Biomechanics of sound production in high-pitched classical singing.

Voice production of humans and most mammals is governed by the MyoElastic-AeroDynamic (MEAD) principle, where an air stream is modulated by self-sustained vocal fold oscillation to generate audible air pressure fluctuations. An alternative mechanism is found in ultrasonic vocalizations of rodents, which are established by an aeroacoustic (AA) phenomenon without vibration of laryngeal tissue. Previously, some authors argued that high-pitched human vocalization is also produced by the AA principle. Here, we investigate the so-called "whistle register" voice production in nine professional female operatic sopranos singing a scale from C6 (≈ 1047 Hz) to G6 (≈ 1568 Hz). Super-high-speed videolaryngoscopy revealed vocal fold collision in all participants, with closed quotients from 30 to 73%. Computational modeling showed that the biomechanical requirements to produce such high-pitched voice would be an increased contraction of the cricothyroid muscle, vocal fold strain of about 50%, and high subglottal pressure. Our data suggest that high-pitched operatic soprano singing uses the MEAD mechanism. Consequently, the commonly used term "whistle register" does not reflect the physical principle of a whistle with regard to voice generation in high pitched classical singing.

Male and female syringeal muscles exhibit superfast shortening velocities in zebra finches.

Vocalisations play a key role in the communication behaviour of many vertebrates. Vocal production requires extremely precise motor control, which is executed by superfast vocal muscles that can operate at cycle frequencies over 100 Hz and up to 250 Hz. The mechanical performance of these muscles has been quantified with isometric performance and the workloop technique, but owing to methodological limitations we lack a key muscle property characterising muscle performance, the force-velocity relationship. Here, we quantified the force-velocity relationship in zebra finch superfast syringeal muscles using the isovelocity technique and tested whether the maximal shortening velocity is different between males and females. We show that syringeal muscles exhibit high maximal shortening velocities of 25L0 s-1 at 30°C. Using Q10-based extrapolation, we estimate they can reach 37-42L0 s-1 on average at body temperature, exceeding other vocal and non-avian skeletal muscles. The increased speed does not adequately compensate for reduced force, which results in low power output. This further highlights the importance of high-frequency operation in these muscles. Furthermore, we show that isometric properties positively correlate with maximal shortening velocities. Although male and female muscles differ in isometric force development rates, maximal shortening velocity is not sex dependent. We also show that cyclical methods to measure force-length properties used in laryngeal studies give the same result as conventional stepwise methodologies, suggesting either approach is appropriate. We argue that vocal behaviour may be affected by the high thermal dependence of superfast vocal muscle performance.

Evolutionary novelties underlie sound production in baleen whales.

Baleen whales (mysticetes) use vocalizations to mediate their complex social and reproductive behaviours in vast, opaque marine environments1. Adapting to an obligate aquatic lifestyle demanded fundamental physiological changes to efficiently produce sound, including laryngeal specializations2-4. Whereas toothed whales (odontocetes) evolved a nasal vocal organ5, mysticetes have been thought to use the larynx for sound production1,6-8. However, there has been no direct demonstration that the mysticete larynx can phonate, or if it does, how it produces the great diversity of mysticete sounds9. Here we combine experiments on the excised larynx of three mysticete species with detailed anatomy and computational models to show that mysticetes evolved unique laryngeal structures for sound production. These structures allow some of the largest animals that ever lived to efficiently produce frequency-modulated, low-frequency calls. Furthermore, we show that this phonation mechanism is likely to be ancestral to all mysticetes and shares its fundamental physical basis with most terrestrial mammals, including humans10, birds11, and their closest relatives, odontocetes5. However, these laryngeal structures set insurmountable physiological limits to the frequency range and depth of their vocalizations, preventing them from escaping anthropogenic vessel noise12,13 and communicating at great depths14, thereby greatly reducing their active communication range.

