Barkan CL , Kelley DB , Zornik E .

R15 NS091977 NINDS NIH HHS , R01 NS023684 NINDS NIH HHS

             male courtship behavior

	Fig 1 A vocal CPG underlies divergent vocalizations in X. laevis and X. petersii. A, Schematic representation of the known hindbrain vocal central pattern generator nuclei. The vocal motor nucleus (n) IX–X contains vocal motor neurons. These neurons send their axons via the vocal (laryngeal) nerve to the larynx, the vocal effector organ. Fictive calling can be recorded from the vocal nerve using a suction electrode. Premotor neurons in the Xenopus PBX project monosynaptically to the vocal motor nucleus. In PBX, whole-cell electrodes can record activity associated with fictive calling. B, Representative example of X. laevis fictive calling consisting of a series of long, fast-rate CAP trills (∼60 Hz; blue box, Nerve) and a simultaneously recorded premotor vocal neuron (Neuron). Temporally expanded recording (dashed box) of a single X. laevis call with introductory trill (gray box) and fast trill (blue box) and corresponding premotor neuron activity (right). C, Representative example of X. petersii fictive calling consisting of a series of brief, fast-rate CAP trills (∼60 Hz, blue box, Nerve) and a simultaneously recorded premotor vocal neuron (Neuron). Temporally expanded recording (dashed box) of a single X. petersii fictive fast trill (blue box) and corresponding premotor neuron activity (right) are also shown.
	Fig 2 Two populations of premotor vocal cells in PBX. A–E, Schematics representing temporal characteristics of premotor vocal neuron activity (top traces, dark blue lines) relative to fictive fast trill (bottom traces, light blue lines) included in Ward's hierarchical clustering and PCA: the maximum depolarization during fast trill (A, arrow), the time between fast trill onset and the first spike in each burst of premotor firing (B, arrow), the percentage of spikes during fast trill (C, green arrows) as opposed to shortly before or after fast trill (C, black arrow), the percentage of spikes that preceded the nearest CAP (D, green arrows), and the times between each spike peak and the nearest CAP peak (positive times: E, green arrows; negative times: black arrow). F, Ward's hierarchical clustering separates cells into two populations in both species. G, FTNs tend to spike during fast trill (blue box), whereas EVNs tend to begin spiking before fast trill onset; in X. laevis, this occurs during introductory trill (gray box). Dashed boxes depict temporally expanded recordings of single calls for each cell type.
	Fig 3 PCA also separates PBX neurons into two groups: FTNs and EVNs. PC1 accounts for 52.5% of the variability, whereas PC2 accounts for 21.1%. FTNs (shaded in blue) are more tightly clustered than EVNs. The two cell types overlap along PC2 but not along PC1.
	Fig 4 FTN spikes precede CAPs during fast trill, but EVN spikes occur during or after CAPs. A, Representative examples of X. laevis (right) and X. petersii (left) FTN activity during fictive fast trill (blue box) and introductory trill (gray box; X. laevis only). Temporally expanded traces of individual spikes and corresponding CAPs are depicted in dashed boxes in bottom panel. Spikes are demarcated by blue dashed lines. In both species, FTN spikes occur during fictive fast trill and spikes precede CAPs. B, Representative examples of X. laevis (right) and X. petersii (left) EVN activity during fictive fast trill (blue box) and introductory trill (gray box; X. laevis only). Temporally expanded traces of individual spikes and corresponding CAPs are depicted in dashed boxes in the bottom panels. EVN spikes occur before fast trill onset and precede nerve CAPs during introductory trill. During fast trill, EVN spikes follow or coincide with associated nerve CAPs. EVNs cease spiking before fast trill offset. C, In FTNs (closed circles) the percentage of spikes that precede CAPs during fast trill is significantly higher than in EVNs (open triangles) for X. laevis [purple; FTNs (n = 14) 82.1 ± 13.2%; EVNs (n = 7), 41.7 ± 24.5) and X. petersii (green; FTNs (n = 11), 90.1 ± 12.7; EVNs (n = 11); LM: p < 0.0001 for all FTN vs EVN comparisons]. There is no effect of species (p > 0.05). In this and subsequent figures, data labeled with different lowercase letters are significantly different, black horizontal lines represent median values, and whiskers represent interquartile range. D, Spike to nearest CAP time is significantly more positive in FTNs (∼4 ms) than EVNs (∼0 ms) [X. laevis: FTNs (n = 14) 3.6 ± 1.4 ms; EVNs (n = 7), 0.68 ± 1.4; X. petersii: FTNs (n = 11), 4.0 ± 1.3; EVNs (n = 11), −0.03 ± 1.5; LM: p < 0.0001 for all FTN vs EVN comparisons]. There is no effect of species (p > 0.05).
