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Abstract Gastropod molluscs such asAplysia,Lymnaea, andTritoniahave been important for determining fundamental rules of motor control, learning, and memory because of their large, individually identifiable neurons. Yet only a small number of gastropod neurons have known molecular markers, limiting the ability to establish brain‐wide structure–function relations. Here we combine high‐throughput, single‐cell RNA sequencing with in situ hybridization chain reaction in the nudibranchBerghia stephanieaeto identify and visualize the expression of markers for cell types. Broad neuronal classes were characterized by genes associated with neurotransmitters, like acetylcholine, glutamate, serotonin, and GABA, as well as neuropeptides. These classes were subdivided by other genes including transcriptional regulators and unannotated genes. Marker genes expressed by neurons and glia formed discrete, previously unrecognized regions within and between ganglia. This study provides the foundation for understanding the fundamental cellular organization of gastropod nervous systems.
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Molecular characterization of frog vocal neurons using constellation pharmacology
Identification and characterization of neuronal cell classes in motor circuits are essential for understanding the neural basis of behavior. It is a challenging task, especially in a non-genetic model organism, to identify cell-specific expression of functional macromolecules. Here, we performed constellation pharmacology, calcium imaging of dissociated neurons to pharmacologically identify functional receptors expressed by vocal neurons in adult male and female African clawed frogs, Xenopus laevis. Previously we identified a population of vocal neurons called fast trill neurons (FTNs) in the amphibian parabrachial nucleus (PB) that express NMDA receptors and GABA and/or glycine receptors. Using constellation pharmacology, we identified four cell classes of putative fast trill neurons (pFTNs, responsive to both NMDA and GABA/glycine applications). We discovered that some pFTNs responded to the application of substance P (SP), acetylcholine (ACh), or both. Electrophysiological recordings obtained from FTNs using an ex vivo preparation verified that SP and/or ACh depolarize FTNs. Bilateral injection of ACh, SP, or their antagonists into PBs showed that ACh receptors are not sufficient but necessary for vocal production, and SP receptors play a role in shaping the morphology of vocalizations. Additionally, we discovered that the PB of adult female X. laevis also contains all the subclasses of neurons at a similar frequency as in males, despite their sexually distinct vocalizations. These results reveal novel neuromodulators that regulate X. laevis vocal production, and demonstrate the power of constellation pharmacology in identifying the neuronal subtypes marked by functional expression of cell-specific receptors in non-genetic model organisms.
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- Award ID(s):
- 1934386
- PAR ID:
- 10213949
- Date Published:
- Journal Name:
- Journal of Neurophysiology
- ISSN:
- 0022-3077
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1152/jn.00105.2020