Quantitative assessment of food safety interventions for Campylobacter spp. and Salmonella spp. along the chicken meat supply chain in Burkina Faso and Ethiopia.

Rural and small-scale chicken farming is a major source of income in most African countries, and chicken meat is an important source of nutrients. However, chicken meat can be contaminated with Campylobacter spp. and Salmonella spp., pathogens with a high reported burden of foodborne illnesses. Therefore, it is essential to control these pathogens in chicken meat. Quantitative microbial risk assessments (QMRA) can aid the development of effective food safety control measures and are currently lacking in chicken meat supply chains in the African context. In this study, we developed stochastic QMRA models for Salmonella spp. and Campylobacter spp. in the chicken meat supply chain in Burkina Faso and Ethiopia employing the modular process risk model in @Risk software. The study scope covered chicken farming, transport, slaughtering, consumer handling, and consumption. Effectiveness of candidate interventions was assessed against baseline models' outputs, which showed that the mean annual Campylobacter spp. risk estimates were 6482 cases of illness per 100,000 persons and 164 disability adjusted life years (DALYs) per 100,000 persons in Burkina Faso, and 12,145 cases and 272 DALYs per 100,000 persons in Ethiopia. For Salmonella spp., mean annual estimates were 2713 cases and 1212 DALYs per 100,000 persons in Burkina Faso, and 4745 cases and 432 DALYs per 100,000 persons in Ethiopia. Combining interventions (improved hand washing plus designated kitchen utensils plus improved cooking) resulted in 75 % risk reduction in Burkina Faso at restaurants and 93 to 94 % in Ethiopia at homes for both Salmonella spp. and Campylobacter spp. For Burkina Faso, adding good hygienic slaughter practices at the market to these combined interventions led to over 91 % microbial risk reduction. Interventions that involved multiple food safety actions in a particular step of the supply chain or combining different interventions from different steps of the supply chain resulted in more risk reduction than individual action interventions. Overall, this study demonstrates how diverse and scanty food supply chain information can be applied in QMRA to provide estimates that can be used to stimulate risk-based food safety action in African countries.

An adaptable, reusable, and light implant for chronic Neuropixels probes.

Electrophysiology has proven invaluable to record neural activity, and the development of Neuropixels probes dramatically increased the number of recorded neurons. These probes are often implanted acutely, but acute recordings cannot be performed in freely moving animals and the recorded neurons cannot be tracked across days. To study key behaviors such as navigation, learning, and memory formation, the probes must be implanted chronically. An ideal chronic implant should (1) allow stable recordings of neurons for weeks; (2) be light enough for use in mice; (3) allow reuse of the probes after explantation. Here, we present the "Apollo Implant", an open-source and editable device that meets these criteria and accommodates up to two Neuropixels 1.0 or 2.0 probes. The implant comprises a "payload" module that is attached to the probe and is recoverable, and a "docking" module that is cemented to the skull. The design is adjustable, making it easy to change the distance between probes, the angle of insertion, and the depth of insertion. We tested the implant across seven labs in head-fixed mice, freely moving mice, and freely moving rats. The number of neurons recorded across days was stable, even after repeated implantations of the same probe. The Apollo implant provides an inexpensive, lightweight, and flexible solution for reusable chronic Neuropixels recordings.

Daily vocal exercise is necessary for peak performance singing in a songbird.

Vocal signals, including human speech and birdsong, are produced by complicated, precisely coordinated body movements, whose execution is fitness-determining in resource competition and mate choice. While the acquisition and maintenance of motor skills generally requires practice to develop and maintain both motor circuitry and muscle performance, it is unknown whether vocal muscles, like limb muscles, exhibit exercise-induced plasticity. Here, we show that juvenile and adult zebra finches (Taeniopygia castanotis) require daily vocal exercise to first gain and subsequently maintain peak vocal muscle performance. Experimentally preventing male birds from singing alters both vocal muscle physiology and vocal performance within days. Furthermore, we find females prefer song of vocally exercised males in choice experiments. Vocal output thus contains information on recent exercise status, and acts as an honest indicator of past exercise investment in songbirds, and possibly in all vocalising vertebrates.