	Fig 5 PBX neuron depolarizations differ across cell type and species. A, Representative recordings of FTNs, which all produced long-lasting depolarizations [purple in X. laevis (10 Hz low-pass filter), green in X. petersii (20 Hz low-pass filter)] coinciding with fictive fast trill. B, Representative recordings of EVNs, in which only 50% (X. laevis) or 42% (X. petersii) of cells produced long-lasting depolarizations. EVNs that produced a long-lasting depolarization are illustrated. For examples of EVNs without an associated long-lasting depolarization, see Figures 2G (bottom) and 4B. C, Depolarization amplitude (“C” in Fig. 5A) is significantly higher (LM: p < 0.0001) in FTNs (X. laevis n = 14, X. petersii n = 11) than EVNs (X. laevis n = 7, X. petersii n = 11) for all FTN versus EVN comparisons; there is no effect of species (p > 0.05). D, Depolarization duration (“D” in Fig. 5A) is significantly longer in X. laevis PBX neurons than X. petersii PBX neurons (GLM: p < 0.0001 for all comparisons). In X. laevis EVNs that exhibited depolarizations (n = 4), durations were significantly longer than X. laevis FTN depolarizations (n = 14; p < 0.001). Depolarization duration did not differ between FTNs (n = 11) and EVNs (n = 5) in X. petersii (p > 0.05). E, Depolarization period (“E” in Fig. 5A) is significantly longer in X. laevis PBX neurons than in X. petersii PBX neurons (GLM: p < 0.0001 for all comparisons). FTN and EVN depolarization periods did not differ between species (p > 0.05).
	Fig 6 Current injections reveal that FTN spike burst durations, but not other intrinsic properties, differ between species. A, Representative FTN responses to current steps in X. laevis (left) and X. petersii (right). Four current steps are shown: the most extreme negative step, rheobase, medium current levels (>110–150% rheobase) and high current levels (>150–200% rheobase). B, Burst duration of X. petersii FTNs is significantly shorter than X. laevis FTNs at medium and high current steps [medium: >110–150% rheobase; X. laevis FTNs (n = 11): 559.9 ± 738.8 ms; X. petersii FTNs (n = 9): 64.0 ± 31.7; GLM: p < 0.01 for all species comparisons; high: >150–200% rheobase; X. laevis FTNs (n = 8): 898.0 ± 782.2 ms; X. petersii FTNs (n = 7): 56.2 ± 29.8; GLM: p ≤ 0.006]. Asterisks (*) indicate statistically significant difference between species. C, Representative EVN responses to current steps in X. laevis and X. petersii EVNs. Four current steps are shown: the most extreme negative step, rheobase, medium current levels (>110–150% rheobase), and high current levels (>150–200% rheobase). D, Burst duration of EVNs in X. laevis and X. petersii do not differ across species at medium or high current steps [medium: X. laevis EVNs (n = 7): 1643.4 ± 545.7 ms; X. petersii EVNs (n = 9), 721.4 ± 853.4; GLM: p > 0.05; high: X. laevis EVNs (n = 8): 1260.6 ± 733.0; X. petersii EVNs (n = 7), 690.5 ± 823.3; GLM p > 0.05 for all species comparisons]. E, Input resistance does not differ across PBX neuron type or species [X. laevis: FTNs (n = 14), 240.3 ± 149.0 MOhm; EVNs (n = 8), 278.6 ± 162.6; X. petersii: FTNs (n = 11), 200.3 ± 58.1; EVNs (n = 12), 229.2 ± 97.6; GLM: p > 0.05 for all comparisons]. F, Resting potential