Two conserved vocal central pattern generators broadly tuned for fast and slow rates generate species-specific vocalizations in Xenopus clawed frogs

  1. Ayako Yamaguchi
  2. Manon Peltier

  • Curated by eLife

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    This important paper compares the neural basis for different calling songs in five species of clawed Xenopus frogs using neural activity recordings combined with lesions of pathways and stimulation of specific parts of the circuit. The evidence supporting the claims is mostly solid but in part incomplete. The work will be of broad interest to neurophysiologists beyond the vocalization topic.

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Abstract

Across phyla, males often produce species-specific vocalizations to attract females. Although understanding the neural mechanisms underlying behavior has been challenging in vertebrates, we previously identified two anatomically distinct central pattern generators (CPGs) that drive the fast and slow clicks of male Xenopus laevis, using an ex vivo preparation that produces fictive vocalizations . Here, we extended this approach to four additional species, X. amieti, X. cliivi, X. petersii, and X. tropicalis, by developing ex vivo brain preparation from which fictive vocalizations are elicited in response to a chemical or electrical stimulus. We found that even though the courtship calls are species-specific, the CPGs used to generate clicks are conserved across species. The fast CPGs, which critically rely on reciprocal connections between the parabrachial nucleus and the nucleus ambiguus, are conserved among fast-click species, and slow CPGs are shared among slow-click species. In addition, our results suggest that testosterone plays a role in organizing fast CPGs in fast-click species, but not in slow-click species. Moreover, fast CPGs are not inherited by all species but monopolized by fast-click species. The results suggest that species-specific calls of the genus Xenopus have evolved by utilizing conserved slow and/or fast CPGs inherited by each species.

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  1. Version published to 10.7554/elife.86299 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    “The abstract does not adequately summarize the content of the paper. There is no mention of stimulation, or bilateral connectivity, which is a large part of the paper. The names of all five species should appear in the abstract, not just X. laevis.”

    In the revised manuscript, we have included all the names of the species and types of stimuli used to elicit fictive vocalizations in the abstract. In regard to bilateral connectivity, we believe that the reviewer was referring to the rostral-caudal connections between the parabrachial nucleus and nucleus ambiguus, which are critical for fast, but not for slow trill production. We have added this piece of information in the abstract. Furthermore, we have clarified the bilateral nature of the two central pattern generators (CPGs) in male X. laevis. In our previous study (Yamaguchi et al., 2017), we demonstrated that transections of the two commissures (one at the parabrachial nuclei level and the other at the nucleus ambiguus level) did not eliminate fictive advertisement calls in male X. laevis brains, indicating the presence of fast and slow trill CPGs in left and right hemi-brains of male X. laevis. This information was originally included in the results section (“Unilateral transection desynchronizes the fast clicks, but not the slow clicks across species”). We have now added this information to the introduction section to provide a clearer description of the anatomical organization of the two CPGs (p5, ln10 – 14).

    “The conclusion that the "fast and slow CPGs identified in male X. laevis are conserved across species." is contradicted by the last paragraph, which states, "Fast trill-like CPGs are likely present only in fast clickers..." This inherent contradiction needs to be resolved.”

    To resolve this contradiction, we have revised sentences in the abstract to clarify our findings. Specifically, we now state that “We found that even though the courtship calls of different Xenopus species vary in their click rates and duration, the CPGs used to generate clicks are conserved across species. The fast CPGs found in male X. laevis, which critically rely on reciprocal connections between the parabrachial nucleus and the nucleus ambiguus, are conserved among species that produce fast clicks. Similarly, the slow CPGs found in the caudal brainstem of male X. laevis are shared among species that produce slow clicks” (p2, ln 10 – 15) By making this change, we hope to provide greater clarity regarding our findings and help to resolve any contradictions.

    “The testosterone results are over-emphasized.” “The conclusion that there is differential expression of testosterone receptors in the brain of each species is completely speculative and not supported by the data presented here.”