Peak performance singing requires daily vocal exercise in songbirds.

Vocal signals mediate much of human and non-human communication. Key performance traits - such as repertoire size, speed and accuracy of delivery - affect communication efficacy in fitness-decisive contexts such as mate choice and resource competition 1 . Specialized fast vocal muscles 2,3 are central to accurate sound production 4 , but it is unknown whether vocal, like limb muscles 5,6 , need exercise to gain and maintain peak performance 7,8 . Here, we show that for song development in juvenile songbirds, the closest analogue to human speech acquisition 9 , regular vocal muscle exercise is crucial to achieve adult peak muscle performance. Furthermore, adult vocal muscle performance reduces within two days of abolishing exercise, leading to downregulation of critical proteins transforming fast to slower muscle fibre types. Daily vocal exercise is thus required to both gain and maintain peak vocal muscle performance, and if absent changes vocal output. We show that conspecifics can detect these acoustic changes and females prefer the song of exercised males. Song thus contains information on recent exercise status of the sender. Daily investment in vocal exercise to maintain peak performance is an unrecognized cost of singing and could explain why many birds sing daily even under adverse conditions 10 . Because neural regulation of syringeal and laryngeal muscle plasticity is equivalent, vocal output may reflect recent exercise status in all vocalizing vertebrates.

Toothed whales use distinct vocal registers for echolocation and communication.

Echolocating toothed whales (odontocetes) capture fast-moving prey in dark marine environments, which critically depends on their ability to generate powerful, ultrasonic clicks. How their supposedly air-driven sound source can produce biosonar clicks at depths of >1000 meters, while also producing rich vocal repertoires to mediate complex social communication, remains unknown. We show that odontocetes possess a sound production system based on air driven through nasal passages that is functionally analogous to laryngeal and syringeal sound production. Tissue vibration in different registers produces distinct echolocation and communication signals across all major odontocete clades, and thus provides a physiological basis for classifying their vocal repertoires. The vocal fry register is used by species from porpoises to sperm whales for generating powerful, highly air-efficient echolocation clicks.

Rare case of an acute liver torsion.

Torsion of an abdominal organ often leads to an acute abdomen requiring emergency surgery. This report describes the rare case of an acute liver torsion in a 76-year-old man. Surgery revealed dislocation of the left liver lobe, which was flipped over to the right upper abdomen. There was a hypermobile and long falciform ligament and absence of the triangular ligaments. The liver was manually flipped back with subsequent fixation of the umbilical ligament to the diaphragm to prevent reoccurrence. The patient had an uneventful recovery and is doing well 3 months after surgery with good liver function.

Dynamic System Coupling in Voice Production.

Voice is a major means of communication for humans, non-human mammals and many other vertebrates like birds and anurans. The physical and physiological principles of voice production are described by two theories: the MyoElastic-AeroDynamic (MEAD) theory and the Source-Filter Theory (SFT). While MEAD employs a multiphysics approach to understand the motor control and dynamics of self-sustained vibration of vocal folds or analogous tissues, SFT predominantly uses acoustics to understand spectral changes of the source via linear propagation through the vocal tract. Because the two theories focus on different aspects of voice production, they are often applied distinctly in specific areas of science and engineering. Here, we argue that the MEAD and the SFT are linked integral aspects of a holistic theory of voice production, describing a dynamically coupled system. The aim of this manuscript is to provide a comprehensive review of both the MEAD and the source-filter theory with its nonlinear extension, the latter of which suggests a number of conceptual similarities to sound production in brass instruments. We discuss the application of both theories to voice production of humans as well as of animals. An appraisal of voice production in the light of non-linear dynamics supports the notion that voice production can best be described with a systems view, considering coupled systems rather than isolated contributions of individual sub-systems.

