how neuropeptides control specific neurons and provides a flexible tool for manipulating targeted behaviors. The system's design centers on a Hydra-derived Ramide-related peptide (HyRamide I/I) and its receptor (HyRac C/79), which are distinct from those in other organisms, ensuring orthogonality and minimizing interference with endogenous systems. By utilizing conserved neuropeptide processing, transport, and release mechanisms, HySyn aims for minimal components and broad applicability across biological contexts. The divergent evolution of this neuropeptide-receptor pair offers a unique advantage: while leveraging conserved cell biology, it produces a biophysically synthetic neocircuit where components remain functional outside their native context.

The authors review existing chemogenetic and optogenetic tools, highlighting their limitations in creating new synaptic connections. They then discuss previous research on neuropeptides and their role in neuromodulation, emphasizing the conserved mechanisms of neuropeptide synthesis, transport, and release across species. This background establishes the rationale for using a Hydra-derived neuropeptide-receptor pair for creating a novel, orthogonal neuromodulatory system. The literature review also touches upon prior approaches to labeling neuropeptides, specifically mentioning the use of natriuretic neuropeptide precursors. The authors build upon this knowledge to develop the HyPep system, a synthetic pre-pro-peptide carrier designed to utilize the endogenous neuropeptide processing pathway for efficient and targeted delivery of the heterologous neuropeptide.

The researchers designed HyPep, a pre-pro-peptide carrier, to facilitate heterologous expression, processing, and transport of the Hydra-derived neuropeptide. HyPep incorporates a signal peptide for trafficking, acidic spacers with enzymatic recognition sites, and the neuropeptide sequence itself. The signal peptide is based on neuropeptide Y, ensuring compatibility with the endogenous neuropeptide processing pathway. Artificial neuropeptide scaffolds with consensus cleavage sites for PC2 (prohormone convertase 2) were included. The efficacy of HyPep was tested by expressing it in Neuro2A cells, observing intracellular transport of a fluorescent reporter to expected compartments and release sites. Electrophysiology was used to test HySyn's functionality. 'Presynaptic' Neuro2A cells were co-expressed with HyPep and ChR2M (channelrhodopsin 2), and 'postsynaptic' cells with HyCal (Hydra neuropeptide receptor). Optogenetic stimulation of 'presynaptic' cells resulted in measurable 'postsynaptic' currents, demonstrating the creation of novel neuropeptidergic synaptic relationships. Calcium imaging confirmed the functional neuromodulation of postsynaptic cells, even with solution transfer experiments. In vivo studies in *C. elegans* involved expressing HySyn components in specific neurons (AIB interneurons) and muscle tissues. Confocal microscopy confirmed the subcellular localization of HySyn components to appropriate synaptic sites. Behavioral assays examined the effects of HySyn on worm locomotion using DeepLabCut, which quantifies worm postures. The study also investigated HySyn's potential for reconfiguring circuits by targeting the serotonergic NSM neuron and muscle tissues in *C. elegans*, which regulates the behavioral switch between roaming and dwelling states. The researchers used various assays to assess locomotion and dwelling behavior in different genotypes and HySyn configurations. The methodology includes detailed information on molecular biology techniques (plasmid construction), cell culture and transfection protocols, electrophysiology procedures, *C. elegans* transgenics and behavioral assays, off-food and on-food exploration assays, and head thrashing analysis using DeepLabCut.

The study successfully demonstrated the creation of functional synthetic neuromodulatory connections using HySyn in both in vitro and in vivo settings. In Neuro2A cells, optogenetic stimulation of HyPep-expressing cells induced distinct postsynaptic currents in co-cultured cells expressing HyCal, confirming the formation of novel neuropeptidergic synapses. Calcium imaging further validated this finding, showing that optogenetic stimulation increased intracellular calcium levels in HyCal-expressing cells. The localization of HySyn components to appropriate synaptic compartments in *C. elegans* was verified through confocal microscopy. Behaviorally, HySyn expression resulted in altered locomotion, with animals exhibiting severely uncoordinated movement and reduced velocity. The specificity of the system was confirmed by showing that the paralysis phenotype was suppressed in neuropeptide processing mutants. Furthermore, the ability of HySyn to reconfigure neural circuits in vivo was demonstrated by restoring a food-mediated dwelling state in *C. elegans* serotonin biosynthesis mutants by 'repairing' a broken neuromodulatory connection. The persistence of HySyn effects on locomotion was characterized, observing a gradual decay consistent with known neuropeptide half-lives. This finding shows how HySyn could be used to bias the relationship between synaptic input and postsynaptic neuronal calcium. The researchers note, however, that the neuromodulatory aspect could be a limitation in specific applications and suggest further investigation into this and other factors. Overall, HySyn effectively reconfigured neural circuits, altering animal behavior and offering a novel approach to dissect the role of neuromodulation in establishing neural circuit logic and connectivity.

The successful creation and characterization of HySyn offer a significant advance in neuroscience research. The system's ability to reconstitute synthetic neuromodulatory connections in both in vitro and in vivo models allows for unprecedented control and manipulation of neural circuits. HySyn's modularity and compatibility with existing techniques enhance its utility, and this methodology opens the door for investigating the genetic requirements of neuropeptide signaling and neuromodulation in various systems. Furthermore, HySyn's ability to reconfigure specific circuits provides a powerful tool to explore complex behavioral mechanisms. While the study showcases HySyn's potential, the authors acknowledge limitations, particularly concerning the neuromodulatory nature of the system and the need for further investigation into the kinetics of HySyn's effects in different contexts. This opens several avenues for future research, including a more in-depth examination of the temporal dynamics of HySyn's effects, its applications in different neuronal circuits and organisms, and its integration with other technologies for even more sophisticated circuit manipulations.

HySyn represents a significant advancement in the field of neuroscience, providing a novel tool for precisely manipulating and understanding the intricate workings of neural circuits. This work demonstrates the successful development and application of a system capable of creating functional connections between neurons, leading to altered behaviors in a living organism. Future studies could focus on exploring HySyn's potential in diverse neuronal systems and disease models, expanding the application of this technology in neuroscience research. The modularity and versatility of HySyn further enhance its potential for addressing many significant questions in circuit neuroscience and neurobiology.

The study's primary limitation is the focus on two model systems (Neuro2A cells and *C. elegans*). While these models provide valuable insights, further research is needed to determine HySyn's efficacy and applicability across other, more complex organisms. Additionally, the long-lasting effects of HySyn-mediated neuromodulation could be a limitation in applications where precise temporal control is crucial. Future studies should carefully evaluate and address this aspect to optimize HySyn's utility in various experimental paradigms. The neuromodulatory nature of HySyn means that its effects may be less specific than direct synaptic connections, potentially affecting other circuits or processes in unexpected ways. Careful controls and experimental designs are essential to fully understand the scope of HySyn's effects and to avoid misinterpretations.