    We have extensively revised our manuscript to ensure a more accurate interpretation of the results regarding testosterone experiments. Revised conclusions are outlined below:

    Abstract: “In addition, our results suggest that testosterone plays a role in organizing fast CPGs in fast-click species, but it does not appear to have the same effect in slow-click species.” (p2, ln 15 – 17)

    Introduction: “Additionally, we found that fast trill-like CPGs are present only in species that produce fast clicks and their presence appears to be regulated by testosterone in these species. “ (p6, ln 2 – 4)

    Discussion: “However, this effect of testosterone appears to be limited to the fast clicker species. Male X. tropicalis, a slow clicker species, has been shown to have comparable plasma levels of testosterone to male X. laevis (mean plasma levels of testosterone of male X. laevis: 13 to 22ng/ml (Hecker et al., 2005; Hayes et al., 2010), male X. tropicalis: ~20ng/ml, (Olmstead et al., 2009)), yet the synapses between the PBN and laryngeal motoneurons in male X. tropicalis remained weak, and PBN showed little activity during fictive advertisement calls. These results suggest that testosterone acts differently on the central vocal pathways of fast and slow clickers, promoting the emergence of fast trill-like CPGs in X. laevis but not in X. tropicalis. Although further experiments with controlled testosterone levels are necessary to validate these results, we hypothesize that changes in the androgen receptors (e.g., expression patterns, ligand affinity) may have contributed to the divergence of fast and slow clickers.“ (p26, ln 13 – 24)

    ”The use of the word "development" implies embryology. Here, adults were treated and looked at 13 weeks later. There is no data presented about development. ”

    In our revised manuscript, we have replaced the term “development” with “presence” or “acquisition” of neural circuitry to enhance the clarity and help to prevent any potential misunderstandings.

  3. eLife

    eLife assessment

    This important paper compares the neural basis for different calling songs in five species of clawed Xenopus frogs using neural activity recordings combined with lesions of pathways and stimulation of specific parts of the circuit. The evidence supporting the claims is mostly solid but in part incomplete. The work will be of broad interest to neurophysiologists beyond the vocalization topic.

  4. eLife

    Reviewer #1 (Public Review):

    The authors compared the neural mechanisms of calling song in five Xenopus species. Two (X. laevis and X. petersii) were previously shown to produce fictive calls. This paper developed the techniques to evoke fictive calls for three additional species: X. cliivi, X. amieti, and X. tropicalis. The authors compared fast and low components of the calls and determined that the fast components in all species required bilateral coordination in the parabrachial nucleus (PBN), but the slow components were produced in the nucleus ambiguous (presumably with bilateral control, but that was not tested.

    The abstract does not adequately summarize the content of the paper. There is no mention of stimulation, or bilateral connectivity, which is a large part of the paper. The names of all five species should appear in the abstract, not just X. laevis.

    The conclusion that the "fast and slow CPGs identified in male X. laevis are conserved across species." is contradicted by the last paragraph, which states, "Fast trill-like CPGs are likely present only in fast clickers..." This inherent contradiction needs to be resolved.

    The abstract also over-emphasizes the testosterone results. It states, "the development of fast CPGs [central pattern generators] depends on testosterone in a species-specific manner: testosterone facilitates the development of fast CPGs in a species with a courtship call containing fast clicks, but not in a species with a courtship call made entirely of slow clicks." The use of the word "development" implies embryology. Here, adults were treated and looked at 13 weeks later. There is no data presented about development. The effects of T could be simply to upregulate certain receptors of a circuit that was already present.

    The concluding sentence of the abstract is, "The results suggest that species-specific calls of the genus Xenopus have evolved by utilizing conserved fast or slow CPGs that are broadly tuned to generate fast or slow trains of clicks, the development of which appear to be regulated by a strategic expression of testosterone receptors in the brain of each species." However, testosterone treatment was only applied to X. laevis females. The conclusion is based on plasma levels of testosterone in X. tropicalis. The conclusion that there is differential expression of testosterone receptors in the brain of each species is completely speculative and not supported by the data presented here.

  5. eLife

    Reviewer #2 (Public Review):

    This study by Yamaguchi and Peltier provides a detailed investigation of the brainstem CPG functional organization that rules vocal behaviors in several Xenopus species, from an evolutionary perspective. The main conclusion of the paper reveals that vocal CPGs, located in the brainstem, generating fast and slow clicks in Xenopus male courtship calls are conserved across various Xenopus species. But the development of the fast CPG depends on testosterone only in species producing fast-click courtship calls.

  6. Version published to 10.1101/2023.01.27.525835v1 on bioRxiv