A flexible carbon nanotube electrode array for acute in vivo EMG recordings.

Executing complex behaviors requires precise control of muscle activity. Our understanding of how the nervous system learns and controls motor skills relies on recording electromyographic (EMG) signals from multiple muscles that are engaged in the motor task. Despite recent advances in tools for monitoring and manipulating neural activity, methods for recording in situ spiking activity in muscle fibers have changed little in recent decades. Here, we introduce a novel experimental approach to recording high-resolution EMG signals using parylene-coated carbon nanotube fibers (CNTFs). These fibers are fabricated via a wet spinning process and twisted together to create a bipolar electrode. Single CNTFs are strong, extremely flexible, small in diameter (14-24 µm), and have low interface impedance. We present two designs to build bipolar electrode arrays that, due to the small size of CNTF, lead to high spatial resolution EMG recordings. To test the EMG arrays, we recorded the activity of small (4 mm length) vocal muscles in songbirds in an acute setting. CNTF arrays were more flexible and yielded multiunit/bulk EMG recordings with higher SNR compared with stainless steel wire electrodes. Furthermore, we were able to record single-unit recordings not previously reported in these small muscles. CNTF electrodes are therefore well-suited for high-resolution EMG recording in acute settings, and we present both opportunities and challenges for their application in long-term chronic recordings.NEW & NOTEWORTHY We introduce a novel approach to record high-resolution EMG signals in small muscles using extremely strong and flexible carbon nanotube fibers (CNTFs). We test their functionality in songbird vocal muscles. Acute EMG recordings successfully yielded multiunit recordings with high SNR. Furthermore, they successfully isolated single-unit spike trains from CNTF recordings. CNTF electrodes have great potential for chronic EMG studies of small, deep muscles that demand high electrode flexibility and strength.

Patient-related healthcare costs for diarrhoea, Guillain Barré syndrome and invasive non-typhoidal salmonellosis in Gondar, Ethiopia, 2020.

BACKGROUND: Globally, foodborne diseases result in a significant disease burden with low- and middle-income countries disproportionately affected. Estimates of healthcare costs related to foodborne disease can aid decision makers to take action to mitigate risks and prevent illness. However, only limited data on the African continent are available, especially related to more severe sequelae. We provide estimates of direct and indirect (non)-medical costs of patients with diarrhoea, Guillain-Barré syndrome (GBS), and invasive non-typhoidal salmonellosis (iNTS) in three healthcare facilities in Gondar, Ethiopia. METHODS: We used healthcare data from patient records, interviews with family caregivers and 2020 healthcare resource unit costs. Descriptive statistical analysis was performed. For diarrhoea, differences in mean and median transformed costs between healthcare facilities and etiologies (Campylobacter spp., enterotoxigenic Escherichia coli, non-typhoidal Salmonella enterica) were analysed with ANOVA and chi squared tests. Contribution of healthcare facility, dehydration severity, sex, age and living area to transformed costs was identified with linear regression. Results are in 2020 USD per patient. To extrapolate to national level, 2017 national incidence estimates were used. RESULTS: Mean direct medical costs were 8.96 USD for diarrhoea (health centre 6.50 USD, specialised hospital 9.53 USD, private clinic 10.56 USD), 267.70 USD for GBS, and 47.79 USD for iNTS. Differences in costs between diarrhoea patients were mainly associated with healthcare facility. Most costs did not differ between etiologies. Total costs of a diarrhoea patient in the specialised hospital were 67 USD, or 8% of gross national income per capita. For direct medical plus transport costs of a GBS and iNTS patient in the specialised hospital, this was 33% and 8%, respectively. Of the 83.9 million USD estimated national non-typhoidal Salmonella enterica related cost, 12.2% was due to iNTS, and of 187.8 million USD related to Campylobacter spp., 0.2% was due to GBS. CONCLUSION: Direct medical costs per patient due to GBS and iNTS were 30 respectively five times those due to diarrhoea. Costs of a patient with diarrhoea, GBS or iNTS can be a substantial part of a household's income. More severe sequalae can add substantially to cost-of-illness of foodborne hazards causing diarrheal disease.