does not differ across PBx neuron type or species [X. laevis: FTNs (n = 14), −74.2 ± 5.6 mV; EVNs (n = 8), −68.3 ± 4.2; X. petersii: FTNs (n = 11), −68.2 ± 5.0; EVNs (n = 12), −69.8 ± 3.5; LM: p > 0.05 for all comparisons]. G, Capacitance does not differ across PBX neuron type or species [X. laevis: FTNs (n = 9), 305.8 ± 130.1 pF; EVNs (n = 3), 480.1 ± 256.4; X. petersii: FTNs (n = 6), 286.3 ± 125.4; EVNs (n = 7), 334.4 ± 149.4; LM: p > 0.05 for all comparisons]. H, Sag index does not differ across PBX neuron type or species [X. laevis: FTNs (n = 15), 0.094 ± 0.051; EVNs (n = 8), 0.143 ± 0.142; X. petersii: FTNs (n = 11), 0.096 ± 0.051; EVNs (n = 12), 0.134 ± 0.066; LM: p > 0.05 for all comparisons]. I, Spike threshold does not differ across PBX neuron type or species [X. laevis: FTNs (n = 13), 680.8 ± 467.8 pA; EVNs (n = 8), 363.8 ± 178.3; X. petersii: FTNs (n = 10), 463.2 ± 282.1; EVNs (n = 11), 478.6 ± 242.4; GLM: p > 0.05 for all comparisons). FTNs (n = 6), 286.3 ± 125.4; EVNs (n = 7), 334.4 ± 149.4; LM p > 0.05 for all comparisons]. J, Action potential half-width does not differ across PBX neuron type or species [X. laevis: FTNs (n = 15), 0.67 ± 0.18 ms; EVNs (n = 8), 0.86 ± 0.54; X. petersii: FTNs (n = 11), 0.91 ± 0.27; EVNs (n = 9), 0.70 ± 0.14; LM: p > 0.05 for all comparisons].
	Fig 7 FTNs, but not EVNs, intrinsically encode species-specific call duration and period. A, Representative FTN activity (Neuron) in X. laevis and X. petersii during fictive calling (Nerve). All FTNs displayed characteristic long-lasting depolarizations (X. laevis, n = 14; X. petersii, n = 11; see Fig. 5). Representative oscillations of the same FTN in response to NMDA in the presence of TTX (+TTX/NMDA; X. laevis, n = 7; X. petersii, n = 5). B, As observed for long-lasting depolarizations during fictive calling, NMDA-induced oscillation durations were significantly longer in X. laevis than in X. petersii (GLM: p < 0.0001 for all comparisons). Asterisks (*) indicate statistically significant difference between species. C, As observed for long-lasting depolarizations during fictive calling, the NMDA-induced oscillation periods were significantly longer in X. laevis than in X. petersii (GLM: p < 0.0001 for all comparisons). Asterisks (*) indicate statistically significant difference between species. D, Representative EVN activity (Neuron) in X. laevis and X. petersii during fictive calling (Nerve) and NMDA-induced oscillations of the same EVN (+TTX/NMDA; X. laevis, n = 3; X. petersii, n = 5). EVNs displayed long-lasting depolarizations ∼50% of the time (X. laevis, n = 4/7; X. petersii, n = 5/11), so we were only able to compare EVN oscillations directly with long-lasting depolarizations in two cells (one in each species). However, all EVNs held stably throughout the protocol, regardless of whether they produced a long-lasting depolarization during fictive calling, oscillated in response to NMDA in the presence of TTX. E, NMDA-induced oscillation duration of EVNs did not differ across species (GLM: p > 0.05 for all comparisons). F, NMDA-induced oscillation period of EVNs did not differ across species (GLM: p > 0.05 for all comparisons).

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