Syringeal Emgs and Synthetic Stimuli Reveal a Switch-Like Activation of the Songbird's Vocal Motor Program
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Syringeal EMGs and synthetic stimuli reveal a switch- like activation of the songbird’s vocal motor program Alan Busha,b,1, Juan F. Döpplera,b, Franz Gollerc, and Gabriel B. Mindlina,b aDepartamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires CP 1428, Argentina; bInstituto de Física de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires CP 1428, Argentina; and cDepartment of Biology, University of Utah, Salt Lake City, UT 84112 Edited by Harvey J. Karten, University of California, San Diego, La Jolla, CA, and approved July 10, 2018 (received for review January 23, 2018) The coordination of complex vocal behaviors like human speech BOS (11). This suggests that these synthetic songs are ideally and oscine birdsong requires fine interactions between sensory suited to study the activation dynamics of the song system by and motor programs, the details of which are not completely auditory stimuli: good enough to evoke the highly specific re- understood. Here, we show that in sleeping male zebra finches sponse, yet sufficiently different as to produce measurable (Taeniopygia guttata), the activity of the song system selectively changes in that response. Moreover, the parameters in the model evoked by playbacks of their own song can be detected in the used to generate the synthetic songs can be continuously varied syrinx. Electromyograms (EMGs) of a syringeal muscle show to explore changes in the activation dynamics under a smooth playback-evoked patterns strikingly similar to those recorded dur- degradation of the stimulus. ing song execution, with preferred activation instants within the Measuring the sparse firing pattern of few individual neurons song. Using this global and continuous readout, we studied the gives limited information on the overall activity of the system. We activation dynamics of the song system elicited by different audi- can overcome this difficulty by measuring efferent motor com- tory stimuli. We found that synthetic versions of the bird’s song, mands that provide a global, continuous, and graded readout of ’ rendered by a physical model of the avian phonation apparatus, the song system s activity. In this regard, the electrical activity of A evoked very similar responses, albeit with lower efficiency. Mod- syringealis ventralis (vS), the largest muscle in the syrinx (Fig. 1 ), ifications of autogenous or synthetic songs reduce the response has been measured during song execution (12, 13). Remarkably, probability, but when present, the elicited activity patterns match this muscle spontaneously activates during sleep, showing variable execution patterns in shape and timing, indicating an all-or-nothing patterns which sometimes resemble the execution pattern (14). activation of the vocal motor program. In this work, we measure electromyograms (EMGs) of the vS muscle during song execution and nocturnal auditory playback experiments. This enables the study of the overall activation zebra finch | song system | sensory–motor integration | syrinx | dynamics of the song system elicited by autogenous and synthetic electromyogram stimuli with high temporal resolution. he cooperation between different individuals, or the ability to Results Tperform coordinated and even synchronic actions, is at the We implanted electrodes in the syrinx of male zebra finches foundation of many species’ success. This requires a delicate obtaining EMG signals with amplitudes of [0.4–1.7] mV before interaction between sensory and motor programs, at specific amplification and dynamic ranges around 30 dB compared with regions of the nervous system. In this work, we explore some baseline fluctuations. Implants lasted from a few days up to 3 wk, aspects of this link, in the framework of oscine song production. showing a mild reduction of EMG amplitude over time in some Our model will be the elicitation of motor-like patterns in the cases. We confirmed electrode placement in vS by postmortem oscine song system when exposed, during sleep, to their own examinations. In some animals we observed an electrocardiogram- song. The avian auditory pathway indirectly connects to the song like (ECG) component in the signal (SI Appendix,Fig.S1A and B), system, a set of neural nuclei necessary to generate the motor gestures required for phonation (Fig. 1A; reviewed in ref. 1). Significance Neurons of HVC and RA (two of these nuclei) show a stereo- typed sparse spiking pattern elicited by playback of autogenous song in sleeping or anesthetized animals, firing at few specific The study of the integration between sensory inputs and mo- instants of the song (2–6). In zebra finches and white-crowned tor commands has greatly