Bats expand their vocal range by recruiting different laryngeal structures for echolocation and social communication.

Echolocating bats produce very diverse vocal signals for echolocation and social communication that span an impressive frequency range of 1 to 120 kHz or 7 octaves. This tremendous vocal range is unparalleled in mammalian sound production and thought to be produced by specialized laryngeal vocal membranes on top of vocal folds. However, their function in vocal production remains untested. By filming vocal membranes in excised bat larynges (Myotis daubentonii) in vitro with ultra-high-speed video (up to 250,000 fps) and using deep learning networks to extract their motion, we provide the first direct observations that vocal membranes exhibit flow-induced self-sustained vibrations to produce 10 to 95 kHz echolocation and social communication calls in bats. The vocal membranes achieve the highest fundamental frequencies (fo's) of any mammal, but their vocal range is with 3 to 4 octaves comparable to most mammals. We evaluate the currently outstanding hypotheses for vocal membrane function and propose that most laryngeal adaptations in echolocating bats result from selection for producing high-frequency, rapid echolocation calls to catch fast-moving prey. Furthermore, we show that bats extend their lower vocal range by recruiting their ventricular folds-as in death metal growls-that vibrate at distinctly lower frequencies of 1 to 5 kHz for producing agonistic social calls. The different selection pressures for echolocation and social communication facilitated the evolution of separate laryngeal structures that together vastly expanded the vocal range in bats.

Are We Facing the End of Gross Anatomy Teaching as We Have Known It for Centuries?

The status of anatomy education in undergraduate medical education has dramatically changed over the course of the past century. From the most important and time-consuming component of the preclinical program, anatomy education has reduced in size and status, and yielded in curricular space to accommodate other disciplines and topics. Meanwhile, radiology has become more prominent, as a means to visualize anatomy, not only in clinical care but also in education. For this perspective paper, the authors, all with backgrounds in anatomy, radiology and/or medical education, conducted structured conversations with several academic colleagues with similar backgrounds, reviewed pertinent literature and analyzed the causes of the historical decline of a knowledge domain of medical education, that nevertheless is widely considered essential for medical students and graduates. After this analysis, the authors propose four ways forward. These directions include systematic peer teaching and development of anatomy education as a scholarly domain, further vertical integration with postgraduate medical education, full integration with radiology education, and capitalizing on educational technology. Schools in several industrialized countries have made steps in these directions, which can be further strengthened. In less affluent countries, and in countries with curricula strongly determined by tradition, these steps are less easy to make. To respond to changes in global health and health care, combined with the inevitable technological progress, and international mobility, we believe all schools will move in these directions, slower or faster.

Driving singing behaviour in songbirds using a multi-modal, multi-agent virtual environment.

Interactive biorobotics provides unique experimental potential to study the mechanisms underlying social communication but is limited by our ability to build expressive robots that exhibit the complex behaviours of birds and small mammals. An alternative to physical robots is to use virtual environments. Here, we designed and built a modular, audio-visual 2D virtual environment that allows multi-modal, multi-agent interaction to study mechanisms underlying social communication. The strength of the system is an implementation based on event processing that allows for complex computation. We tested this system in songbirds, which provide an exceptionally powerful and tractable model system to study social communication. We show that pair-bonded zebra finches (Taeniopygia guttata) communicating through the virtual environment exhibit normal call timing behaviour, males sing female directed song and both males and females display high-intensity courtship behaviours to their mates. These results suggest that the environment provided is sufficiently natural to elicit these behavioral responses. Furthermore, as an example of complex behavioral annotation, we developed a fully unsupervised song motif detector and used it to manipulate the virtual social environment of male zebra finches based on the number of motifs sung. Our virtual environment represents a first step in real-time automatic behaviour annotation and animal-computer interaction using higher level behaviours such as song. Our unsupervised acoustic analysis eliminates the need for annotated training data thus reducing labour investment and experimenter bias.

Potential risk factors for the presence of anti-Toxoplasma gondii antibodies in finishing pigs on conventional farms in the Netherlands.

BACKGROUND: The parasite Toxoplasma gondii (T. gondii) causes a substantial human disease burden worldwide. Ingesting improperly cooked pork containing T. gondii is considered one of the major sources of human infection in Europe and North America. Consequently, control of T. gondii infections in pigs is warranted. The European Food Safety Authority advised to perform serological monitoring of pigs and to conduct farm audits for the presence of risk factors. Serological monitoring was implemented in several Dutch slaughterhouses, one to six blood samples (a total of 5134 samples) were taken from each delivery of finishing pigs and samples were tested for the presence of anti-T. gondii antibodies. Using these test results, a cross-sectional study was initiated to assess the association between the within-herd T. gondii seroprevalence and the presence of risk factors for T. gondii infections at 69 conventional finishing pig farms in the Netherlands. RESULTS: A multivariable model showed significant (P ≤ 0.05) association with twelve potential risk factors: type of farm, presence of dogs, presence of ruminants, use of boots, use of shower and farm clothing, mode of rodent control, bedding accessibility for rodents, presence of cats, type of drinking water, heating of the feed, use of goat whey and shielding of birds. CONCLUSIONS: Serological monitoring of finishing pigs for T. gondii in slaughterhouses can be used to identify the presence of T. gondii risk factors on Dutch conventional finishing pig farms and seems a valuable tool to guide and monitor the control of T. gondii in pork production.

Aerodynamics and motor control of ultrasonic vocalizations for social communication in mice and rats.

BACKGROUND: Rodent ultrasonic vocalizations (USVs) are crucial to their social communication and a widely used translational tool for linking gene mutations to behavior. To maximize the causal interpretation of experimental treatments, we need to understand how neural control affects USV production. However, both the aerodynamics of USV production and its neural control remain poorly understood. RESULTS: Here, we test three intralaryngeal whistle mechanisms-the wall and alar edge impingement, and shallow cavity tone-by combining in vitro larynx physiology and individual-based 3D airway reconstructions with fluid dynamics simulations. Our results show that in the mouse and rat larynx, USVs are produced by a glottal jet impinging on the thyroid inner wall. Furthermore, we implemented an empirically based motor control model that predicts motor gesture trajectories of USV call types. CONCLUSIONS: Our results identify wall impingement as the aerodynamic mechanism of USV production in rats and mice. Furthermore, our empirically based motor control model shows that both neural and anatomical components contribute to USV production, which suggests that changes in strain specific USVs or USV changes in disease models can result from both altered motor programs and laryngeal geometry. Our work provides a quantitative neuromechanical framework to evaluate the contributions of brain and body in shaping USVs and a first step in linking descending motor control to USV production.

Effectiveness and costs of interventions to reduce the within-farm Toxoplasma gondii seroprevalence on pig farms in the Netherlands.