benefited from the finding that in sparrows these responses are highly specific to the bird’s own sleeping oscine birds, playback of their own song evokes highly song (BOS) because they are not evoked by a reversed version of specific firing patterns in neurons also involved in the pro- BOS, and similar conspecific songs, tone bursts, or combinations duction of that song. Nevertheless, the sparse spiking patterns of simple stimuli only evoke weak responses, if any (3, 7, 8). that can be recorded from few single neurons gives limited Furthermore, the spiking times are almost identical to those of information of the overall activity of the song system. Here, we the same neurons measured during song execution (4, 6). Se- show that this response is not restricted to the central nervous lective responses have also been observed in motor neurons of system but reaches vocal muscles. Combining this integrated the hypoglossal motor nucleus (nXIIts) that innervate the syrinx, A measure of the activity of the system with surrogate synthetic the avian vocal organ (9, 10) (Fig. 1 ). The highly selective re- songs, we found an all-or-nothing or switch-like activation of sponse of the song system to playbacks provides an excellent the song system. experimental opportunity to study its activation by auditory stimuli and will be our tool to explore the integration between Author contributions: A.B., F.G., and G.B.M. designed research; A.B. and J.F.D. performed the auditory and motor programs. research; A.B. analyzed data; and A.B. and G.B.M. wrote the paper. One way to understand how the song system is activated by The authors declare no conflict of interest. this stimulus is to study the response to slight variations of BOS that maintain the capacity to activate the system but in a slightly This article is a PNAS Direct Submission. deteriorated way. In this regard, synthetic songs generated with a Published under the PNAS license. mathematical model of the avian vocal organ (SYN) have been 1To whom correspondence should be addressed. Email: [email protected]. shown to evoke firing patterns in HVC similar to those evoked by This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. BOS (11). Nevertheless, the probability of any given neuron of 1073/pnas.1801251115/-/DCSupplemental. responding to SYN was at most 60% of that of responding to Published online August 1, 2018. 8436–8441 | PNAS | August 14, 2018 | vol. 115 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1801251115 Downloaded by guest on October 2, 2021 AC also capable of eliciting selective vS activity, consistent with the re- HVC Nif LMAN vS motif overlays sponse observed in HVC (11). L EMG 10 RA X 8 6 Correlation Analysis Reveals Short Delays of the Evoked Syringeal DLM 4 Uva (kHz) Freq. 2 Response. For each stimulus, we approximated the response pat- Ov DM 40 terns as the 95th percentile of vS activity at each time point of the MLd 30 A nXIIts LL 20 motif (referred to as BOS95,SYN95,CON95, and REV95;Fig.3 ). 10 CN We then cross-correlated these patterns to the median vS activity vS act. (dB) 0 -0.1 0.0 0.1 0.2 0.3 0.4 0.50.6 0.7 during motif execution (EXE50;Fig.3B). BOS95 and SYN95 had Syrinx Hair Time (s) Pearson’s correlation coefficient ranging from 0.63 to 0.83 (Fig. 3C cells and SI Appendix,TableS1), with no significant difference among B motif 1 motif 2 motif 3 motif 4 D them (P = 0.20), but both were significantly higher than those of 10 0.2 −5 8 CON95 and SYN95 (P < 10 , ANOVA with orthogonal contrasts). 6 baseline 4 0.1 ThetimedelaysofBOS95 and SYN95, with respect to the execution Density Freq. (kHz) Freq. 2 pattern EXE , were less than 30 ms in all instances, with some 0.0 50 40 -10 0 10 20 30 40 50 C 30 cases showing no detectable delay (Fig. 3 ). There was no signif- vS activity (dB) 20 icant difference in delay times between SYN95 and BOS95 (P = 10 t vS act. (dB) 0 0.43, paired test). 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Upon closer examination, we noticed that for some animals the Time (s) time delay of individual responses was not constant (Fig. 3 D and E Fig. 1. vS EMGs during song execution. (A, Top Left) Schematic represen- ). To visualize how the delay varies along the motif we con- structed correlation spectrograms, in which local correlation co- tation of the nuclei of the auditory pathway (violet), song motor pathway D (blue), and anterior forebrain pathways (red). (Bottom Right) Schematic efficients to EXE50 are shown for all times and delays (Fig. 3 , representation of the syrinx and associated muscles with approximate im- Bottom). In this particular example, the delay of maximal corre- plantation point of the bipolar electrodes in vS (red). (B, Top) Spectrogram lation increases linearly with time, reaching values close to 40 ms, of a typical song of a male zebra finch.