BACKGROUND: The parasite Toxoplasma gondii (T. gondii) is recognized as one of the major foodborne pathogens with a high human disease burden. To control T. gondii infections in pigs, European Food Safety Agency (EFSA) advises serological testing of pigs and audits of pig farms to identify risk factors for T. gondii infection. In line with this approach, the aim of the current study was to assess the effectiveness and costs of intervention measures implemented to reduce the T. gondii seroprevalence on finishing pig farms in the Netherlands. A crossover clinical trial was conducted at five case farms were their own control and the cross-over moment was the implementation of interventions to reduce risk factors. Each of the case farms had a farm-specific intervention strategy with one principal intervention measure (neutering of cats, professional rodent control or covering food storage). RESULTS: All finishing pig farms (n = 5) showed a reduction in T. gondii seroprevalence within one year of implementing the intervention strategy. Cat neutering (n = 3) and feed coverage (n = 1) showed statistically significant reductions in seroprevalence. Rodent control (n = 1) did not show a statistically significant reduction. The estimated reduction in seroprevalence in response to the neutering of cats and feed coverage were 67 and 96 %, respectively. CONCLUSIONS: Our work demonstrates that it is possible to reduce the within-farm T. gondii seroprevalence within one year after interventions were implemented to reduce T. gondii risk factors. This information is essential and encouraging for policy makers, food business operators, and farmers to implement in their risk assessment and to apply to food safety control systems.

Quarter of pig farmers readily viewed information on Toxoplasma gondii infection in pigs provided in a data exchange system of a Dutch slaughter company.

Toxoplasma gondii (T. gondii) is a food safety hazard causing a substantial human disease burden. Because infected pig meat is estimated to attribute 12 % to this disease burden, it is important to control T. gondii infection in pigs. Providing pig farmers with information on T. gondii infection in general, and more specific on the status on their farm, could motivate them to take actions. In this study, we analysed the strategy pig farmers used to view specific T. gondii information provided for the first time on webpages in an existing data exchange system of a Dutch pig slaughter company. The available information for farmers comprised a webpage displaying the farm-level T. gondii seroprevalence and a webpage with information on risk sources and control measures for T. gondii infection in pigs and on human health consequences of a T. gondii infection. A total of 1404 owners of pig farms logged in the data exchange system. Of these, a quarter viewed the webpage with information on T. gondii seroprevalence, and about of third of them also viewed the webpage with the information on risk sources and control measures. T. gondii seroprevalence exceeded 2.0 % at only 0.6 % of these 1404 farms. The seroprevalence level on a particular farm neither influenced the likelihood of the farmer viewing the webpage with the T. gondii seroprevalence, nor the likelihood of them continuing to the webpage with the additional information. In the days when the pop-up message was included, the number of views registered on the seroprevalence and the additional information webpages rose nine and two times, respectively. Since the majority of views was in the period with a pop-up message pointing to this information we conclude that a targeted pop-up might help to transfer needed information to farmers with higher T. gondii seroprevalence at farm-level. More general, our study provides valuable insight into pig farmers' viewing strategies of new information on food safety hazards provided in a slaughter data exchange system.

One-to-one innervation of vocal muscles allows precise control of birdsong.

The motor control resolution of any animal behavior is limited to the minimal force step available when activating muscles, which is set by the number and size distribution of motor units (MUs) and muscle-specific force. Birdsong is an excellent model system for understanding acquisition and maintenance of complex fine motor skills, but we know surprisingly little about how the motor pool controlling the syrinx is organized and how MU recruitment drives changes in vocal output. Here we developed an experimental paradigm to measure MU size distribution using spatiotemporal imaging of intracellular calcium concentration in cross-sections of living intact syrinx muscles. We combined these measurements with muscle stress and an in vitro syrinx preparation to determine the control resolution of fundamental frequency (fo), a key vocal parameter, in zebra finches. We show that syringeal muscles have extremely small MUs, with 40%-50% innervating ≤3 and 13%-17% innervating a single muscle fiber. Combined with the lowest specific stress (5 mN/mm2) known to skeletal vertebrate muscle, small force steps by the major fo controlling muscle provide control of 50-mHz to 7.3-Hz steps per MU. We show that the song system has the highest motor control resolution possible in the vertebrate nervous system and suggest this evolved due to strong selection on fine gradation of vocal output. Furthermore, we propose that high-resolution motor control was a key feature contributing to the radiation of songbirds that allowed diversification of song and speciation by vocal space expansion.